SURFACE MODIFIED CARBON NANOTUBES AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/429,769, filed December 2, 2022, the entire contents of which are herein incorporated by reference.

BACKGROUND

Field of the Disclosure

[0002] In various embodiments, the present disclosure generally relates to carbon nanotube compositions, which are useful for delivering active agents, such as proteins, peptides, nucleic acids, antigens, vaccines, etc.

Government's Interests

[0003] The invention was made with government support under the following contract numbers: Contract No. W81XWH-17-C -0047 awarded by the US Army (US Army Medical Research Acquisition Activity (USAMRAA)); and 75N93019C00034 awarded by the National Institute of Health, NIH/NIAID. The U.S. government has certain rights in the invention.

Background

[0004] Newly emerging and reemerging infectious viral diseases or cancers have threatened humanity throughout history. An effective therapeutic or vaccine is required to end these diseases. However, one limitation for newly discovered drugs and therapeutics is a lack of optimized delivery systems or platforms. Before the drug or vaccine is effective in vivo, many critical barriers must be faced. This includes rapid filtration in the kidney and clearance via the reticulo-endothelial system (RES) — particularly for drugs that spend a lot of time in the bloodstream — as well as transport from the bloodstream to target cells within tissues. As a result, methods to increase the short in vivo half-life of drugs before action must be developed. Subsequently, at the tissue or cellular target, the drug must cross the plasma membrane, and within the cell, it must escape the harsh acidic environment of endolysosomes, within which biomolecules such as proteins and oligonucleotides may be inactivated or degraded. Other barriers are the nuclear membrane and multiple drug resistance mechanisms [1]. Recent studies illustrate some particularly promising ways in which nanomatcrials as drug or vaccine carriers can assist in navigating these banners, with a focus on administration a solution-based formulation by injection. Further, reducing or eliminating the toxicity and inflammatory response caused by delivery system/platform such as lipid-based delivery vehicles (used for COVID- 19 vaccinations) is critical. As a result, there is a necessity to formulate delivery systems with no or minimal toxicity to overcome these obstacles and provide functional vaccinations and therapeutic delivery.

[0005] The site for the administration is also very important. In the mucosal site, for example, intranasal administration not only can stimulate an immune response in saliva, nasal secretions or in the respiratory tract, but can also elicit a strong vaginal mucosal immune response. The efficacy of current vaccine delivery systems has been limited by mucosal barriers (e.g., pH and degrading enzymes) that limit the delivery of antigens to target tissues. Further delivery platforms need to be designed and developed for muco-adhesion, biodegradation, and immune cell-specific targeting; together, these characteristics will need to be developed for enhancing biological responses and prolonging the retention time in the biological systems.

BRIEF SUMMARY

[0006] The present disclosure is based, in part, on the discovery of a carbon nanotube-based delivery platform that is safe and biocompatible, and can be used to deliver various active agents such as proteins, peptides, antigens, nucleic acids, vaccines, etc. As detailed herein, the carbon nanotube delivery platform has numerous advantages and desired features, which include but not limited to the following:

1 ) The carbon nanotube delivery platform can be surface functionalized or internally encapsulated with biologies (multiple copies of the same or controlled ratios of different ones) such as drugs or antigens (nucleic acids, proteins, glycoproteins, cellular components, targeting peptides, etc.). This feature enables the intracellular uptake of multiple copies of antigens per targeted membrane permeation, which reduces the ratio between delivery vehicle and biologies to maximize the effective dose of antigens and the delivery efficiency. 2) Surface functionalization with chemistries highlighted in Table 1 also increases the solubility and biocompatibility of the carbon nanotube delivery vehicle.

3) A synthesis approach that can precisely control the carbon nanotubc size and aspect ratio (FIG. 2), allowing us to systemically investigate the dimensional effect on delivery efficiency and biological response was developed. The relevant biological responses include immune responses such as IgA, IgG, and IgM antibody responses generated locally in mucosal sites or tissues or systematically, any responses generated at the cellular level such as T cell responses, and related.

4) The formulations can be developed for all routes of administration, such as intramuscular, subcutaneous, intranasal administration, intraperitoneal or intradermal.

5) Multiple forms of formulations can be developed such as in a lyophilized powder form, solution based, gel based, etc.

6) Finally, the formulation can be functionalized for stabilization of the active agents such as small molecules, proteins, peptides, enzymes, nucleic acids, or other biologies from degradation. This includes stability of mRNA that is immobilized or bound to the surface of the delivery vehicle.

[0007] The present disclosure provides the following numbered exemplary embodiments 1-95:

Embodiment 1. A pharmaceutical composition comprising a surface functionalized carbon nanotube and an active agent, wherein the surface functionalized carbon nanotube is a carbon nanotube which is surface functionalized with one or more surface molecules selected from (i) a polyaminc, (ii) a hypcrbranchcd polymer or dendrimer, which has one or more groups that are charged or chargeable at pH of 7, (iii) a cationic lipid, which has one or more groups that are positively charged or positively chargeable at pH of 7, (iv) a surfactant, and (v) a hydrophilic linker, and wherein the carbon nanotube has carboxylic acid groups, preferably, has about 4-12% weight percentage of carboxylic acid groups, and wherein the carbon nanotube has an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio (length/diameter) of about 0.2: 1 to about 12,500: 1, wherein the average length and diameter are measured by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM), respectively. Embodiment 2. The pharmaceutical composition of Embodiment 1, wherein the carbon nanotube is a multi-walled carbon nanotube.

Embodiment 3. The pharmaceutical composition of Embodiment 1 or 2, wherein the carbon nanotube has an average diameter of about 30 nm to about 150 nm, such as about 60 nm to about 100 nm.

Embodiment 4. The pharmaceutical composition of any of Embodiments 1-3, wherein the carbon nanotube has an average length of about 40 nm to about 1000 nm, such as about 100 nm to about 200 nm, about 200 nm to about 400 nm, about 200 nm to about 500 nm, about 400 nm to about 800 nm, etc.

Embodiment 5. The pharmaceutical composition of any of Embodiments 1-4, wherein the length of the carbon nanotube is substantially uniform, for example, the carbon nanotube is monodisperse as measured by DLS.

Embodiment 6. The pharmaceutical composition of any of Embodiments 1-5, wherein the carbon nanotube has an average aspect ratio of about 1 : 1 to about 7: 1, such as about 1 : 1 to about 5: 1, or about 2: 1 to about 4: 1.

Embodiment 7. The pharmaceutical composition of any of Embodiments 1-6, wherein the surface functionalized carbon nanotube has a zeta potential with an absolute value of greater than 20 mV, preferably, greater than 40 mV, prior to binding the active agent.

Embodiment 8. The pharmaceutical composition of any of Embodiments 1-7, which is storage stable at 0°C for 1 month or more, preferably, for 3 months or more.

Embodiment 9. The pharmaceutical composition of any of Embodiments 1-8, wherein the surface functionalized carbon nanotubc is surface functionalized with the polyaminc.

Embodiment 10. The pharmaceutical composition of Embodiment 9, wherein the poly amine is a polyalkyleneimine, preferably, polyethyleneimine.

Embodiment 11. The pharmaceutical composition of Embodiment 10, wherein the polyethyleneimine is a branched or linear polyethyleneimine, for example, those having a number or weight average molecular weight (Mn or Mw) of about 500 to about 50,000 Daltons, such as about 600, about 1,000, about 2,000, about 2,500, about 10,000, about 25,000, about 35,000, about 50,000 Daltons, or any range or value between the recited values, preferably, the Mn or Mw is about 2,500 Daltons for the linear- polyethyleneimine, preferably, the Mn or Mw is about 25,000 Daltons for the branched polyethyleneimine.

Embodiment 12. The pharmaceutical composition of Embodiment 10 or 11, wherein the polyethyleneimine is a branched polyethyleneimine.

Embodiment 13. The pharmaceutical composition of any of Embodiments 9-12, wherein the polyamine is covalently bound to the carbon nanotube, e.g., through an amide formation between an amine group of the polyamine and a carboxylic acid group of the carbon nanotube.

Embodiment 14. The pharmaceutical composition of any of Embodiments 9-12, wherein the polyamine is non-covalently bound to the carbon nanotube.

Embodiment 15. The pharmaceutical composition of any of Embodiments 9-14, wherein the weight ratio of the polyamine to the carbon nanotube (prior to any functionalization with the one or more surface molecules) ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values.

Embodiment 16. The pharmaceutical composition of any of Embodiments 1-15, wherein the surface functionalized carbon nanotube is surface functionalized with the cationic lipid.

Embodiment 17. The pharmaceutical composition of Embodiment 16, wherein the cationic lipid is a multivalent cationic lipid.

Embodiment 18. The pharmaceutical composition of Embodiment 17, wherein the multivalent cationic lipid is selected from MVL-5 (Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3- amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide), DOSPA (2,3- diolcyloxy-N-[2-(spcrminccarboxamido)cthyl]-N,N-dimcthyl-l-p ropanaminium salt, such as chloride salt), and GL67 (N4-Cholesteryl-Spermine Salt, such as HC1 salt), preferably, the multivalent cationic lipid is MVL-5.

Embodiment 19. The pharmaceutical composition of Embodiment 16, wherein the cationic lipid is a phosphocholine lipid.

Embodiment 20. The pharmaceutical composition of Embodiment 19, wherein the cationic lipid is an ethyl phosphocholine lipid, such as 16:0 EPC (l,2-dipalmitoyl-sn-glycero-3- ethylphosphocholine), 12:0 EPC, 18:0 EPC, 18: 1 EPC, 14: 1 EPC, 16:0-18: 1 EPC (1- palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine), etc., preferably, the phosphocholine lipid is l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

Embodiment 21. The pharmaceutical composition of any of Embodiments 16-20, wherein the weight ratio of the cationic lipid to the carbon nanotube (prior to any functionalization with the one or more surface molecules) ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values.

Embodiment 22. The pharmaceutical composition of any of Embodiments 9-21, wherein the surface functionalized carbon nanotube is surface functionalized with both the polyamine and the cationic lipid.

Embodiment 23. The pharmaceutical composition of Embodiment 22, wherein the weight ratio of (a) the combined amount of the polyamine and the cationic lipid to (b) the carbon nanotube (prior to any functionalization with the one or more surface molecules), (a)/(b), ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values.

Embodiment 24. The pharmaceutical composition of any of Embodiments 1-23, wherein the surface functionalized carbon nanotube is surface functionalized with a polyamidoamine dendrimer, such as a G0-G10 polyamidoamine dendrimer.

Embodiment 25. The pharmaceutical composition of any of Embodiments 1-24, wherein the surface functionalized carbon nanotube is surface functionalized with the surfactant.

Embodiment 26. The pharmaceutical composition of Embodiment 25, wherein the surfactant is a PEGlated lipid, which comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, e.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, methoxy.

Embodiment 27. The pharmaceutical composition of Embodiment 25, wherein the surfactant is a phospholipid, such as a neutral or negatively charged phospholipid, containing a polyethylene glycol chain, such as l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000].

Embodiment 28. The pharmaceutical composition of any of Embodiments 25-27, wherein the surfactant is in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization), for example, about 0.025% to about 0.1% by weight of the carbon nanotube.

Embodiment 29. The pharmaceutical composition of any of Embodiments 1-28, wherein the surface functionalized carbon nanotube is surface functionalized with the hydrophilic linker.

Embodiment 30. The pharmaceutical composition of Embodiment 29, wherein the hydrophilic linker comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc.

Embodiment 31. The pharmaceutical composition of Embodiment 29 or 30, wherein the hydrophilic linker has an NH or NH2 group and is capable of attaching to the carbon nanotube through an amide bond formation between the NH or NH2 group of the hydrophilic linker and a carboxylic acid group of the carbon nanotube.

Embodiment 32. The pharmaceutical composition of any of Embodiments 29-31, wherein the hydrophilic linker attaches to the active agent, such as a small molecule, peptide, protein, or nucleic acid, through an amide bond, an ester bond, an ether bond, or a thioether bond or through a non-covalent interaction such as electrostatic interaction.

Embodiment 33. The pharmaceutical composition of any of Embodiments 1-32, wherein the active agent is covalently attached to the surface functionalized carbon nanotube, e.g., through the hydrophilic linker.

Embodiment 34. The pharmaceutical composition of any of Embodiments 1-33, wherein the active agent binds to the surface functionalized carbon nanotube noncovalently.

Embodiment 35. The pharmaceutical composition of any of Embodiments 1 -34, wherein the active agent is a nucleic acid, such as a DNA or RNA molecule, wherein the weight ratio of the nucleic acid to the carbon nanotube (weight prior to any functionalization) ranges from about 20:1 to 1:20, such as about 1: 1 to about 1:5 or about 1:5 to about 1:10.

Embodiment 36. The pharmaceutical composition of any of Embodiments 1-34, wherein the active agent is a peptide or protein, wherein the molar ratio of the peptide or protein to the carbon nanotube ranges from about 4000: 1 to 1:20, such as about 20: 1 to about 300:1; or the active agent is a small molecule, wherein the molar ratio of the small molecule to the carbon nanotube ranges from about 4000: 1 to 1 :20, such as about 20: 1 to about 300: 1. Embodiment 37. The pharmaceutical composition of any of Embodiments 1-34, wherein the active agent is an antigen, wherein the molar ratio of the antigen to the carbon nanotube ranges from about 4000: 1 to 1:20, such as about 20: 1 to about 300: 1.

Embodiment 38. The pharmaceutical composition of any of Embodiments 1-37, formulated for intramuscular or subcutaneous injection.

Embodiment 39. The pharmaceutical composition of any of Embodiments 1-37, formulated for intranasal administration.

Embodiment 40. The pharmaceutical composition of any of Embodiments 1-37, in the form of a lyophilized powder, a solution, a gel, or a suspension.

Embodiment 41. A carbon nanotube conjugate of Formula I:

(I), wherein:

CNT represents a carbon nanotube,

SPC represents a hydrophilic spacer,

LNK represents a group which connects SPC to D,

D represents a residue of an active agent of D-Y, wherein Y is a group (c.g., amino, carboxy, hydroxy, or SH group) that is capable of linking D to the SPC, preferably, the active agent represents a small molecule, peptide, protein, or nucleic acid; and n is the number of carboxy groups of the carbon nanotube that are functionalized with NH- SPC-LNK-D.

Embodiment 42. The carbon nanotube conjugate of Embodiment 41, wherein CNT represents a multi-walled carbon nanotube.

Embodiment 43. The carbon nanotube conjugate of Embodiment 41 or 42, wherein the carbon nanotube has an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio of about 0.2: 1 to about 12,500: 1, wherein the average length and diameter are measured by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM), respectively. Embodiment 44. The carbon nanotube conjugate of any of Embodiments 41-43, wherein the carbon nanotube has an average diameter of about 40 nm to about 150 nm, such as about 60 nm to about 100 nm.

Embodiment 45. The carbon nanotube conjugate of any of Embodiments 41-44, wherein the carbon nanotube has an average length of about 50 nm to about 1000 nm, such as about 100 nm to about 200 nm, about 200 nm to about 400 nm, about 200 nm to about 500 nm, about 400 nm to about 800 nm, etc.

Embodiment 46. The carbon nanotube conjugate of any of Embodiments 41-45, wherein the length of the carbon nanotube is substantially uniform, for example, the carbon nanotube is monodisperse as measured by DLS.

Embodiment 47. The carbon nanotube conjugate of any of Embodiments 41-46, wherein the functionalized carbon nanotube has an average aspect ratio of about 1: 1 to about 7: 1, such as about 1 : 1 to about 5: 1 , or about 2: 1 to about 4: 1.

Embodiment 48. The carbon nanotube conjugate of any of Embodiments 41-47, wherein the hydrophilic spacer comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc.

Embodiment 49. The carbon nanotube conjugate of any of Embodiments 41-48, wherein LNK is an amide group,

Embodiment 50. The carbon nanotube conjugate of any of Embodiments 41-49, wherein the active agent is a small molecule, peptide or protein.

Embodiment 51. The carbon nanotube conjugate of any of Embodiments 41-49, wherein the active agent is an antigen, such as an HIV antigen, such as an envelope glycoprotein antigen, e.g., gpl20 or V1V2 region of HIV-1 gpl20 protein/peptide.

Embodiment 52. The carbon nanotube conjugate of any of Embodiments 41-51, wherein the molar ratio of D to CNT ranges from about 10: 1 to about 1000: 1, such as about 20: 1 to about 300: 1, about 27: 1 to about 470:1, etc.

Embodiment 53. The carbon nanotube conjugate of any of Embodiments 41-52, further comprising a surfactant attached to the surface of the carbon nanotube noncovalently. Embodiment 54. The carbon nanotube conjugate of Embodiment 53, wherein the surfactant is a PEGlated lipid comprising a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, c.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, methoxy.

Embodiment 55. The carbon nanotube conjugate of Embodiment 54, wherein the surfactant is a phospholipid, such as a neutral or negatively charged phospholipid, containing the polyethylene glycol chain, such as l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000].

Embodiment 56. The carbon nanotube conjugate of any of Embodiments 53-55, wherein the surfactant is in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (exclusive of any functionalization), for example, about 0.025% to about 0.1% by weight of the carbon nanotube.

Embodiment 57. A pharmaceutical composition comprising the carbon nanotube conjugate of any of Embodiments 41-56.

Embodiment 58. The pharmaceutical composition of Embodiment 57, formulated for intramuscular injection or subcutaneous injection.

Embodiment 59. The pharmaceutical composition of Embodiment 57, formulated for intranasal administration.

Embodiment 60. The pharmaceutical composition of Embodiment 57, in the form of a lyophilized powder, a solution, a gel, or a suspension.

Embodiment 61. A method of preparing a vaccine composition for treating or preventing an infection with a microorganism (c.g., virus), the method comprising: a) surface functionalizing a carbon nanotube with one or more surface molecules to produce a surface functionalized carbon nanotube, wherein the one or more surface molecules are selected from (i) a polyamine, (ii) a hyperbranched polymer or dendrimer, which has one or more groups that are charged or chargeable at pH of 7, (iii) a cationic lipid, which has one or more groups that are positively charged or positively chargeable at pH of 7, (iv) a surfactant, and (v) a hydrophilic linker; b) combining the surface functionalized carbon nanotube with an antigen of the microorganism to form an antigen bound carbon nanotube; c) formulating the antigen bound carbon nanotube to provide the vaccine composition, wherein the carbon nanotube has carboxylic acid groups, preferably, has about 4-12% weight percentage of carboxylic acid groups, and wherein the carbon nanotubc has an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio of about 0.2:1 to about 12,500: 1, wherein the average length and diameter are measured by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM), respectively.

Embodiment 62. The method of Embodiment 61 , wherein the carbon nanotube is a multiwalled carbon nanotube.

Embodiment 63. The method of Embodiment 61 or 62, wherein the carbon nanotube has an average diameter and/or average length within 5-fold (e.g., within 1-fold, 2-fold, or within 3- fold) of that of the microorganism, such as a virus, respectively.

Embodiment 64. The method of any of Embodiments 61-63, wherein the length of the carbon nanotube is substantially uniform, for example, the carbon nanotube is monodisperse as measured by DLS.

Embodiment 65. The method of any of Embodiments 61-64, wherein the carbon nanotube has an average aspect ratio of about 1 : 1 to about 7: 1, such as about 1 : 1 to about 5: 1 , or about 2: 1 to about 4: 1.

Embodiment 66. The method of any of Embodiments 61-65, wherein the surface functionalization is carried out such that the surface functionalized carbon nanotube has an absolute zeta potential of greater than 20 mV, preferably, greater than 40 mV.

Embodiment 67. The method of any of Embodiments 61-66, wherein the carbon nanotubc is surface functionalized with one or more of the surface molecules as described in any of Embodiments 9-32.

Embodiment 68. The method of any of Embodiments 61-67, wherein the antigen is covalently attached to the carbon nanotube, e.g., through the hydrophilic linker.

Embodiment 69. The method of any of Embodiments 61-68, wherein the antigen binds to the carbon nanotube noncovalently.

Embodiment 70. The method of any of Embodiments 61-69, wherein the antigen is a nucleic acid, such as a DNA or RNA molecule, wherein the weight ratio of the nucleic acid to the carbon nanotube ranges from about 20: 1 to 1:20, such as about 1: 1 to about 1:5 or about 1:5 to about 1: 10.

Embodiment 71. The method of any of Embodiments 61-69, wherein the antigen is a peptide or protein, wherein the molar ratio of the peptide or protein to the carbon nanotube ranges from about 4000: 1 to 1:20, such as about 20: 1 to about 300: 1.

Embodiment 72. A method of preparing a vaccine composition for treating or preventing an infection with a microorganism (e.g., virus), the method comprising: a) conjugating a carbon nanotube having carboxylic acid groups with a compound having a Formula II: ^H 2 ^N ~-— ■— -^ structure of SPG ^G (II), wherein SPC represents a hydrophilic spacer, and G represents a group capable of forming a covalent bond with an antigen of the microorganism, preferably, the antigen is a peptide, protein, or nucleic acid, to form a functionalized carbon nanotube having a structure of Formula III:

(III); wherein:

CNT represents a carbon nanotube,

SPC and G are as defined above, and n is the number of carboxy groups of the carbon nanotube that are functionalized with the compound of Formula II; b) coupling the functionalized carbon nanotube of Formula III with the antigen of the microorganism to form a carbon nanotube conjugate of Formula IV:

(IV); wherein:

A represents (i) the antigen covalently attached to SPC through a bond formed with

G, or (ii) G or a derivative thereof; and CNT, SPC, G, and n are as defined above; c) formulating the carbon nanotube conjugate to provide the vaccine composition.

Embodiment 73. The method of Embodiment 72, wherein the carbon nanotubc is a multiwalled carbon nanotube.

Embodiment 74. The method of Embodiment 72 or 73, wherein the carbon nanotube has an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio of about 0.2: 1 to about 12,500: 1, wherein the average length and diameter are measured by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM), respectively.

Embodiment 75. The method of any of Embodiments 72-74, the carbon nanotube has an average diameter and/or average length within 5-fold (e.g., within 1-fold; 2-fold, or within 4- fold) of that of the microorganism, such as a virus, respectively.

Embodiment 76. The method of any of Embodiments 72-75, wherein the length of the carbon nanotube is substantially uniform, for example, the carbon nanotube is monodisperse as measured by DLS.

Embodiment 77. The method of any of Embodiments 72-76, wherein the carbon nanotube has an average aspect ratio of about 1 : 1 to about 7: 1, such as about 1 : 1 to about 5: 1 , or about 2: 1 to about 4: 1.

Embodiment 78. The method of any of Embodiments 72-77, wherein the carbon nanotube has about 4-12% carboxylic acid groups, by weight.

Embodiment 79. The method of any of Embodiments 72-78, wherein greater than 30% (such as greater than 50%, greater than 60%, up to all) of the carboxylic acid groups of the carbon nanotube are functionalized with the compound of Formula II.

Embodiment 80. The method of any of Embodiments 72-79, wherein greater than 30% (such as greater than 50%, greater than 60%, up to all) of the G group in Formula III is coupled with the antigen to form the carbon nanotube conjugate of Formula IV.

Embodiment 81. The method of any of Embodiments 72-80, wherein the hydrophilic spacer comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc.

Embodiment 82. The method of any of Embodiments 72-81, wherein G is NH2 or COOH. Embodiment 83. The method of any of Embodiments 72-82, wherein the antigen and G are coupled to form an amide bond.

Embodiment 84. The method of any of Embodiments 72-83, wherein the antigen is a peptide or protein.

Embodiment 85. The method of any of Embodiments 72-83, wherein the antigen is an HIV antigen, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V1V2 region of gpl20, or a nucleic acid encoding the same.

Embodiment 86. The method of any of Embodiments 72-85, wherein the molar ratio of the antigen to the carbon nanotube (exclusive of any functionalization), ranges from about 10: 1 to about 1000: 1, such as about 20: 1 to about 300: 1, about 27: 1 to about 470: 1, etc.

Embodiment 87. The method of any of Embodiments 72-86, further comprising absorbing a surfactant on the surface of the carbon nanotube.

Embodiment 88. The method of Embodiment 87, wherein the surfactant is a PEGlated lipid, which comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100. PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, e.g.. with an alkoxy group, such as a Ci-io alkoxy group, preferably, methoxy.

Embodiment 89. The method of Embodiment 88, wherein the surfactant is a phospholipid, such as a neutral or negatively charged phospholipid, containing the polyethylene glycol chain, such as l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000].

Embodiment 90. The method of Embodiment 88 or 89, wherein the surfactant is in an amount of about 0.01% to about 0.5% by weight of the carbon nanotubc (exclusive of any functionalization), for example, about 0.025% to about 0.1% by weight of the carbon nanotube.

Embodiment 91. The vaccine composition produced by any of the methods according to Embodiments 61-90.

Embodiment 92. The vaccine composition of Embodiment 91, formulated for intramuscular or subcutaneous injection.

Embodiment 93. The vaccine composition of Embodiment 91, formulated for intranasal administration. Embodiment 94. The vaccine composition of Embodiment 91, in the form of a lyophilized powder, a solution, a gel, or a suspension.

Embodiment 95. A method of treating or preventing an infection with a microorganism (c.g., virus), the method comprising administering an effective amount of the pharmaceutical composition of any of Embodiments 1-40 and 57-60, the carbon nanotube conjugate of any of Embodiments 41-56, or the vaccine composition of any of Embodiments 91-94.

[0008] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention herein.

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 shows a design of delivery vehicle optimized to mimic virion size and structure.

[0010] FIG. 2 presents schematic illustrations of CNT designed to have variable length, different diameters and tunable density of biological (mRNA, protein, peptide, etc.) loading.

[0011] FIG. 3 shows SEM images of carbon nanotubes before cutting and separation SO with length of 50 nm - 2 pm and after separation with different size range SI: 100-200 nm; S2: 200- 500nm and S3:400-800nm. Photograph of CNT solution of various length ranges from left to the right (1-4) is different range of short to long carbon nanotubes with different color. CNT solution is highly stable and purified.

[0012] FIG. 4 A shows surface functionalization and cutting of CNT process (Left); multiple step filtrations for CNT collection and purifications (right).

[0013] FIG. 4B shows a representative DLS analysis of a CNT composition.

[0014] FIG. 4C shows a representative zeta potential analysis of a CNT composition.

[0015] FIG. 5 shows images of ThPl DC cells incubated with 10 pg/ml of iFluor 594 dye labelled CNT- gpl20 A244 antigen (top panel) and antigen only (bottom panel) after 24 hours of incubation.

[0016] FIG. 6 shows microscope images of control THP-1 human monocyte cells and CNT- gpl20 A244 antigen uptaken by monocyte derived immature dendritic cells (DCs) by time and dose dependent manner. (The arrows showed the CNT uptake). Note: CNTs appear dark in microscopic images. [0017] FIG. 7 shows fluorescence microscopic images of cells transfected with GFP mRNA at 0.5 pg/ml, and CNT: RNA ratio at 10: 1, 20: 1 and 5: 1 for GFP production after 48 hours using different formulation.

[0018] FIG. 8 shows an optimized CNT-mRNA formulation.

[0019] FIG. 9 shows (A) The trimer of HIV Env design for the bNAbs binding epitopes PG9,

PGT128, etc. (B) The surface of V2i mAb 83OA epitope is illustrated on Env trimer. V1V2 regions are colored in magenta. The 830A-binding site surface is colored in orange. The surface of the PG9 epitope (colored in blue) is shown as a reference. (C) A zoom-in of the 830A epitope surface from an angle of view about 90 degree rotated from that in B.

[0020] FIG. 10 shows ELISA studies of broadly neutralizing antibodies PG9 (A), PGT128 (B) and mAb 83OA (C) responses to the two modified gpl20 antigens 6240A11 (1) and A244A11 (2) and their conjugates with long CNT (LCNT) and short CNT (SCNT) respectively.

[0021] FIG. 11 shows Antigenicity test of CNT-V 1 V2 formulations with and without PEG- Lipid final coating at low (0.025%), medium (0.05%) and high (0.075%) concentration of CNTs against PG9, a VlV2-specific mAb. PG9 was serially diluted and titration of PG9 to these formulations was analyzed to evaluate the antigenicity of immunogen VI V2 (ZM53)- 2F5K, CNT formulations, and multiple controls (CNT alone, PEG-Lipid alone, and CNT- PEG-Lipid mixture).

[0022] FIG. 12 shows Short carbon nanotube-based HIV vaccine. This novel bioinspired nanodelivery platform can be optimized for better mimicking HIV virion particles. It can be achieved by tuning the CNT properties (length, diameter, surface linker and functionalities).

[0023] FIG. 13 shows TEER changes were monitored using Calu-3 cells after exposure to different formulations at 0-24 hours. (B) A dye FITC labelled VI V2 protein flux across Calu- 3 monolayers and the concentrations in basolateral culture medium were measured at each time intervals 1, 2, 4, and 24 hours.

[0024] FIG. 14 shows Titration of immune mouse sera IgG (A) and IgA (B) against HIV-1 antigen VI V2 (ZM53)-2F5K in ELISA test for murine intranasal route immunogenicity study administrated using powder (Al, B l) and solution based (A2, B2) CNT-VlV2+PEG-Lipid formulations. Serum was drawn 2 weeks after each boost from individual animals and pooled into appropriate groups (5 animals per group). mAb 830 served as the positive control, while secondary human and mouse Abs were negative controls.

[0025] FIG. 15 shows Rabbits (5 rabbits per group) were intramuscularly administered with VI V2 protein alone (group A) and CNT-VlV2+PEG-Lipid formulation (group B) for three doses (1 dose every two weeks). ELISA tests were performed by titrations of rabbit immune sera (collected every 2 weeks) against VI V2 (ZM53)-2F5K to monitor VI V2-specific immune response.

[0026] FIG. 16 shows Rabbits (5 rabbits per group) were intranasally administered with VI V2 and CNT-VlV2+PEG-Lipid formulations for three doses (one does every two weeks). ELISA tests were carried out to evaluate VlV2-specific immune response by titration of sera IgG (Al: V1V2 alone, A2: CNT-VlV2+PEG-Lipid) every 2 weeks, IgA in urine (Bl) and vaginal washes (B2), and IgM in serum (Cl) and vaginal washes (C2) at the end of administration against V1V2 (ZM53)-2F5K.

[0027] FIG. 17 shows (A) ELISA sera evaluation of IgG responses for each group of vaccine candidates immunized HIS mouse against HIV-1 VI V2 antigen by either IM or IN administration routes. (B) Human IFN-gamma ELISpot Assay to evaluate CNT formulations vaccinated HIS-A2/DR4 mice. Each data point was averaged from triplicates subtracting from no antigen and naive mouse as control.

[0028] FIG. 18 shows ELISA sera evaluation of IgG responses for CNT-mRNA immunized rabbits against HIV-1 V1V2 (ZM53)-2F5K antigen.

[0029] FIG. 19 shows (A) Detection of Luciferase-labeled carbon nanotube (CNT) in Male BALB/c Mice by IVIS Imaging; (B) Whole body and snout region of interest (ROI) total flux measurements. A statistically significant increase in whole body and snout ROI total flux (p/s) was noted 4 hours post-dose. G indicates group number; *p<0.05, n=4.

[0030] FIG. 20 shows (A) CNTs in mice organs following intranasal injection after 4 and 48 hours. (B) Percent body weight change compared to Day 1 baseline weights. Mean values ± standard deviation is shown for each group and time point. * Significant difference (n-4). Statistical analysis of the data was performed using GraphPad Prism 6.04. Statistical comparisons of percent body weight change from Day 1 were compared by a Kruskal- Wallis one-way analysis of variance test followed by post-hoc testing using Dunn’s multiple comparisons test. In all cases, a p<0.05 was considered statistically significant.

[0031] FIG. 21 shows body weight and organ weight changes of rats during the experiment from day 2 to 42 days after administrated CNT at different doses by subcutaneous administration.

DET AILED DESCRIPTION

[0032] Delivery of nucleic acid or protein/peptide antigens is critical for vaccination and advanced therapeutic development in infectious diseases and treatment or prevention of diseases such as cancer, cardiovascular diseases or disorders, neurological disease or disorder such as Alzheimer’s disease, etc. To enhance delivery efficiency and biological response (e.g., immune response generated by vaccination), the present disclosure provides a carbon nanomaterial-based delivery platform that can deliver a broad range of drugs, antigens, and nucleic acids to treat diseases or prevent infections. The delivery platform can be designed to mimic virus particles, i.c. the dimension (length, diameter), surface protein density, orientation and presentation (Figure 1). This delivery platform can be easily recognized and efficiently uptaken by cells for the indue tion/stimulation and enhancement of biological responses such as immunogenicity and cell signaling. Further, these responses are stronger than when compared to delivery without the vehicle or using other commercial adjuvants/delivery vehicles such as Incomplete Freund's adjuvant (IFA).

[0033] Adjuvants or delivery systems are capable of stimulating different arms of the immune system and are vital components of subunit vaccines, especially in the case of poorly immunogenic envelope glycoprotein (Env) [2]. In accordance with their tailored structures, modular compositions, and controlled length scales, vaccine nanotechnologies possess versatile properties, including multivalent target delivery to lymphoid tissues and specific immune cells, multistage stimulating control release of immune components, key immune pathway engagement, and perfect iterative design systems. The rational structural designs of various types of nano vaccines are ranging from polymeric nanoparticles, nanogels, inorganic materials [3]. Antigen delivery systems optimize the presentation of antigens, and they also play a major role in solving the problem of presentation of multivalent vaccine mainly due to the viral mutants. Nanomaterials, with their defined compositions, commonly modular construction, and length scales allowing the engagement of key immune pathways, collectively facilitate the iterative design process necessary to identify such protective immune responses and achieve them reliably. Nanomaterials also provide strategies for engineering the trafficking and delivery of vaccine components to key immune cells and lymphoid tissues, and they can be highly multivalent, improving their engagement with the immune system [4],

[0034] The nucleoside-modified mRNA-LNP vaccine platform used by Pfizer/BioNTech and Modema in their SARS-CoV-2 vaccines has been widely tested in preclinical studies. These vaccines’ mRNA component is nucleoside modified to decrease potential innate immune recognition. The LNP was chosen as a carrier vehicle to protect the mRNA from degradation and aid intracellular' delivery and endosomal escape [5]. The LNPs consist of a mixture of phospholipids, cholesterol, PEGylated lipids, and cationic or ionizable lipids. The phospholipids and cholesterol have structural and stabilizing roles, whereas the PEGylated lipids support prolonged circulation. The cationic/ionizable lipids are included to allow the complexing of the negatively charged mRNA molecules and enable the exit of the mRNA from the endosome to the cytosol for translation [6]. However, the potential inflammatory nature of these LNPs was assessed recently that they were highly inflammatory in mice. Intradermal and intramuscular injection of these LNPs led to rapid and robust inflammatory responses. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate. Thus, the mRNA-LNP platforms’ potency in supporting the induction of adaptive immune responses and the observed side effects may stem from the LNPs’ highly inflammatory nature [7].

[0035] Luna Labs has pioneered the medical application of carbon nanomaterials including CNTs and fullerenes (Patent US7758889B1) for use as therapeutic agents. The pristine carbon nanotubes are chemically inert and aggregate in aqueous solution under physiological conditions. In this disclosure we describe effective chemical surface modifications that were made to increase the solubility and biocompatibility of CNTs, control surface antigen presentation for enhanced presentation to cells, provide stabilization of biologies on the CNT surface, and more. Chemical functionalization provides a way to covalently attach antigens to CNTs which leads to better dispersion and stability of the vaccine and consequently improves antigen pharmacokinetic and pharmacodynamic properties in vivo. To achieve such complex systems, it is imperative to master the intcrmolccular forces between CNTs and antigens, including geometry effects, e.g., CNT length, diameter, curvature and chirality (helical arrangement of carbon hexagons in the longitudinal direction) and how they translate into changes in the local environment (e.g. water or biological entropy). This type of interaction between antigens (proteins) and CNTs has important consequences for the preservation of their structure and, in turn, function.

[0036] In addition, CNTs produced by most of methods are a complicated mixture of nanotubes with various diameters and lengths as well as varying chirality for single walled nanotubes. It is important to use highly purified CNTs for biomedical applications to achieve batch to batch reproducible results. Another consideration of using CNT for delivery is its biosafety. Biodegradation of carbon-based nanomaterials has been pursued intensively and their long-term toxicity has attracted specific concern. The surface functionalization is another factor that will eventually reduce the CNT based delivery platform toxicity concerns. In this disclosure, we have modified the CNT by strong oxidants to accelerate their degradation [8].

[0037] The present disclosure generally relates to pharmaceutical compositions comprising a surface functionalized carbon nanotube and an active agent. As used herein, unless otherwise specified or contrary from context, the term “surface functionalized” includes surface functionalization through covalent bonding and/or noncovalent force, such as hydrophobic interactions, n-Tt interactions, electrostatic interactions, such as hydrogen bonding, ionic interactions, etc. The terms “attach”, “bind”, “immobilize”, and the like, can include both covalent bonding and noncovalent interactions, unless otherwise indicated. Typically, the term “adsorb”, “absorb”, or “encapsulate” and the like is used to indicate that the interaction is noncovalent interaction, unless otherwise specified or contrary from context. On the other hand, the terms “link”, “form a bond”, “conjugate”, “couple”, and the like generally refer to forming a covalent bond, unless otherwise indicated or contrary from context.

[0038] As discussed herein, the surface functionalized carbon nanotubes herein can be used for delivering a broad range of active agents, which are not particularly limited. Thus, the term “active agent” as used herein, unless otherwise specified or contrary from context, should be broadly interpreted to include but not limited to therapeutic agents, prophylactic agents such as vaccines, antigens, imaging agents, diagnostic agents, adjuvants, etc. In addition, unless otherwise specified or contrary from context, the term “active agent” is not particularly limited to any particular classes of compounds and can encompass small molecules, peptides, proteins, nucleic acids (herein broadly referring to any DNA, RNA, oligonucleotide, or polynucleotide types of compounds), and other classes of compounds. Exemplary useful active agents include without limitation, small molecules, biologies, antigens, nucleic acids such as oligonucleotides, polynucleotides, DNA, RNA, a silencing RNA (e.g., small interfering RNA (siRNA) and microRNA (miRNA), and short hairpin RNA (shRNA), antisense RNA and ribozymes), mRNA, proteins, polypeptides such as antibodies, antigen binding fragments of antibodies, single domain antibodies (VHH), aptamers, proteins having alternative binding scaffolds, peptides, glycosaminoglycans, oligosaccharides, and polysaccharides, and derivatives or analogs thereof. As used herein, the term "antigen" refers to a substance that can induce an immune response in a subject. Suitable antigens for the pharmaceutical composition or carbon nanotube conjugates herein include those that are capable of inducing a humoral immune response in a subject. "Antigen" also includes a polynucleotide that encodes the polypeptide that functions as an antigen. Nucleic acid-based vaccination strategies are known, wherein a vaccine composition that contains a polynucleotide is administered to a subject. The antigenic polypeptide encoded by the polynucleotide is expressed in the subject, such that the antigenic polypeptide is ultimately present in the subject, just as if the vaccine composition itself had contained the polypeptide.

[0039] The surface functionalized carbon nanotubc herein can be active-agent loaded or active-agent free. In some embodiments, the surface functionalized carbon nanotube herein can include an active agent covalently attached to the carbon nanotube either directly or through a surface molecule that is covalently attached to the carbon nanotube, such as a hydrophilic linker herein. These surface functionalized carbon nanotubes would be understood as always in an active-agent loaded state with respect to the covalently attached active agent. It should be clear that such surface functionalized carbon nanotubes can further adsorb, bind, encapsulate, and/or otherwise associate an additional active agent, which can be the same or different from the covalently attached active agent, through covalent (e.g., through a different covalent link) or non-covalent interactions with the carbon nanotube directly and/or through a surface molecule that is covalently or noncovalently attached to the carbon nanotubc.

[0040] In some embodiments, the surface functionalized carbon nanotube herein can be in an active-agent free state, which can be formulated with an active agent to adsorb, bind, encapsulate, and/or otherwise associate the active agent through non-covalent interactions with the carbon nanotube directly and/or through a surface molecule that is covalently or noncovalently attached to the carbon nanotube.

Surface Functionalization of Carbon Nanotubes

[0041] In some embodiments, the present disclosure relates to novel surface functionalization of carbon nanotubes with one or more surface molecules described herein.

A. Carbon nanotubes

[0042] Carbon nanotubes are well known in the art and generally refer to an allotrope of carbon having a cylindrical or tube shape. Unless otherwise specified, the term “carbon nanotube” as used herein refers to both single- walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). SWCNTs often have a diameter of about 0.3 nm to about 8 nm. Multi-wall carbon nanotubes often have a diameter of about 30 nm to about 200 nm. Useful carbon nanotubes are not particularly limited. In some embodiments, the carbon nanotube can be a SWCNT. In some embodiments, the carbon nanotube is a multiwalled carbon nanotube (including double walled carbon nanotube). In some embodiments, a multiwalled carbon nanotube is preferred.

[0043] It should be noted that while many of the embodiments herein are directed to carbon nanotubes, it is also contemplated that other forms of carbons, such as graphene spheres or “carbon nanosphere,” which arc commonly referred to as buckyballs or fullerene, may be similarly functionalized and used.

[0044] The carbon nanotubes that are functionalized with one or more surface molecules herein typically have COOH groups, for example, on the surface of the carbon nanotubes. The COOH groups of the carbon nanotubes can facilitate the surface functionalization with the one or more surface molecules described herein, for example, through covalent bond (e.g., amide bond) formations with the one or more surface molecules, by electrostatic interactions with a positively charged group(s) of the one or more surface molecules, etc. Carbon nanotubes having COOH groups can be readily obtained in view of the present disclosure. For example, in some embodiments, the carbon nanotubc having COOH groups can be obtained from oxidation or other functionalization methods of readily available carbon nanotube, such as those commercially available unfunctionalized carbon nanotubes, to introduce COOH groups.

[0045] The amount of COOH groups of the carbon nanotubes that are surface functionalization with one or more surface molecules herein is not particularly limited. In preferred embodiments, the carbon nanotubes are characterized as having about 1-20% by weight of COOH groups, such as about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 15%, about 20%, or any range or value between the recited values, such as about 2-15%, about 3-14%, about 3-12%, or about 4-12% by weight of COOH groups. The amount of COOH group in a carbon nanotube composition can be determined by methods well known in the art, for example, by the titration method described herein. Depending on the surface functionalization and/or the active agent to be loaded, the amount of COOH groups of the carbon nanotubes can be adjusted and controlled. The present disclosure provides methods for controlling the extent of COOH group of the carbon nanotubes, for example, through controlling the reaction conditions of reacting carbon nanotubes with HNO3 and H2SO4, see e.g., the Examples section.

[0046] In some embodiments, prior to surface functionalization with one or more surface molecules herein, the carbon nanotube can have an amount of COOH groups such that the zeta potential of the carbon nanotubc can be less than -10 mv, more preferably, less than -20 mv, such as about -25 mv. For example, FIG. 4C shows an exemplary carbon nanotube with a zeta potential of -25.1 mv.

[0047] The carbon nanotubes herein can have various diameters and length. Typically, the carbon nanotubes herein have an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio (length/diameter) of about 0.2: 1 to about 12,500: 1. The average diameter and length of the carbon nanotubes can be measured and determined by Dynamic Light Scattering (DLS) and SEM methods known in the art and are also exemplified herein. [0048] In some embodiments, the carbon nanotubes herein can be characterized as having certain size and/or shape, for example, to mimic that of a microorganism, such as a virus. For example, in some embodiments, the carbon nanotubc can be a multi-walled carbon nanotubc, characterized as having an average diameter of about 20 nm to about 200 nm, such as about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 80 nm, about 100 nm, about 120 nm, about 150 nm, about 200 nm, or any range or value between the recited values, such as about 30 nm to about 150 nm, or about 60 nm to about 100 nm. In some embodiments, the carbon nanotube can also be characterized as having an average length of about 10 nm to about 1000 nm, about 40 nm, about 100 nm, about 120 nm, about 150 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 800 nm, about 1000 nm, or any range or value between the recited values, such as about 100 nm to about 200 nm, about 200 nm to about 500 nm, about 200 nm to about 400 nm, about 400 nm to about 800 nm, etc. In some specific embodiments, the carbon nanotube has an average length of about 100 nm to about 200 nm. In some specific embodiments, the carbon nanotube has an average length of about 200 nm to about 400 nm. In some embodiments, the carbon nanotube can also have an average length of above 1000 nm and up to 5000 nm and beyond, such as about 2000 nm, about 4000 nm, about 5000 nm, or any range or value between the recited values. In some embodiments, the carbon nanotube can also be characterized as having an average aspect (length/diameter) ratio of about 0.2:1 to about 10: 1, such as about 1: 1 to about 7: 1, such as about 0.2: 1, about 0.5: 1, about 0.8: 1, about 1: 1, about 2: 1, about 3:1, about 4: 1, about 5: 1, about 6: 1 , about 7: 1 , or any range or value between the recited values, such as about 1 : 1 to about 5: 1, or about 2: 1 to about 4:1. For example, as shown herein, in some embodiments, the carbon nanotube is designed to mimic a virus which is substantially spherical, and the average aspect ratio of the carbon nanotube can be about 0.5: 1, about 0.8: 1, about 1:1, about 1.2: 1, about 1.5: 1, about 2: 1, or any range or value between the recited values.

[0049] The carbon nanotubes can typically be characterized as having a large surface area. For example, in some embodiments, the carbon nanotubes can have a surface area of about 800-1000 m ² /g. Without wishing to be bound by theories, it is believed that the high surface area can allow the carbon nanotube to effectively interact with active agents herein, such as biomolecules. For example, the carbon nanotubes can have high surface area which can enable it to bind 1-100’s of proteins per CNT, leading to significantly improved binding capacity and delivery efficiency.

[0050] In some embodiments, the carbon nanotubc can also be a single-walled carbon nanotube or a multi-walled such as double-walled carbon nanotube having a smaller diameter than 20 nm. For example, in some embodiments, the carbon nanotube can have an average diameter of about 0.4 nm to about 20 nm. The length of the carbon nanotube with a diameter in this range is not particularly limited, which include any of those described herein. In some embodiments, the carbon nanotube can have an aspect ratio ranging from about 1: 1 to about 12,500: 1, such as about 10: 1, about 50: 1, about 100:1, about 200: 1, about 500: 1, about 1,000: 1, about 5,000: 1, about 10,000: 1, about 12,500: 1, or any range or value between the recited values.

[0051] In some preferred embodiments, the carbon nanotube has a substantially uniform length. For example, in some embodiments, the carbon nanotube can be characterized as being monodisperse as determined by DLS. In some embodiments, the carbon nanotube can be characterized as having a polydisperse index (PDI) of less than 1, more preferably, less than 0.75, less than 0.5, less than 0.25, or even less than 0.1. Exemplified methods for preparing carbon nanotubes having a substantially uniform length are described herein. For example, in the Examples section, a method of successive filtration with different pore size can be used to prepare carbon nanotubes with a narrow size distribution, see e.g., FIG. 4B, which has an average size of 112.8 nm, and a PDI of 0.25.

B. Surface Molecules and Functionalization

[0052] The carbon nanotubes herein can typically be surface functionalized with one or more surface molecules herein. Useful surface molecules for the surface functionalized carbon nanotubes are not particularly limited. However, surface molecules are typically biocompatible. Typically, the surface molecules are such that in comparison with non-surface functionalized carbon nanotubes, the surface functionalization of the carbon nanotubes can (1) enhance aqueous solubilities of the carbon nanotubes, (2) enhance the capability of the carbon nanotubes in loading an active agent herein, for example, by introducing an appropriate surface charge to bind the active agent or introducing an appropriate linker to covalently attach the active agent, (3) improve efficacy of the loaded active agent, for example, by potentially mimicking the distribution of the loaded active agent to that of a protein (and/or another biomolecule) on a microorganism, such as a virus, and/or by maintaining the loaded active agent in a preferred orientation such that the active agent can have its desired function, (4) improve stability of the loaded active agent, (5) enhance cell permeation of the loaded active agent, (6) improve versatility in formulating the loaded active agent, and/or (7) improve the ADME profiles of the carbon nanotubes so as to render the carbon nanotubes safe for use in mammals (particularly humans).

[0053] In some preferred embodiments, the surface molecules are such that the surface functionalized carbon nanotubes have a zeta potential with an absolute value of greater than 20 mV, preferably, greater than 40 mV, prior to binding the active agent. For example, in some embodiments, the surface functionalized carbon nanotubes have a positive zeta potential greater than 20 mV, preferably, greater than 40 mV, prior to binding the active agent. In some embodiments, the surface functionalized carbon nanotubes have a negative zeta potential less than -20 mV, preferably, less than -40 mV, prior to binding the active agent.

[0054] Typically, the surface molecules are such that the surface functionalized carbon nanotubes are storage stable at 0°C for 1 month or more, preferably, for 3 months or more, prior to or after binding the active agent. By storage stable, it is meant that the surface functionalized carbon nanotubes, with or without the active agent, upon storage at the recited condition, are substantially unchanged, for example, with substantially the same amount of active agent, having substantially the same physical appearances and/or other physical properties, substantially the same impurities if any, etc. A person of ordinary skill in the art would understand the term “substantially the same” in this context. As used herein, for profiles and/or properties that can be quantified, “substantially the same” should be understood as having a value that is within 80-125% of a reference value, or within experimental/measurement errors acceptable to a person of ordinary skill in the art.

[0055] Typically, in embodiments herein, the carbon nanotube is surface functionalized with one or more surface molecules selected from (i) a polyamine, (ii) a hyperbranched polymer or dendrimer, which has one or more groups that are charged or chargeable at pH of 7, (iii) a cationic lipid, which has one or more groups that are positively charged or positively chargeable at pH of 7, (iv) a surfactant, and (v) a hydrophilic linker. As used herein, the categorization of (i)-(v) herein is not meant to be mutually exclusive, as a surface molecule may fit into more than one category of surface molecules (i)-(v) herein. For example, if a surface molecule S 1 can be characterized as both a polyaminc and a hydrophilic linker, when a surface functionalized carbon nanotube is said to be surface functionalized with a hydrophilic linker, such surface functionalized carbon nanotube is meant to include a carbon nanotube surface functionalized with the surface molecule S 1. Further, with respect to the amount of a surface molecule that fits into more than one category of surface molecules (i)- (v), the amount of the surface molecule is considered as within a recited range herein as long as under one category, the amount of the surface molecule is within the recited range for that category. For example, if a surface molecule S2 can be both a cationic lipid and a surfactant, a surface molecule S3 is a surfactant, when a surface functionalized carbon nanotube is said to be surface functionalized with a cationic lipid and a surfactant, with the cationic lipid in an amount of 0.5-2% by weight of the carbon nanotube and the surfactant of 0.1-3% by weight of the carbon nanotube, such surface functionalized carbon nanotube is meant to include a carbon nanotube that is surface functionalized with S2 and S3, with S2 in an amount of 0.7% by weight of the carbon nanotube and S3 in an amount of 0.25% by weight of the carbon nanotube. Other circumstances should be interpreted similarly.

[0056] In some embodiments, the surface molecule can include a polyamine. Polyamines generally refer to compounds having two or more amino groups, including primary, secondary, tertiary, and quaternary amino groups. Preferably, the polyamines herein refer to those compounds having two or more amino groups that are primary amino (NH2) or secondary amino (NHR) groups, such that the polyamincs arc capable of conjugating with another molecule through one or more of the NH2 and/or NHR groups. More preferably, the polyamines herein are hydrophilic and have an aqueous solubility of at least 1 mg/mL at pH 7, such as at least 10 mg/mL, at least 30 mg/mL, etc. In some embodiments, the polyamine has carbon, hydrogen, and nitrogen atoms, and can be characterized by an average ratio of (the number of nitrogen atoms)/(the number of carbon atoms) of the polyamine of about 1:4 or higher, such as about 1:3, about 1:2, or about 1: 1. For example, a polyethylene imine having a formula of (C2HsN)n, in which the ratio of the number of nitrogen atoms to the number of carbon atoms would be 1:2. In some embodiments, the polyamine has carbon, hydrogen, oxygen, and nitrogen atoms, and can be characterized by an average ratio of (the number of nitrogen and oxygen atoms)/(the number of carbon atoms) of the polyamine of about 1:4 or higher, such as about 1:3, about 1:2, or about 1: 1.

[0057] Typically, the polyamines herein have a molecular weight above 500 Daltons. For example, in some embodiments, the poly amines can have an average number or weight molecular weight of (Mn or Mw), preferably, an average weight molecular weight of (Mw), ranging from about 500 to about 100,000 Daltons, such as about 500, about 600, about 1,000, about 2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 7,500, about 10,000, about 25,000 Daltons, about 50,000 Daltons, or any range or value between the recited values, such as about 500-50,000, about 500-5,000, about 1,000-10,000, about 2,000-7,500, etc. In some embodiments, the polyamines can have an Mn or Mw of about 2,000-3,000, such as about 2,500 Daltons. In some embodiments, the polyamine can have an Mn or Mw ranging from about 500-50,000, such as about 1,000-5,000 Daltons, about 2,000-3,000 Daltons, about 600 Daltons, about 2,000 Daltons, about 2,500 Daltons, about 10,000 Daltons, about 25,000 Daltons, about 35,000 Daltons, about 50,000 Daltons, or any range or value between the recited values. In some embodiments, the polyamines can also be a low molecular weight polyamine having a molecular weight less than 500 Daltons.

[0058] In some embodiments, the polyamines are alkyl polyamines, such as polyalkyleneimine, which can be linear or branched. Useful polyalkyleneimines include polypropyleneimine (PPI) and polyethyleneimine (PEI) polymers, as well those co-polymers containing the PPI or PEI polymers. In some preferred embodiments, the polyamine herein is a branched or linear polycthylcnciminc, for example, those having a number or weight average molecular weight (Mn or Mw), preferably, an average weight molecular weight of (Mw), ranging from about 500 to about 50,000 Daltons, such as about 500, about 600, about 1,000, about 2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 7,500, about 10,000, about 25,000 Daltons, about 35,000 Daltons, about 50,000 Daltons, or any range or value between the recited values, such as about 500-50,000, about 500-5,000, about 1,000- 10.000, about 2,000-7,500, about 10,000 to 35,000 etc. In some embodiments, the polyamines can have an Mn or Mw of about 2,000-3,000 Daltons, such as about 2,500 Daltons. In some preferred embodiments, the poly amine herein is a branched polyethyleneimine, having an Mn or Mw ranging from about 500-50,000, such as about 1,000-5,000 Daltons, about 2,000-3,000 Daltons, about 600 Daltons, about 2,000 Daltons, about 2,500 Daltons, about 10,000 Daltons, about 25,000 Daltons, about 35,000 Daltons, about 50,000 Daltons, or any range or value between the recited values. In some embodiments, the Mn or Mw of the branched polyethyleneimine is about 25,000 Daltons. Polyethyleneimines, linear or branched, are commercially available. In some embodiments, a branched polyethyleneimine can be generally represented by the structure below:

[0059] In some embodiments, the polyamine can be characterized as a hyperbranched polymer, such as a dendrimer. The term “hyperbranched polymer” is well understood in the art to refer to a molecule having a tree like branching structure in which all bonds converge to a focal point or core, and which have a plurality of reactive chain-ends, see e.g., Jeon, I. et al, Molecules 23:657 (2018). A dendrimer is an example of a “hyperbranched polymer”. Dendrimers are well known and are hyperbranched polymer molecules in which the degree of branching is 100% (occasionally referred to herein as “perfectly branched” i.e. 100% of functional groups capable of branching are branched) and which are therefore highly symmetrical about the core. Hyperbranched polymers consist of three basic architectural components, (i) the core, (ii) the interior or branches, and (iii) the functional end groups or terminal groups. The core is positioned at the center of the molecule and to it branched wedges, called dendrons, are attached. Dendrimers are perfectly branched molecules and for a given starting material, the only variable is the number of layers or generations in the dendrimer. Other hyperbranched molecules also contain a high number of branches, for example, having a degree of branching of at least 30%, 40% or 50%, or having a degree of branching of at least 60%, 70%, 80% or 90%. Unlike dendrimers, the structure of such hyperbranched molecules will not be completely regular but they may also adopt a generally globular structure. [0060] In some embodiments, the polyamine can be a dendrimer comprising repeating alkylene amine units, such as repeating ethylene amine, propylene amine units, which has terminal amino groups, preferably, terminal NH2 groups. For example, in some embodiments, the dendrimer can be a polyethyleneimine with a core of diaminoethane, which can be G0-G10 dendrimer, such as a G2, G3, or G4 dendrimer. Dendrimers having repeating ethylene amine units with a different core, such as N, diaminobutane core, etc. can also be used. Polypropylene based dendrimers that terminate with amino groups are also known in the art and can be used as a polyamine herein. In some embodiments, the polyamine is a polyamidoamines (PAMAMs) in which a nitrogen atom, a di- or tri-amine (e.g. ethylenediamine) can be the core and the branches can be built up by reacting the ammonia or the free amine groups with e.g. methyl acrylate followed by ethylene diamine leading to a structure having a number of NH2 groups at the terminal. When the polyamine is a dendrimer, different generations of dendrimer can be used, which without limitation include GO to GIO dendrimers, preferably, G2, G3, or G4. An example of useful polyamine dendrimer can be a GO to GIO polyamidoamine (PAMAM)dendrimer, such as a G2, G3, or G4 polyamidoamine dendrimer with terminal NH2 groups.

[0061] In some embodiments, the polyamine herein can be attached to the carbon nanotube through a covalent bond. For example, in some embodiments, the poly amine can bind to the carbon nanotube through an amide bond formed between an amine group of the polyamine and a carboxylic acid function of the carbon nanotube. In some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with an amine group of the polyaminc. However, in some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%, or any range or value between the recited value, form an amide bond with an amine group of the polyamine.

[0062] In some embodiments, the polyamine herein can be attached to the carbon nanotube through noncovalent interactions, such as hydrophobic interactions, electrostatic interactions, such as hydrogen bonding or ionic interactions, or 71-71 interactions, etc.

[0063] In some embodiments, the same polyamine can be attached to the carbon nanotube both covalently and through noncovalent interactions. In other words, some of the polyamine is covalently attached whereas the remaining polyamine is attached to the carbon nanotube through nonco valent interactions.

[0064] In some embodiments, more than one types of polyamincs can be attached to the carbon nanotube, wherein each type independently can be either covalently attached or attached to the carbon nanotube through noncovalent interactions.

[0065] The amount of poly amine attached to the carbon nanotube is not particularly limited. However, in preferred embodiments, the weight ratio of the polyamine (e.g., the branched polyethyleneimine herein) to the carbon nanotube (prior to any functionalization with the one or more surface molecules herein) ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values. In some embodiments, the amount of polyamine attached to the carbon nanotube is such that the surface functionalized carbon nanotube has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. Without wishing to be bound by theories, it is believed that the highly positively charged carbon nanotubes are particularly suited for loading and delivering active agents that are negatively charged, such as a nucleic acid, e.g., oligonucleotides, polynucleotides, DNA, RNA, a silencing RNA (e.g., small interfering RNA (siRNA) and microRNA (miRNA), and short hairpin RNA (shRNA), antisense RNA and ribozymes), mRNA etc. For example, in some embodiments, the active agent can be an RNA, such as a mRNA.

[0066] In some embodiments, the surface molecule includes a hyperbranched polymer, such as a dendrimer, which has one or more groups that are charged or chargeable at pH of 7. Hypcrbranchcd polymer, such as dendrimers, useful for the present disclosure arc not particularly limited. Typically, the hyperbranched polymer is hydrophilic and has a net charge, positive or negative, at pH of 7. As used herein, a hydrophilic molecule refers to a molecule that has (i) at least one, preferably, at least 2 or at least 3, neutral hydrophile group (e.g., O. OH, etc.) per 5 carbons, and/or (ii) at least one, preferably, at least 2, at least 3, or at least 4, electrically charged hydrophile group (e.g., charged amine groups such as quaternary amin groups, chargeable amine groups, COOH groups, SO3H groups, etc.) per 7 carbons. In some embodiments, the hyperbranched polymer has an aqueous solubility of greater than 1 mg/mL at pH 7, preferably, greater than 10 mg/mL, or greater than 30 mg/mL at pH 7. Hyperbranched polymers such as dendrimers may contain a number of functionalities, for example, the hyperbranched polymer may be polyamines (e.g., those having primary amines as functional end groups), polyamidoamincs (c.g., those having amide groups and secondary and tertiary amine groups and with primary amines as functional end groups), or polyethers with amine functionality (e.g. polyethers such as PEGs in which end groups have been transformed into primary amine groups). Hyperbranched polymers that are polyamines are described above. In some embodiments, the surface molecule can include a dendrimer that has one or more groups that are charged or chargeable at pH of 7. In some embodiments, the core for the dendrimer can be a N atom, a diamine or triamine, such as diaminoethane, diaminobutane, etc.; the branch points can be tertiary amines; and terminal residues can be NH2, OH, COOH, SO3H, or SH groups etc. An example of useful dendrimer can be a GO to GIO polyamidoamine (PAMAM)dendrimer, such as a G2, G3, or G4 polyamidoamine dendrimer.

[0067] In some embodiments, hyperbranched polymers such as dendrimers that can become negatively charged are desired, such as those having carboxylic or SO3H functionalities, such as a hyperbranched polymer having terminal COOH or SO3H groups. Without wishing to be bound by theories, it is believed that by introducing negatively charged groups to the carbon nanotubes, the carbon nanotubes can be particularly suited for loading and delivering active agents that are positively charged, such as a positively charged protein. In some embodiments, the amount of the hyperbranched polymer having negatively charged groups such as COOH or SO3H groups, which are attached to the carbon nanotube is such that the surface functionalized carbon nanotubc has a negative zeta potential of less than -20 mV, preferably, less than -40 mV, or lower.

[0068] Similarly, the hyperbranched polymers such as dendrimers can be attached to the carbon nanotube through covalent bond or through noncovalent interactions, such as hydrophobic interactions, electrostatic interactions, such as hydrogen bonding or ionic interactions, or 7t-7t interactions, etc. In some embodiments, the same hyperbranched polymers can be attached to the carbon nanotube both covalently and through noncovalent interactions. In other words, some of the hyperbranched polymers are covalently attached whereas the remaining hyperbranched polymers are attached to the carbon nanotube through noncovalent interactions. In some embodiments, more than one types of hyperbranched polymers can be attached to the carbon nanotube, wherein each type can independently be covalently attached and/or attached to the carbon nanotubc through noncovalent interactions. Typically, in such embodiments, the different types of hyperbranched polymers have the same types of charges, e.g., all are positively charged or all are negatively charged.

[0069] The amount of hyperbranched polymers such as dendrimers attached to the carbon nanotube is not particularly limited. In some embodiments, the weight ratio of the hyperbranched polymer such as a dendrimer herein to the carbon nanotube (prior to any functionalization with the one or more surface molecules herein) ranges from about 10: 1 to about 1: 10, such as about 2:1, about 1: 1, about 1:2, or any range or value between the recited values. In some embodiments, the amount of the hyperbranched polymer attached to the carbon nanotube is such that the surface functionalized carbon nanotube has a positive or negative zeta potential with an absolute value of greater than 20 mV, preferably, greater than 40 mV, or higher.

[0070] In some embodiments, the surface molecule can include a cationic lipid. Cationic lipids generally refer to positively charged amphiphiles having three basic chemical functional domains: a cationic headgroup, such as a quaternary ammonium headgroup, a hydrophobic domain, such as a fatty acid chain, and a linker that tethers the cationic headgroup and hydrophobic tail domain. Suitable cationic lipids for the present disclosure are not particularly limited. In some preferred embodiments, the cationic lipid is a monovalent cationic lipid, i.e., having a cationic headgroup with a single positive charge. In some preferred embodiments, the cationic lipid is a multivalent cationic lipid, i.e., having a cationic headgroup with two or more positive charges, such as having two or more quaternary amine groups. In some embodiments, the multivalent cationic lipid is selected from MVL-5 (Nl-[2- ((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]bu tylcarboxamido)ethyl]-3,4- di[oleyloxy]-benzamide), DOSPA (2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N- dimethyl-l-propanaminium salt, such as chloride salt), and GL67 (N4-Cholesteryl-Spermine Salt, such as HC1 salt). In some preferred embodiments, the multivalent cationic lipid is MVL-5. [0071] In some embodiments, the cationic lipid can be a phospholipid, such as a phosphocholine lipid. In some embodiments, the phosphocholine lipid is a monovalent cationic lipid. In some embodiments, the phosphocholinc lipid is an alkyl ester of phosphotidy Icholine, i.e., the anionic charge of the phosphate oxygen has been eliminated by forming an alkyl phosphate ester. For example, in some embodiments, the cationic lipid can be an ethyl phosphotidylcholine (EPC), which may be represented by the formula below

(counterion not shown) , wherein R ¹ and R ² can be the same or different and each can be hydrogen or an acyl group derived from a fatty acid (e.g., those having 8-30 carbons with 0-6 double bonds, typically, 12-20 carbons with 0 or 1 double bond), such as lauroyl, palmitoyl, oleoyl, myristoyl, stearoyl, etc., wherein at least one of R ¹ and R ² is not hydrogen. In some embodiments, the cationic lipid can be 16:0 EPC (i.e., 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine), 12:0 EPC, 18:0 EPC, 18: 1 EPC, 14: 1 EPC, 16:0-18:1 EPC (l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine), etc., preferably, the phosphocholine lipid is l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

[0072] The cationic lipid is typically bound to the carbon nanotube through noncovalent interactions, such as hydrophobic interactions or electrostatic interactions (e.g., hydrogen bonding or ionic interactions). When present, the weight ratio of the cationic lipid to the carbon nanotube (prior to any functionalization with the one or more surface molecules) ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1:1, about 1:2, or any range or value between the recited values.

[0073] In some embodiments, the carbon nanotube is surface modified with both a polyamine and a cationic lipid. In some embodiments, the carbon nanotube is surface modified with both a polyalkylene imine (e.g., polyethylene imine) and a monovalent cationic lipid. In some embodiments, the carbon nanotube is surface modified with both a poly alkylene imine (e.g., polyethylene imine) and a multivalent cationic lipid. In some embodiments, the carbon nanotube can be surface modified with the polyamine through covalent bond(s) and the cationic lipid through non-covalent interactions. In some embodiments, the carbon nanotube can be surface modified with both the polyamine and cationic lipid through noncovalent interactions.

[0074] For example, in some embodiments, the carbon nanotubc can be surface modified with a polyethylene imine (e.g., any of those described herein) and an ethyl phosphocholine lipid, such as l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine. In some embodiments, the carbon nanotube can be surface modified with a branched polyethylene imine (e.g., any of those described herein) through covalent amide bond formation between the carboxylic acid group of the carbon nanotube with the amino group of the branched polyethylene imine, and with the ethyl phosphocholine lipid through electrostatic interactions and/or hydrophobic interactions. Without wishing to be bound by theories, it is believed that the presence of the cationic lipid on the surface of the carbon nanotube can help to stabilize a loaded active agent, such as a negatively charged active agent, e.g., a nucleic acid such as oligonucleotides, polynucleotides, DNA, RNA, a silencing RNA (e.g., small interfering RNA (siRNA) and microRNA (miRNA), and short hairpin RNA (shRNA), antisense RNA and ribozymes), mRNA etc., and can help to increase transfection efficiencies of a loaded nucleic acid such as mRNA.

[0075] The amount of the polyamine and cationic lipid for the surface functionalized carbon nanotube is not particularly limited and include any of those described herein. In some embodiments, the weight ratio of (a) the combined amount of the polyamine and the cationic lipid to (b) the carbon nanotube (prior to any functionalization with the one or more surface molecules), (a)/(b), ranges from about 10: 1 to about 1 : 10, such as about 2:1 , about 1 : 1 , about 1:2, or any range or value between the recited values. In some embodiments, the weight ratio of the polyamine to the cationic lipid can range from about 10: 1 to about 1: 10, such as about 10:1, about 5: 1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1:10, or any range or value between the recited values. For example, in some embodiments, the carbon nanotube can be surface modified with a branched polyethylene imine (e.g., any of those described herein) and the ethyl phosphocholine lipid, l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, wherein the weight ratio of the combined amount of the branched polyethylene imine and 1- palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine to that of the carbon nanotube ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1:1, about 1:2, or any range or value between the recited values, and the weight ratio of the branched polyethylene imine to 1- palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine can also range from about 10: 1 to about 1: 10, such as about 10:1, about 5: 1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1:10, or any range or value between the recited values. In some embodiments, the amount of the polyamine and cationic lipid is such that the surface functionalized carbon nanotube has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher.

[0076] In some embodiments, the surface molecule can include a surfactant. For example, in some embodiments, the carbon nanotube can be surface functionalized with both the polyamine and cationic lipid as described hereinabove, and further functionalized with the surfactant. In some embodiments, the carbon nanotube can be surface functionalized with a hydrophilic linker herein and the surfactant. Other combinations are also suitable.

[0077] Useful surfactant is not particularly limited, which can be neutral, zwitterionic, cationic, or anionic. Without wishing to be bound by theories, it is believed that in some embodiments, the surfactant can be used as a conformation protector to present the loaded active agent, such as a protein, peptide, or antigen, in a correct orientation to achieve its desired effect, such as to elicit an immune response. Thus, in some embodiments, the surfactant can be included after the active agent is loaded to the carbon nanotube through another surface molecule, wherein the surfactant can occupy or be absorbed on the surface of the carbon nanotube at available locations. Thus, the surfactant herein is typically a surfactant that can be absorbed on the surface of the carbon nanotube through non-covalent interactions, such as hydrophobic interactions, 7t-7t interactions, electrostatic interactions, etc. In some embodiments, the surfactant may resemble a membrane lipid and can form a bilay er structure. In some embodiments, the surfactant used herein is hydrophilic, with a HLB value of 8 and above, preferably, 10 and above, such as 12-20, e.g., 13-16, or 16-18.

[0078] In some embodiments, the surfactant comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000. etc., which is optionally capped, e.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, a methoxy capping group. In some embodiments, the surfactant is a PEGlated lipid comprising the polyethylene glycol chain. In some embodiments, the surfactant is a PEGlated lipid, in which a lipid moiety (e.g., a fatty ether or fatty ester) is linked to a polyethylene glycol chain. PEGlated lipids are known in the art. For example, in some embodiments, the PEGlated lipid is a glycerol-based lipid, in which the polyethylene glycol chain is attached to one of the glycerol hydroxy group through a linking group, which may be represented by the structure: , wherein R ¹ and R ² can be the same or different and each can be hydrogen or an acyl group derived from a fatty acid (e.g., those having 8-30 carbons with 0-6 double bonds, typically, 12-20 carbons with 0 or 1 double bond), such as lauroyl, palmitoyl, oleoyl, myristoyl, stearoyl, etc., wherein at least one of R ¹ and R ² is not hydrogen, wherein R ³ is a linking group, such as a carbonyl group, an alkylene group, or other linking group such as a phosphoethanoamine group, etc., and PEG is a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, e.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, a methoxy capping group. In some embodiments, the surfactant is a phospholipid, such as a glycerol-based phospholipid containing the polyethylene glycol chain. In some embodiments, the surfactant can be a glycerol-based phospholipid, in which at least one of the glycerol hydroxy groups forms a fatty ether or fatty ester, and one of the glycerol hydroxy group is linked with a polyethylene glycol chain through a phosphate containing group. For example, in one embodiment, the surfactant can be represented by the structure: , wherein R ¹ and R ² can be the same or different and each can be hydrogen or an acyl group derived from a fatty acid (e.g., those having 8-30 carbons with 0-6 double bonds, typically, 12-20 carbons with 0 or 1 double bond), such as lauroyl, palmitoyl, oleoyl, myristoyl, stearoyl, etc., wherein at least one of R ¹ and R ² is not hydrogen, wherein PEG represents a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, e.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, a methoxy capping group. In one preferred embodiment, the surfactant is l,2-dipalmitoyl-sn-glycero-3- phosphocthanolaminc-N-[mcthoxy(polycthylcncglycol)-2000]. [0079] The amount of the surfactant for the surface functionalized carbon nanotube is not particularly limited. In some embodiments, the surfactant can be present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotubc (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube.

[0080] In some embodiments, the surface molecule can include a hydrophilic linker. The hydrophilic linker typically has a group, such as an amine group, that allows it to form a covalent bond with the carbon nanotube and is capable of covalently or non-covalently attaching an active agent, such as a small molecule, peptide, protein, or nucleic acid. In some embodiments, the hydrophilic linker has carbon, hydrogen, oxygen, and nitrogen atoms, and optionally sulfur and halogen atoms, wherein the ratio of the total number of oxygen and nitrogen atoms to the total number of carbon atoms is at least 1:5, preferably, at least 1:4, at least 1:3, such as about 1:2. In some embodiments, the hydrophilic linker has an aqueous solubility of greater than 1 mg/mL at pH 7, preferably, greater than 10 mg/mL, or greater than 30 mg/mL at pH 7. For example, in some embodiments, the hydrophilic linker has an NH or NH2 group and is capable of attaching to the carbon nanotube through an amide bond formation between an amine group of the hydrophilic linker and a carboxylic acid function of the carbon nanotube. In some embodiments, the hydrophilic linker comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000. etc.

[0081] In some embodiments, the hydrophilic linker can have a general formula of G1-L-G2, wherein G1 is a group, such as amine group, that can form a covalent bond with a carboxylic acid function of the carbon nanotube, L is an linking group that connects G1 and G2, such as a polyethylene glycol chain described herein, and G2 is a group that can attach to an active agent, such as a small molecule, peptide, protein, or nucleic acid through a covalent bond such as an amide bond, an ester bond, an ether bond, or a thioether bond. In some more specific embodiments, the hydrophilic linker can have a general formula of G1-(PEG)-G2, wherein G1 is a group, such as amine group, that can form a covalent bond with a carboxylic acid function of the carbon nanotube, PEG is a polyethylene glycol chain with end groups suitable for attaching to G1 or G2, such as a chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., and G2 is a group that can attach to an active agent, such as a small molecule, peptide, protein, or nucleic acid through a covalent bond such as an amide bond, an ester bond, an ether bond, or a thiocthcr bond. The connection of G1 or G2 to the polyethylene glycol chain is not particularly limited. For example, in some embodiments, the PEG has an alkylene group at both ends of the polyethylene glycol chain, with one end alkylene bound to G1 and the other end alkylene bound to G2. The two end alkylene groups can be the same or different. For example, in some embodiments, G1 is NH2, and the NH2 group can be connected to the polyethylene glycol chain through an end alkylene linker of various length, such as ethylene. In some embodiments, G2 is COOH group, which can be connected to the polyethylene glycol chain through an end alkylene linker of various length, such as methylene. In some embodiments, G1 can be NH2, and G2 can be COOH. In some embodiments, both G1 and G2 can be NH2. In some embodiments, G1 can be NH2, and G2 can be OH group. In one preferred embodiments, the hydrophilic linker can be NH2-PEG-

COOH having a formula of: O , wherein m represents the average number of oxyethylene groups of a polyethylene glycol chain, m typically ranges from 1-200, such as 10-100, 20-60, 30-50, 42-48, such as about 44 or 45. In one preferred embodiments, the hydrophilic linker can be NH2-PEG2K-COOH. For example, in some embodiments, the hydrophilic linker can be represented by a formula of: , wherein (CH2CH2O) _m represents a PEG2000 chain. PEG chains of other length such as PEG100, PEG500, PEG1000, PEG2000, PEG3000 etc. can also be used for the hydrophilic linker.

[0082] The amount of hydrophilic linker attached to the carbon nanotube is not particularly limited. For example, in some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with an amine group of the hydrophilic linker. In some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%, or any range or value between the recited value, form an amide bond with an amine group of the hydrophilic linker.

[0083] In some embodiments, the carbon nanotubc is surface functionalized with both the hydrophilic linker and the surfactant. For example, in some embodiments, the carbon nanotube is surface functionalized with a hydrophilic linker of formula G1-(PEG)-G2 as defined herein and a glycerol-based phospholipid containing a polyethylene glycol chain. In some embodiments, the carbon nanotube is surface functionalized with a hydrophilic linker of formula NH2-(PEG)-COOH, wherein PEG is a PEG2000 chain, and a surfactant which is 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethyleneglycol)-2000]. In some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with an amine group of the hydrophilic linker and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%, or any range or value between the recited value, form an amide bond with an amine group of the hydrophilic linker, and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, the carbon nanotube is covalently linked to an active agent, such as a small molecule, peptide, protein, or nucleic acid, through the hydrophilic linker, for example, through a COOH group of the hydrophilic linker. In some embodiments, the active agent is a protein, peptide, or antigen, which is covalently linked to the carbon nanotube through the hydrophilic linker, and the surfactant functions as a conformation protector to present the protein, peptide, or antigen in a correct orientation to achieve its desired effect, such as to induce immune response. In some embodiments, the active agent, such as a small molecule, peptide, protein, or nucleic acid, can be attached to the carbon nanotube through a noncovalent interaction with the hydrophilic linker. [0084] As discussed herein, the surface functionalized carbon nanotubes herein can be used in connection with a broad range of active agents, which are not particularly limited. In some embodiments, the active agent is covalently attached to the carbon nanotubc cither directly or through a surface molecule that is covalently attached to the carbon nanotube, such as a hydrophilic linker herein. In some embodiments, the active agent binds to the surface functionalized carbon nanotubes through non-covalent interactions. In some embodiments, some of the active agent is covalently attached to the carbon nanotube and some of the active agent is attached to the carbon nanotube through non-covalent interactions.

[0085] In some embodiments, more than one active agents are included in the pharmaceutical composition herein. The more than one active agents can be associated with the carbon nanotube and/or independent of the carbon nanotube. In some embodiments, more than one active agents, e.g., two or more antigens, can be bound to the carbon nanotube. For example, in some embodiments, the carbon nanotube can have 1, 2, 3, 4, 5, or more than 5 different active agents. In some embodiments, the carbon nanotube can bind to 1-5, 1-10, 1- 15. 1-20, 1-25, 1-30, 2-10. 2-15. 2-20, 2-25, 3-10, 3-15. 3-20. 3-25, 4-10, 4-15, 4-20. 4-25. 5- 10, 5-15, 5-20, or 5-25 different active agents. When more than one active agents are attached to the carbon nanotube, each active agent can be independently attached to the carbon nanotube through covalent and/or noncovalent interactions. The different active agents can be the same type of agents or different. For example, in some embodiments, the active agents can all be nucleic acids or all peptides/proteins. In some embodiments, some active agents can be nucleic acids, some can be peptides or proteins, and/or some can be small molecules. Other combinations arc also possible.

[0086] The amount of active agent loaded to the carbon nanotubes is not particularly limited. In some embodiments, the amount of active agent loaded to the carbon nanotubes can be optimized according to a desired outcome.

[0087] In some embodiments, the active agent is a nucleic acid, such as an oligonucleotide, polynucleotide, DNA, RNA, a silencing RNA (e.g., small interfering RNA (siRNA) and microRNA (miRNA), and short hairpin RNA (shRNA), antisense RNA and ribozymes), or mRNA, which typically binds to the carbon nanotube through noncovalent interactions. In some preferred embodiments, the active agent is an RNA, such as an mRNA. Typically, the weight ratio of the nucleic acid to the carbon nanotube (weight prior to any functionalization with the one or more surface molecules) ranges from about 20: 1 to 1:20, such as about 1: 1 to about 1:5 or about 1:5 to about 1: 10.

[0088] In some embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with a polyamine herein; and (2) a nucleic acid, such as a DNA or RNA molecule, such as a mRNA, siRNA, or antisense oligonucleotide. In some embodiments, the carbon nanotube is surface modified with a polyalkylene imine (e.g., polyethylene imine). In some embodiments, the carbon nanotube can be surface modified with the polyamine through covalent bond(s). In some embodiments, the carbon nanotube can be surface modified with the polyamine through noncovalent interactions. In some embodiments, the weight ratio of (a) the amount of the polyamine to (b) the carbon nanotube (prior to any functionalization with the one or more surface molecules), (a)/(b), ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values. In some embodiments, the amount of the polyamine is such that the surface functionalized carbon nanotube has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. In some embodiments, the amount of the polyamine can be optimized for transfection efficiency.

[0089] In some more specific embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with a polyethylene imine (e.g., any of those described herein); and (2) a nucleic acid, such as a DNA or RNA molecule, such as a mRNA, siRNA, or antisense oligonucleotide. In some embodiments, the carbon nanotubc can be surface modified with a branched polyethylene imine (e.g., any of those described herein) through covalent amide bond formation between the carboxylic acid group of the carbon nanotube with the amino group of the branched polyethylene imine. In some embodiments, the carbon nanotube can be surface modified with a branched polyethylene imine (e.g., any of those described herein), wherein the weight ratio of the branched polyethylene imine to that of the carbon nanotube ranges from about 10: 1 to about 1: 10. such as about 2: 1, about 1:1, about 1:2, or any range or value between the recited values. In some embodiments, the amount of the branched polyethylene imine is controlled such that the surface functionalized carbon nanotube has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. In some embodiments, the amount of the branched polyethylene imine can be optimized for transfection efficiency.

[0090] In some embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with both a polyamine (e.g., described herein) and a cationic lipid (e.g., described herein); and (2) a nucleic acid, such as a DNA or RNA molecule, such as a mRNA, siRNA, or antisense oligonucleotide. In some embodiments, the carbon nanotube is surface modified with both a poly alkylene imine (e.g., polyethylene imine) and a monovalent cationic lipid herein. In some embodiments, the carbon nanotube is surface modified with both a polyalkylene imine (e.g., polyethylene imine) and a multivalent cationic lipid herein. In some embodiments, the carbon nanotube can be surface modified with the polyamine through covalent bond(s) and the cationic lipid through non-covalent interactions. In some embodiments, the carbon nanotube can be surface modified with both the polyamine and cationic lipid through noncovalent interactions. The amount of the polyamine and cationic lipid for the surface functionalized carbon nanotube is not particularly limited and include any of those described herein. In some embodiments, the weight ratio of (a) the combined amount of the polyamine and the cationic lipid to (b) the carbon nanotube (prior to any functionalization with the one or more surface molecules), (a)/(b), ranges from about 10: 1 to about 1: 10, such as about 2:1, about 1:1, about 1:2, or any range or value between the recited values. In some embodiments, the weight ratio of the polyamine to the cationic lipid can range from about 10: 1 to about 1 : 10, such as about 10:1, about 5:1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1:10, or any range or value between the recited values. In some embodiments, the amount of the polyamine and cationic lipid is controlled such that the surface functionalized carbon nanotube (prior to binding to the nucleic acid) has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. In some embodiments, the amount of the polyamine and cationic lipid can be optimized for transfection efficiency.

[0091] In some more specific embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with a polyethylene imine (e.g., any of those described herein) and an ethyl phosphocholine lipid, e.g., any of those described herein, such as l-palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine; and (2) a nucleic acid, such as a DNA or RNA molecule, such as a mRNA, siRNA, or antisense oligonucleotide. In some embodiments, the carbon nanotubc can be surface modified with (i) a branched polyethylene imine (e.g., any of those described herein) through covalent amide bond formation between the carboxylic acid group of the carbon nanotube with the amino group of the branched polyethylene imine, and (ii) the ethyl phosphocholine lipid (e.g., any of those described herein) through electrostatic interactions and/or hydrophobic interactions. In some embodiments, the carbon nanotube can be surface modified with a branched polyethylene imine (e.g., any of those described herein) and the ethyl phosphocholine lipid, l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, wherein the weight ratio of the combined amount of the branched polyethylene imine and 1-palmitoyl- 2-oleoyl-sn-glycero-3-ethylphosphocholine to that of the carbon nanotube ranges from about 10:1 to about 1:10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values, and the weight ratio of the branched polyethylene imine to l-palmitoyl-2- oleoyl-sn-glycero-3-ethylphosphocholine can also range from about 10: 1 to about 1: 10, such as about 10:1, about 5: 1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1:10, or any range or value between the recited values. In some embodiments, the amount of the branched polyethylene imine andl-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine is such that the surface functionalized carbon nanotube, prior to binding the nucleic acid, has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. In some embodiments, the amount of the branched polyethylene imine and l-palmitoyl-2-oleoyl-sn- glyccro-3-cthylphosphocholinc can be optimized for transfection efficiency.

[0092] In some embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with a polyamine (e.g., described herein), a cationic lipid (e.g., described herein), and a surfactant (e.g., described herein); and (2) a nucleic acid, such as a DNA or RNA molecule, such as a mRNA, siRNA, or antisense oligonucleotide. In some embodiments, the carbon nanotube is surface modified with a polyalkylene imine (e.g., polyethylene imine), a monovalent cationic lipid herein, and a PEGlated lipid. In some embodiments, the carbon nanotube is surface modified with a polyalkylene imine (e.g., polyethylene imine), a multivalent cationic lipid herein, and a PEGlated lipid. In some embodiments, the carbon nanotube can be surface modified with the polyamine through covalent bond(s) and the cationic lipid and surfactant through non-covalcnt interactions. In some embodiments, the carbon nanotubc can be surface modified with the polyamine, cationic lipid, and surfactant through noncovalent interactions. The amount of the polyamine, cationic lipid, and surfactant for the surface functionalized carbon nanotube is not particularly limited and include any of those described herein. In some embodiments, the weight ratio of (a) the combined amount of the poly amine and the cationic lipid to (b) the carbon nanotube (prior to any functionalization with the one or more surface molecules), (a)/(b), ranges from about 10: 1 to about 1: 10, such as about 2:1, about 1:1, about 1:2, or any range or value between the recited values, and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, the weight ratio of the polyamine to the cationic lipid can range from about 10: 1 to about 1: 10, such as about 10: 1, about 5: 1. about 2: 1, about 1: 1, about 1:2, about 1:5, about 1: 10, or any range or value between the recited values. In some embodiments, the amount of the polyamine and cationic lipid is controlled such that the surface functionalized carbon nanotube (prior to binding to the nucleic acid) has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. In some embodiments, the amount of the polyamine, cationic lipid, and the surfactant can be optimized for transfection efficiency.

[0093] In some more specific embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotubc, which is a carbon nanotubc surface modified with a polyethylene imine (e.g., any of those described herein), an ethyl phosphocholine lipid, e.g., any of those described herein, such as l-palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine, and a glycerol-based phospholipid containing a polyethylene glycol chain; and (2) a nucleic acid, such as a DNA or RNA molecule, such as a mRNA, siRNA, or antisense oligonucleotide. In some embodiments, the carbon nanotube can be surface modified with (i) a branched polyethylene imine (e.g., any of those described herein) through covalent amide bond formation between the carboxylic acid group of the carbon nanotube with the amino group of the branched polyethylene imine, and (ii) the ethyl phosphocholine lipid (e.g., any of those described herein) and glycerol-based phospholipid containing a polyethylene glycol chain (e.g., any of those described herein) through electrostatic interactions and/or hydrophobic interactions. In some embodiments, the carbon nanotube can be surface modified with a branched polyethylene imine (e.g., any of those described herein), the ethyl phosphocholine lipid, l-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine, and the surfactant, which is l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000], wherein the weight ratio of the combined amount of the branched polyethylene imine and l-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine to that of the carbon nanotube ranges from about 10:1 to about 1: 10, such as about 2:1, about 1: 1, about 1:2, or any range or value between the recited values, the weight ratio of the branched polyethylene imine to l-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine can also range from about 10: 1 to about 1: 10, such as about 10: 1. about 5: 1, about 2: 1, about 1:1, about 1:2, about 1:5, about 1: 10, or any range or value between the recited values, and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, the amount of the branched polyethylene imine and l-palmitoyl-2-oleoyl- sn-glycero-3 -ethylphosphocholine is such that the surface functionalized carbon nanotube, prior to binding the nucleic acid, has a positive zeta potential of greater than 20 mV, preferably, greater than 40 mV, or higher. In some embodiments, the amount of the branched polyethylene imine, l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, and the surfactant can be optimized for transfection efficiency.

[0094] In some embodiments, the active agent is a peptide or protein, wherein the molar- ratio of the peptide or protein to the carbon nanotube ranges from about 4000: 1 to 1:20, such as about 20: 1 to about 300: 1, such as about 2000:1, about 1000:1, about 750:1. about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 100: 1, about 50: 1, about 20: 1, about 10: 1, about 1: 1, etc. or any range or value between the recited values, such as about 20: 1 to about 300:1, about 100: 1 to about 1000:1, about 200: 1 to about 500: 1, about 300: 1 to about 750:1, etc.

[0095] In some embodiments, the active agent is a small molecule, wherein the molar ratio of the small molecule to the carbon nanotube ranges from about 4000: 1 to 1:20, such as about 20:1 to about 300: 1, such as about 2000: 1, about 1000: 1, about 750: 1, about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 100: 1, about 50: 1, about 20: 1, about 10: 1, about 1: 1, etc. or any range or value between the recited values, such as about 20: 1 to about 300: 1, about 100: 1 to about 1000: 1, about 200: 1 to about 500:1, about 300: 1 to about 750: 1, etc. As used herein, a small molecule refers to an organic compound having a molecular weight of less than 1,000 Daltons, preferably, less than 500 Daltons. Nonlimiting exemplary small molecules include those drugs approved for treating or preventing a human disease or disorder by the U.S. Food and Drug Administration or similar government agencies in non-U.S. countries or regions.

[0096] In some embodiments, the active agent is an antigen. In some embodiments, the pharmaceutical composition comprises 1, 2, 3, 4, 5, or more than 5 different antigens. In some embodiments, the pharmaceutical composition comprises 1-5, 1-10, 1-15, 1-20, 1-25, 1- 30, 2-10, 2-15, 2-20, 2-25, 3-10, 3-15, 3-20, 3-25, 4-10, 4-15, 4-20, 4-25, 5-10, 5-15, 5-20, or 5-25 different antigens. In some embodiments, more than one antigen is bound to the carbon nanotube. In some embodiments, at least some of the more than one antigen included in the pharmaceutical composition are bound to the carbon nanotube and some are independent of the carbon nanotube. In some embodiments, two or more different types of antigens (e.g., one antigen is mRNA based and the other is a peptide), such as those derived from the same infectious agent, can be bound to different surface functionalized carbon nanotubes and formulated together in the pharmaceutical composition herein.

[0097] In some embodiments, the antigen is derived from or corresponding to an infectious agent, such as a microorganism, such as a virus (e.g., HIV virus, HBV virus, HCV virus, influenza, corona virus, such as SARS-COV-2, etc.), a bacteria, a fungus, a protozoan, a parasite, and/or a helminth.

[0098] In some embodiments, the antigen is a Tumor-Associated Antigen (TAA). As used herein, a TAA is an antigen derived from or corresponding to an aberrantly overexpressed self-antigens in a tumor cell compared to a normal cell and might represent a universal antigen among patients with the same malignancy. TAAs can also include: cell lineage differentiation antigens, which are normally not expressed in adult tissue (e.g., tyrosinase, gplOO, MART-1, prostate-specific antigen (PSA); prostatic acid phosphatase (PAP)); and cancer/germline antigens (also known as cancer/testis), which are normally expressed only in immune privileged germline cells (e.g., MAGE-A1, MAGE-A3, NY-ESO-1, and PRAME). In some embodiments, the antigen is a Tumor-Spccific Antigen (TSA). As used herein, a TSA is an antigen that is specific to tumors and is not expressed on the surface of normal cells. A TSA can include for example, a mutated neoantigen as well as an antigen from an oncovirus, and an endogenous retroviral element (HERV). In some embodiments, the drug-loaded nanoparticle comprises one or more antigens that is a TAA and one or more antigens that is a TSA.

[0099] Polypeptides or fragments thereof that may be useful as antigens include, without limitation, those derived from or corresponding to a TAA or TSA expressed in colorectal cancer, gastric cancers, urothelial/bladder cancer, pancreatic cancer, breast cancer (e.g., TNBC) , ovarian cancer, prostate cancer, liver cancer (e.g., HCC), kidney, lung cancer (e.g., NSCLC and SCLC), melanoma, glioblastoma, myeloma (e.g., SPCM), leukemia (lympocytic leukemia), or lypmphoma (ALL, follicular lymphoma.

[0100] Additional polypeptides or fragments thereof that may be useful as antigens include, without limitation, those derived from or corresponding to aldolase, adipophilin, AFP, AIM-2, ART-4, BAGE, a-fetoprotein, BCL-2, Bcr-Abl, BING-4, CEA. CPSF, CT, cyclin DI, Ep- CAM, EphA2, EphA3, ELF-2, FGF-5, G250, Gonadotropin Releasing Hormone, gplOO, HER-2, intestinal carboxyl esterase (iCE), HIF-la, IGF-1R, IGFBP-2, IL13Ra2, MAGE-1, MAGE-2, MAGE-3, MAGE-A1, MAGE-A3, MART-1, MART-2. M-CSF, MDM-2, mesothelin, MMP-2, MUC-1 , NY-ESO-1 , MUM- 1 , MUM-2, MUM-3, PAP, p53, PBF, PRAME, PSA, PSMA, RAGE-1, RNF43, RU1, RU2AS, SART-1, SART-2, SART-3, SAGE- 1, SCRN 1, SOX2, SOX10, STEAP1, survivin, tyrosinase, telomerase, TGFp RII, TRAG-3. TRP-1, TRP-2, hTERT, WT1, and/or a neoantigen.

[0101] In some specific embodiments, the active agent is an HIV antigen, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or VI V2 region of gpl20, or a nucleic acid encoding the same. Suitable HIV antigens include any of those known in the art, such as those that were tested in clinical trials or are currently in clinical trials. See also e.g., Yates, N. L. et al. J Virol 92:e01843-17 (2018). The amount of antigen loaded to the carbon nanotube is not particularly limited. In some embodiments, the molar ratio of the antigen to the carbon nanotube can range from about 4000: 1 to 1:20, such as about 2000: 1, about 1000:1, about 750: 1, about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 100: 1, about 50:1, about 20: 1, about 10:1, about 1: 1, etc. or any range or value between the recited values, such as about 20: 1 to about 300: 1, about 100: 1 to about 1000: 1, about 200: 1 to about 500: 1, about 300: 1 to about 750: 1, etc.

[0102] In some embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with a hydrophilic linker herein; and (2) an antigen, such as an HIV antigen, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V1V2 region of gpl20. In some embodiments, the carbon nanotube is surface modified with a hydrophilic linker having a general formula of G1-(PEG)-G2, as defined herein. In one preferred embodiment, the hydrophilic linker can be NH2-(PEG)-COOH, wherein PEG is a PEG2000 chain. In some embodiments, the carbon nanotube is surface modified with the hydrophilic linker through an amide bond formed between an amine group of the hydrophilic linker and a carboxylic acid function of the carbon nanotube. In some embodiments, the antigen is covalently bonded to the hydrophilic linker, such as through a group of the hydrophilic linker that forms an amide bond, an ester bond, an ether bond, or a thioether bond, with the antigen. In some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with an amine group of the hydrophilic linker. In some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%, or any range or value between the recited value, form an amide bond with an amine group of the hydrophilic linker.

[0103] In some embodiments, the pharmaceutical composition herein can comprise (1) a surface modified carbon nanotube, which is a carbon nanotube surface modified with a hydrophilic linker and a surfactant herein; and (2) an antigen, such as an HIV antigen, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V1V2 region of gpl20. In some embodiments, the carbon nanotube is surface functionalized with a hydrophilic linker of formula G1-(PEG)-G2 as defined herein and a PEGlated lipid such as a glycerol-based phospholipid containing a polyethylene glycol chain as defined herein. In some embodiments, the carbon nanotube is surface functionalized with a hydrophilic linker of formula NH2-(PEG)-C00H, wherein PEG is a chain of PEG2000, and a surfactant which is l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [mcthoxy(polycthylcncglycol)-2000]. In some embodiments, the antigen is covalently bonded to the hydrophilic linker, such as through a group of the hydrophilic linker that forms an amide bond, an ester bond, an ether bond, or a thioether bond, with the antigen. In some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with an amine group of the hydrophilic linker and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%. or any range or value between the recited value, form an amide bond with an amine group of the hydrophilic linker, and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1 % by weight of the carbon nanotube.

[0104] Table 1 below shows some exemplary parameters for carbon nanotube modifications herein. It should be understood that these parameters can be combined in any fashion as applicable, for example, any of the carbon nanotube properties can be combined with any of the surface treatment and any of the surface immobilizations (active agent).

Table 1. Exemplary CNT delivery vehicle modifications

[0105] The pharmaceutical composition herein can be formulated for any suitable route of administration, including for example, orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally. For example, in some embodiments, the pharmaceutical composition herein can be formulated for intramuscular or subcutaneous injection. In some embodiments, the pharmaceutical composition herein can be formulated for intranasal administration.

[0106] The pharmaceutical composition herein is also not limited to a particular form of formulation. For example, in some embodiments, the pharmaceutical composition can be in the form of a solid, semi-solid, gel, liquid, such as a solution, an emulsion, etc. In some embodiments, the pharmaceutical composition can be in the form of a lyophilized powder. In some embodiments, the pharmaceutical composition can be in the form of a solution, typically, an aqueous solution. In some embodiments, the pharmaceutical composition can also be in the form of a gel. In some embodiments, the pharmaceutical composition can also be in the form of a suspension. Techniques for preparing formulations known in the art may be used for formulating the pharmaceutical compositions herein.

Carbon Nanotube Conjugates

[0107] Some embodiments of the present disclosure are directed to carbon nanotube conjugates, in which an active agent is conjugated to a carbon nanotube covalently through a linker.

[0108] In some embodiments, the present disclosure provides a carbon nanotube conjugate of Formula I:

(I), wherein:

CNT represents a carbon nanotube,

SPC represents a hydrophilic spacer,

LNK represents a group which connects SPC to D,

D represents a residue of an active agent of D-Y, wherein Y is a group (e.g., amino, carboxy, hydroxy, or SH group) that is capable of linking D to the SPC, preferably, the active agent represents a small molecule, peptide, protein, or nucleic acid; and n is the number of carboxy groups of the carbon nanotube that are functionalized with NH- SPC-LNK-D, for example, n can typically range from 1-10,000, such as 1-4,000, 1-500, 20- 600, 30-500, etc.

[0109] To be clear, in Formula I, the CNT may contain one or more free COOH groups or functionalized COOH groups that are not covalently functionalized with NH-SPC-LNK-D as defined herein. For example, the CNT may contain one or more moieties represented by -CO- NH-SPC-LNK-X, wherein SPC and LNK are as defined herein, and X is not the D as defined hereinabove, for example, X can be hydrogen or hydroxy group. In some embodiments, the CNT may contain less than 10% of free carboxylic acid group, such as less than 5%, less than 2%, or less than 1% of free COOH group, based on all carboxy groups of the carbon nanotube, i.e., the total of (i) free COOH group; (ii) carboxy group functionalized with NH- SPC-LNK-D as defined herein; and (iii) carboxy group functionalized with other moiety such as those represented by -CO-NH-SPC-LNK-X as defined herein. In some embodiments, the number of carboxy groups of the carbon nanotube that are functionalized with NH-SPC-LNK- D represents at least 30%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, of all carboxy groups of the carbon nanotube.

[0110] Suitable carbon nanotubes for Formula I are not particularly limited, which can be any of those described herein. For example, the carbon nanotube can be single-walled or multi-walled carbon nanotube. In some embodiments, the carbon nanotube can have an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio (length/diameter) of about 0.2:1 to about 12,500: 1, wherein the average length and diameter arc measured by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM), respectively. In some embodiments, the carbon nanotube can have an average diameter of about 40 nm to about 200 nm, and an average length of about 50 nm to about 5,000 nm, with an average aspect ratio of about 1: 1 to about 100: 1. In some embodiments, the carbon nanotube can be characterized as having certain size and/or shape, for example, to mimic that of a microorganism, such as a virus. For example, in some embodiments, the carbon nanotube can be a multi-walled carbon nanotube, characterized as having an average diameter of about 20 nm to about 200 nm, such as about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 80 nm, about 100 nm, about 120 nm, about 150 nm, about 200 nm, or any range or value between the recited values, such as about 30 nm to about 150 nm, about 40 nm to about 150 nm, or about 60 nm to about 100 nm. In some embodiments, the carbon nanotubc can also be characterized as having an average length of about 10 nm to about 1000 nm, about 40 nm, about 50 nm, about 100 nm, about 120 nm, about 150 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 800 nm, about 1000 nm, or any range or value between the recited values, such as about 50 nm to about 1000 nm, about 100 nm to about 200 nm, about 200 nm to about 400 nm, about 200 nm to about 500 nm, about 400 nm to about 800 nm, etc. In some specific embodiments, the carbon nanotube can be characterized as having an average length of about 100 nm to about 200 nm. In some specific embodiments, the carbon nanotube can be characterized as having an average length of about 200 nm to about 400 nm. In some embodiments, the carbon nanotube can also have an average length of above 1000 nm and up to 5000 nm and beyond, such as about 2000 nm, about 4000 nm, about 5000 nm, or any range or value between the recited values. In some embodiments, the carbon nanotube can also be characterized as having an average aspect (length/diameter) ratio of about 0.2: 1 to about 10: 1, such as about 1:1 to about 7:1, such as about 0.2: 1, about 0.5:1, about 0.8:1, about 1: 1, about 2: 1, about 3: 1, about 4:1, about 5:1, about 6: 1, about 7: 1, or any range or value between the recited values, such as about 1: 1 to about 5: 1, or about 2: 1 to about 4: 1. For example, as shown herein, in some embodiments, the carbon nanotube is designed to mimic a virus which is substantially spherical, and the average aspect ratio of the carbon nanotube can be about 0.5: 1 , about 0.8: 1 , about 1 :1 , about 1.2: 1 , about 1.5: 1 , about 2: 1 , or any range or value between the recited values.

[0111] In some embodiments, the carbon nanotube preferably has a substantially uniform length. For example, in some embodiments, the carbon nanotube can be characterized as being monodisperse as determined by DLS. In some embodiments, the carbon nanotube can be characterized as having a polydisperse index of less than 1, more preferably, less than 0.75, less than 0.5, less than 0.25, or even less than 0.1. Exemplified methods for preparing carbon nanotubes having a substantially uniform length are described herein.

[0112] Various hydrophilic spacers can be SPC in formula I. The hydrophilic spacer typically has carbon, hydrogen, and oxygen and/or nitrogen atoms, and optionally sulfur and halogen atoms, wherein the ratio of the total number of oxygen and/or nitrogen atoms to the total number of carbon atoms is at least 1:5, preferably, at least 1:4, at least 1:3, such as about 1:2. In some embodiments, the hydrophilic spacer only contains carbon, hydrogen, and oxygen atoms, wherein the ratio of the total number of oxygen atoms to the total number of carbon atoms is at least 1:5, preferably, at least 1:4, at least 1:3, such as about 1:2. In some embodiments, the hydrophilic spacer contains only carbon, hydrogen, oxygen and nitrogen atoms, wherein the ratio of the total number of oxygen and nitrogen atoms to the total number of carbon atoms is at least 1:5, preferably, at least 1:4, at least 1:3, such as about 1:2. The molecular mass of the SPC typically ranges from 100 to 10,000 Daltons, such as about 100, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 10,000, Daltons, or any range or value between the recited values, for example, about 500-5000 or about 1000-3000 Daltons, etc. In some embodiments, the hydrophilic spacer comprises a polyethylene glycol chain. In some embodiments, the hydrophilic spacer comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., with two suitable end groups, for example, in some embodiments, both end groups of the polyethylene glycol chain can independently be an alkylene of 1-5 carbons. In some embodiments, SPC is a polyethylene glycol chain that is represented by , wherein m represents the average number of oxyethylene groups of a polyethylene glycol chain, m typically ranges from 1-200, such as 10-100, 20-60, 30-50, 42-48, such as about 44 or 45, LNK and NH are included to show direction of connection. In some embodiments, the (CEECHaOlm represents a PEG 100, PEG500, PEG 1000, PEG2000, or PEG3000 chain, preferably, a PEG2000 chain. Polyethylene glycol chain with a different length can also be used.

[0113] The LNK that connects SPC to D is typically an amide group, such However, other connections are also suitable, depending on the structure of the active agent (i.c., D-Y defined herein). [0114] Typically, Y is a group that allows D to form a covalent link to the CNT through

LNK, such as an amine, COOH, OH, SH, etc. For example, in some embodiments, prior to conjugation, D may be attached to an NH2 group, i.e., as in D-NH2, and the hydrophilic spacer bound to the CNT may be attached to a COOH group, i.e., upon conjugation, the conjugate of Formula I can be formed, with LNK being an amide group, see Formula I-a below:

(La).

[0115] In some specific embodiments, the conjugate of Formula I can have a formula according to I-b: wherein CNT, m, n, and D are defined herein. For example, in some embodiments, the (CH ₂ CH ₂ O) _m represents a PEG100, PEG500, PEG1000, PEG2000, or PEG3000 chain, preferably, a PEG2000 chain. Polyethylene glycol chain with a different length can also be used.

[0116] Suitable active agents for the conjugate of formula I (e.g., I-a or I-b) are not particularly limited. In some embodiments, D-Y can be a small molecule. In some embodiments, D-Y can be a peptide or protein. In some embodiments, D-Y can be an antigen, such as an HIV antigen, such as an envelope glycoprotein antigen, e.g., gpl20 or V1V2 region of HIV-1 gpl20 protein/peptide.

[0117] The amount of D conjugated to the CNT is not particularly limited. For example, in some embodiments, the molar ratio of D (such as an antigen described herein) to CNT in the conjugate of formula I (e.g., I-a or I-b) can range from about 10: 1 to about 4000: 1, such as about 2000: 1, about 1000: 1, about 750: 1, about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 100: 1, about 50: 1, about 20: 1, about 10: 1, about 1: 1, etc. or any range or value between the recited values, such as about 20:1 to about 300: 1, about 100: 1 to about 1000: 1, about 200: 1 to about 500:1, about 300: 1 to about 750: 1, about 27:1 to about 470: 1, etc.

[0118] In some embodiments, the carbon nanotube conjugate of formula I (e.g., I-a or I-b) herein further comprises a surfactant attached to the surface of the carbon nanotube noncovalently. In some embodiments, in the carbon nanotube conjugate of formula I (e.g., I-a or I-b), D is a residue of an antigen, such as an HIV antigen, such as an envelope glycoprotein antigen, e.g., gpl20 or V1V2 region of HIV-1 gpl20 protein/peptide. In some embodiments, the surfactant can help to present the antigen in a correct orientation to achieve its desired effect, such as to induce immune response.

[0119] Suitable surfactants include any of those described herein. In some embodiments, the surfactant comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, e.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, a methoxy capping group. In some embodiments, the surfactant is a PEGlated lipid (e.g., any of those described herein) containing the polyethylene glycol chain, such as those glycerol-based lipid with at least one of the glycerol hydroxy groups forms a fatty ether or fatty ester, and one of the glycerol hydroxy groups is linked to a polyethylene glycol chain directly or through a linking group. In some embodiments, the surfactant is a phospholipid, such as a glycerol -based phospholipid containing the polyethylene glycol chain. In some embodiments, the surfactant can be a glycerol-based phospholipid, in which at least one of the glycerol hydroxy groups forms a fatty ether or fatty ester, and one of the glycerol hydroxy groups is linked to a polyethylene glycol chain through a phosphate containing group. For example, in one preferred embodiment, the surfactant is l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000]. The surfactant can exist in various amount. For example, in some embodiments, the surfactant is in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (exclusive of any functionalization), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. [0120] The carbon nanotube conjugate can be typically included in a pharmaceutical composition, which can be formulated for any suitable route of administration, including for example, orally, nasally, transdcrmally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally. For example, in some embodiments, the pharmaceutical composition can be formulated for intramuscular or subcutaneous injection. In some embodiments, the pharmaceutical composition can be formulated for intranasal administration. In some embodiments, the pharmaceutical composition can be formulated for intraperitoneal or intradermal administration. The pharmaceutical composition is also not limited to a particular form of formulation. For example, in some embodiments, the pharmaceutical composition can be in the form of a solid, semi-solid, gel, liquid, such as a solution, an emulsion, etc. In some embodiments, the pharmaceutical composition can be in the form of a lyophilized powder. In some embodiments, the pharmaceutical composition can be in the form of a solution, typically, an aqueous solution. In some embodiments, the pharmaceutical composition can also be in the form of a gel. In some embodiments, the pharmaceutical composition can also be in the form of a suspension. Techniques for preparing formulations known in the art may be used for formulating the pharmaceutical compositions herein.

Method of Preparation

[0121] Some embodiments of the present disclosure are directed to methods of preparing a pharmaceutical composition, such as a vaccine composition.

[0122] In some embodiments, the present disclosure provides a method of preparing a pharmaceutical composition comprising a surface functionalized carbon nanotube and an active agent. In some embodiments, the method comprises (a) surface functionalizing a carbon nanotube with one or more surface molecules (e.g., any of those described herein) to produce a surface functionalized carbon nanotube; (b) combining the surface functionalized carbon nanotube with the active agent (e.g., any of those described herein) to form an active agent bound carbon nanotube; and (c) formulating the active agent bound carbon nanotube to provide the pharmaceutical composition. Suitable carbon nanotube, surface molecule, surface functionalization, and active agent, and amounts thereof, include any of those described herein in any combinations.

[0123] In some particular embodiments, the present disclosure provides a method of preparing a vaccine composition for treating or preventing an infection with a microorganism (e.g., virus). In some embodiments, the method comprises (a) surface functionalizing a carbon nanotube with one or more surface molecules (e.g., any of those described herein) to produce a surface functionalized carbon nanotube; (b) combining the surface functionalized carbon nanotube with an antigen of the microorganism (such as an antigen selected from proteins, peptides, polysaccharides, lipids, or nucleic acids, e.g., any of those described herein) to form an antigen bound carbon nanotube; and (c) formulating the antigen bound carbon nanotube to provide the vaccine composition. Suitable carbon nanotube, surface molecule, surface functionalization, and antigen, and amounts thereof, include any of those described herein in any combinations.

[0124] The carbon nanotube for the surface functionalization in the methods herein is typically a multi-walled carbon nanotube, which has COOH groups, for example, on the surface of the carbon nanotube. In some preferred embodiments, the carbon nanotubes are characterized as having about 1-20% by weight of COOH groups, such as about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 15%, about 20%, or any range or value between the recited values, such as about 2-15%, about 3-14%, about 3-12%, or about 4-12% by weight of COOH groups.

[0125] The carbon nanotubes for the surface functionalization in the methods herein can have various diameters and length as described herein, for example, with an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5.000 nm, with an average aspect ratio (length/diameter) of about 0.2: 1 to about 12,500: 1. In some embodiments, the carbon nanotubes can be characterized as having certain size and/or shape, for example, to mimic that of a microorganism, such as a virus. For example, in some embodiments, the carbon nanotube has an average diameter and/or average length within 5- fold (e.g., within 1-fold, 2-fold, or within 3-fold) of that of the microorganism, such as a virus (e.g., an HIV virus), respectively. In such embodiments, the diameter and/or length of the carbon nanotubes will vary according to the size of a microorganism. Exemplary carbon nanotubes for mimicking an HIV virus are shown herein in the Examples section.

[0126] In some embodiments, the surface functionalizing step in the methods herein can comprise surface functionalizing a carbon nanotube with one or more surface molecules to produce a surface functionalized carbon nanotube, wherein the one or more surface molecules are selected from (i) a polyamine, (ii) a hyperbranched polymer or dendrimer, which has one or more groups that are charged or chargeable at pH of 7, (iii) a cationic lipid, which has one or more groups that are positively charged or positively chargeable at pH of 7, (iv) a surfactant, and (v) a hydrophilic linker. Suitable polyamine, hyperbranched polymer or dendrimers, cationic lipids, surfactants, and hydrophilic linkers, and amounts thereof, include any of those described herein. In some embodiments, the carbon nanotube is surface functionalized with the polyamine. In some embodiments, the carbon nanotube is surface functionalized with the hyperbranched polymer or dendrimer. In some embodiments, the carbon nanotube is surface functionalized with the cationic lipid. In some embodiments, the carbon nanotube is surface functionalized with the surfactant. In some embodiments, the carbon nanotube is surface functionalized with the hydrophilic linker. In some embodiments, the carbon nanotube is surface functionalized with the polyamine and the cationic lipid. In some embodiments, the carbon nanotube is surface functionalized with the hyperbranched polymer or dendrimer and the cationic lipid. In some embodiments, the carbon nanotube is surface functionalized with the polyamine and the surfactant. In some embodiments, the carbon nanotube is surface functionalized with the hyperbranched polymer or dendrimer and the surfactant. In some embodiments, the carbon nanotubc is surface functionalized with the hydrophilic linker and the surfactant. In some embodiments, the carbon nanotube is surface functionalized with the polyamine, the cationic lipid, and the cationic lipid. In some embodiments, the carbon nanotube is surface functionalized with the hyperbranched polymer or dendrimer, the cationic lipid, and the surfactant. In some embodiments, the carbon nanotube is surface functionalized with the hydrophilic linker, the cationic lipid, and the surfactant.

[0127] Surface functionalization methods are not particularly limited. For example, in some embodiments, surface functionalization comprises reacting the carbon nanotube with the surface molecule to form a covalent bond (e.g., an amide bond, ester bond, ether bond, thioether bond, etc.) to attach the surface molecule to the carbon nanotube. In some embodiments, surface functionalization comprises mixing the carbon nano tube with the surface molecule to attach the surface molecule to the carbon nanotube through non-covalent interactions, such as hydrophobic interaction, electrostatic interaction, etc. In some embodiments, some of the surface molecule can be covalently attached to the carbon nanotube and some of the surface molecule can be non-covalently attached to the carbon nanotube.

[0128] Similarly, combining the surface functionalized carbon nanotube with the active agent or antigen for the methods herein is not particularly limited. In some embodiments, the combining step comprises reacting the surface functionalized carbon nanotube with the active agent or antigen to form a covalent bond to attach the active agent or antigen to the carbon nanotube, e.g., through an amide bond formation. In some embodiments, the combining step comprises mixing the surface functionalized carbon nanotube with the active agent or antigen to attach the active agent or antigen to the carbon nanotube through non-covalent interactions, such as hydrophobic interaction, electrostatic interaction, etc. In some embodiments, some of the active agent or antigen can be covalently attached to the carbon nanotube and some of the active agent or antigen can be non-covalently attached to the carbon nanotube.

[0129] In some embodiments, the active agent or antigen is negatively charged, e.g., a nucleic acid including DNA, mRNA, siRNA, antisense oligonucleotides etc., and the surface functionalization can be earned out such that the zeta potential of the surface functionalized carbon nanotube is positive and has a value of at least 20 mV, such as at least 40 mV, or higher, prior to binding the active agent or antigen. For example, in some embodiments, the surface functionalizing step in the methods herein can comprise surface functionalizing the carbon nanotube with a polyamine herein, such as a polyalkylene imine, preferably, polyethylene imine (e.g., any of those described herein), and optionally a cationic lipid herein, such as an ethyl phosphocholine lipid, such as l-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine. In some specific embodiments, the carbon nanotube can be surface modified with (i) a branched polyethylene imine (e.g., any of those described herein) through covalent amide bond formation between the carboxylic acid group of the carbon nanotube with the amino group of the branched polyethylene imine, and (ii) the ethyl phosphocholine lipid through electrostatic interactions and/or hydrophobic interactions. In some embodiments, the weight ratio of (a) the combined amount of the polyamine and the cationic lipid to (b) the carbon nanotubc (prior to any functionalization with the one or more surface molecules), (a)/(b), ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1: 1, about 1:2, or any range or value between the recited values. In some embodiments, the weight ratio of the polyamine to the cationic lipid can range from about 10: 1 to about 1: 10, such as about 10: 1, about 5: 1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1 : 10, or any range or value between the recited values. For example, in some embodiments, the carbon nanotube can be surface modified with a branched polyethylene imine (e.g., any of those described herein) and the ethyl phosphocholine lipid, l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, wherein the weight ratio of the combined amount of the branched polyethylene imine and 1- palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine to that of the carbon nanotube ranges from about 10: 1 to about 1: 10, such as about 2: 1, about 1:1, about 1:2, or any range or value between the recited values, and the weight ratio of the branched polyethylene imine to 1- palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine can also range from about 10: 1 to about 1: 10, such as about 10:1, about 5: 1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1:10, or any range or value between the recited values. In some embodiments, the carbon nanotube can be further surface functionalized with a surfactant herein, such as a PEGlated lipid, for example, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000]. When present, the surfactant is typically in an amount of about 0.01 % to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In embodiments, the negatively charged active agent or antigen is typically attached to the carbon nanotube through noncovalent interactions, such as electrostatic interactions. The amount of the negatively charged active agent or antigen, e.g., a nucleic acid described herein, such as an mRNA herein (e.g., an mRNA that encodes an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V1V2 region of gpl20), is typically in a weight ratio to the carbon nanotube ranging from about 20: 1 to 1:20, such as about 1: 1 to about 1:5 or about 1:5 to about 1: 10. [0130] In some embodiments, the active agent or antigen is a peptide or protein, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V1V2 region of gpl20, and the surface functionalization can be carried out such that at least a portion of the active agent is attached to the carbon nanotube through a covalent link, e.g., through one or more of the NH2, COOH, OH, or SH groups present in the peptide or protein. For example, in some embodiments, the surface functionalizing step in the methods herein can comprise surface functionalizing the carbon nanotube with a hydrophilic linker described herein, optionally with the surfactant described herein. In some specific embodiments, the carbon nanotube is surface functionalized with a hydrophilic linker of formula G1-(PEG)-G2 as defined herein and a PEGlated lipid (e.g., any of those described herein) containing a polyethylene glycol chain, such as those glycerol-based phospholipid containing a polyethylene glycol chain, as defined herein. In some embodiments, the carbon nanotube is surface functionalized with a hydrophilic linker of formula , wherein (CHiCHrO)™ represents a PEG2000 chain, and a surfactant which is l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000]. In some embodiments, the active agent or antigen is covalently bonded to the hydrophilic linker, such as through a group of the hydrophilic linker that forms an amide bond, an ester bond, an ether bond, or a thioether bond, with the active agent or antigen. In some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with an amine group of the hydrophilic linker and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%, or any range or value between the recited value, form an amide bond with an amine group of the hydrophilic linker, and the surfactant is present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube. In some embodiments, the molar ratio of the active agent or antigen to the carbon nanotube can range from about 4000: 1 to 1:20, such as about 2000: 1, about 1000: 1, about 750: 1, about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 100: 1, about 50: 1, about 20: 1, about 10: 1, about 1: 1, etc. or any range or value between the recited values, such as about 20: 1 to about 300:1, about 100: 1 to about 1000:1, about 200: 1 to about 500: 1, about 300: 1 to about 750:1. etc.

[0131] In some embodiments, the present disclosure provides a method of preparing a pharmaceutical composition comprising a carbon nanotube conjugate. In some embodiments, the method comprises: (a) conjugating a carbon nanotube having carboxylic acid groups with a compound having a structure of Formula II: (II) , wherein SPC represents a hydrophilic spacer, and G represents a group capable of forming a covalent bond with an active agent (e.g., any of those described herein), to form a functionalized carbon nanotube having a structure of Formula III: wherein:

CNT represents a carbon nanotube, SPC and G are as defined above, and n is the number of carboxy groups of the carbon nanotube that are functionalized with the compound of Formula II;

(b) coupling the functionalized carbon nanotube of Formula III with the active agent to form a carbon nanotube conjugate of Formula IV: wherein: A represents (i) the active agent covalently attached to SPC through a bond formed with G, or (ii) G or a derivative thereof; and

CNT, SPC, G, and n arc as defined above; and

(c) formulating the carbon nanotube conjugate of Formula IV to provide the pharmaceutical composition.

[0132] In some embodiments, the present disclosure provides a method of preparing a vaccine composition for treating or preventing an infection with a microorganism (e.g., virus). In some embodiments, the method comprises:

(a) conjugating a carbon nanotube having carboxylic acid groups with a compound having a structure of Formula II: (II), wherein SPC represents a hydrophilic spacer, and G represents a group capable of forming a covalent bond with an antigen of the microorganism to form a functionalized carbon nanotube having a structure of Formula III:

(ill); wherein:

CNT represents a carbon nanotube,

SPC and G are as defined above, and n is the number of carboxy groups of the carbon nanotube that are functionalized with the compound of Formula II;

(b) coupling the functionalized carbon nanotube of Formula III with the antigen of the microorganism to form a carbon nanotube conjugate of Formula IV:

(IV); wherein:

A represents (i) the antigen covalently attached to SPC through a bond formed with G, or (ii) G or a derivative thereof; and CNT, SPC, G, and n are as defined above; and (c) formulating the carbon nanotube conjugate of Formula IV to provide the vaccine composition.

[0133] It should be understood that each of steps (a), (b), and (c) discussed hereinabove can independently represent a novel aspect of the present disclosure. For example, the step of preparing a functionalized carbon nanotube having a structure of Formula III is a novel process, independent of steps (b) and/or (c). Similarly, the step of converting a functionalized carbon nanotube having a structure of Formula III to a carbon nanotube conjugate of Formula IV is also a novel process, independent of steps (a) and/or (c). And the step of formulating the carbon nanotube conjugate of Formula IV is independent of steps (a) and/or (b). The functionalized carbon nanotube of Formula III and the carbon nanotube conjugate of Formula IV are also novel compositions of the present disclosure.

[0134] The carbon nanotube for the conjugating steps in the methods herein is typically a multi-walled carbon nanotube, which has COOH groups, for example, on the surface of the carbon nanotube. In some preferred embodiments, the carbon nanotubes are characterized as having about 1-20% by weight of COOH groups, such as about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 15%, about 20%, or any range or value between the recited values, such as about 2-15%, about 3-14%, about 3-12%, or about 4-12% by weight of COOH groups.

[0135] The carbon nanotubes for the conjugating steps in the methods herein can have various diameters and length as described herein, for example, with an average diameter of about 0.4 nm to about 200 nm, and an average length of about 10 nm to about 5,000 nm, with an average aspect ratio (length/diameter) of about 0.2:1 to about 12,500:1. In some embodiments, the carbon nanotubes can be characterized as having certain size and/or shape, for example, to mimic that of a microorganism, such as a virus. For example, in some embodiments, the carbon nanotube has an average diameter and/or average length within 5- fold (e.g., within 1-fold, 2-fold, or within 3-fold) of that of the microorganism, such as a virus (e.g., an HIV virus), respectively. In such embodiments, the diameter and/or length of the carbon nanotubes will vary according to the size of a microorganism. Exemplary carbon nanotubes for mimicking an HIV virus are shown herein in the Examples section. [0136] In some embodiments, substantially all (greater than 90%) of the carboxylic acid function of the carbon nanotube form an amide bond with the NH2 group of the compound of Formula II. In some embodiments, only a portion of the carboxylic acid function of the carbon nanotube, such as about 10%, about 30%, about 50%, about 75%, about 90%, or any range or value between the recited value, form an amide bond with the NH2 group of the compound of Formula II. For example, in some embodiments, greater than 30% (such as greater than 50%, greater than 60%, up to all) of the carboxylic acid groups of the carbon nanotube are functionalized with the compound of Formula II. To be clear, the CNT in Formula III can have carboxylic acid group(s) that is not functionalized with the compound of Formula II. For example, assuming 90% efficiency of the reaction between the carbon nanotube and the compound of Formula II, there would be 10% of the carboxylic acid group that is not functionalized with the compound of Formula II. The extent of functionalization can be controlled, for example, by varying the amount of the compound of Formula II used in the conjugation reaction.

[0137] In some embodiments, SPC in formula II or III can be any of the hydrophilic spacer described herein. For example, in some embodiments, SPC only contains carbon, hydrogen, and oxygen atoms, wherein the ratio of the total number of oxygen atoms to the total number of carbon atoms is at least 1:5, preferably, at least 1:4, at least 1:3, such as about 1:2. In some embodiments, SPC contains only carbon, hydrogen, oxygen and nitrogen atoms, wherein the ratio of the total number of oxygen and nitrogen atoms to the total number of carbon atoms is at least 1 :5, preferably, at least 1 :4, at least 1 :3, such as about 1 :2. The molecular mass of the SPC typically ranges from 100 to 10,000 Daltons, such as about 100, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 10,000, Daltons, or any range or value between the recited values, for example, about 500-5000 or about 1000-3000 Daltons, etc. In some embodiments, SPC comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc. In some embodiments. SPC is a polyethylene glycol chain that is represented by wherein m represents the average number of oxyethylene groups of a polyethylene glycol chain, m typically ranges from 1-200, such as 10-100, 20-60, 30-50, 42-48, such as about 44 or 45, LNK and NH are included to show direction of connection. In some embodiments, the (CH ₂ CH ₂ O) _m represents a PEG100, PEG500, PEG1000, PEG2000, or PEG3000 chain, preferably, a PEG2000 chain. Polyethylene glycol chain with a different length can also be used.

[0138] In some embodiments, G in Formula II or III is (CH ₂ ) _X NH ₂ or (CH ₂ ) _X COOH, wherein x is 0-5 (e.g., 0, 1, 2, 3, or 4), wherein as applicable, one or two of the CH ₂ unit may be replaced with C(O), NH, or O. In some embodiments, x is 0, i.e., G is NH2 or COOH. In some preferred embodiments, G in Formula II or III is COOH. As would be understood in the ait, the NH2 or COOH group in G may be optionally masked/protected during the conjugation step with the carbon nanotube, which can be deprotected to expose the NH2 or COOH for reacting with the active agent or antigen. Protection and deprotection of a NH2 or COOH group are well known in the art.

[0139] In some specific embodiments, the compound of Formula II has a formula according to O , wherein m represents the average number of oxyethylene groups of a polyethylene glycol chain, m typically ranges from 1-200, such as 10-100, 20-60, 30-50, 42-48, such as about 44 or 45. In some embodiments, (CH ₂ CH ₂ O) _m represents a PEG100, PEG500, PEG1000, PEG2000, or PEG3000 chain, preferably, a PEG2000 chain.

[0140] In some specific embodiments, the compound of Formula III has a formula according , wherein m represents the average number of oxyethylene groups of a polyethylene glycol chain, m typically ranges from 1-200. such as 10-100, 20-60, 30-50, 42-48, such as about 44 or 45, and CNT and n are defined herein. In some embodiments, (CH ₂ CH ₂ O) _m represents a PEG100, PEG500, PEG1000, PEG2000, or PEG3000 chain, preferably, a PEG2000 chain. [0141] The coupling of the functionalized carbon nanotube of Formula III with the active agent or antigen is not particularly limited, which can include those known methods for forming a covalent bond, such as those forming an amide bond, an ester bond, an ether bond, or a thioether bond. In some preferred embodiments, the covalent bond is an amide bond, in other words, the active agent or antigen are coupled with G to form an amide bond.

[0142] The efficiency of coupling of formula III with the active agent or antigen can vary. In some embodiments, greater than 30% (such as greater than 50%, greater than 60%, up to all) of the G group in Formula III is coupled with the active agent or antigen to form the carbon nanotube conjugate of Formula IV. The amount of active agent or antigen conjugated to the carbon nanotube can be measured by any suitable methods, including those known in the art, such as by UV-Vis spectroscopy or by Micro BCA assay kit available from Thermo Scientific. Thus, in some embodiments, some of the G group in formula III does not form a bond with the active agent or antigen, and in such embodiments, some of the A group in formula IV would be a G group or a derivative thereof, such as a protected G group, for example, when G is COOH, some of the A group in formula IV can be COOH or an ester of COOH. For example, in some specific embodiments, the carbon nanotube conjugate of Formula IV can be represented by a formula according to , wherein m represents the average number of oxyethylene groups of a polyethylene glycol chain, m typically ranges from 1-200, such as 10-100, 20-60, 30-50, 42-48, such as about 44 or 45, A’-NFh represents the active agent or antigen, nl+n2=n, and CNT and n are defined herein. In some embodiments, (CH2CH ₂ O) _m represents a PEG100. PEG500, PEG1000, PEG2000, or PEG3000 chain, preferably, a PEG2000 chain.

[0143] In some embodiments, the active agent or antigen is a peptide or protein, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20. or VI V2 region of gpl20. In some embodiments, the molar ratio of the active agent or antigen to the carbon nanotube can range from about 4000: 1 to 1:20, such as about 2000: 1, about 1000: 1, about 750: 1, about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 100: 1, about 50:1, about 20: 1, about 10:1, about 1:1, etc. or any range or value between the recited values, such as about 20: 1 to about 300: 1, about 100: 1 to about 1000: 1, about 200: 1 to about 500: 1, about 300: 1 to about 750: 1, etc. In some embodiments, the molar ratio of the antigen to the carbon nanotube (exclusive of any functionalization), ranges from about 10:1 to about 1000:1, such as about 20: 1 to about 300: 1, about 27: 1 to about 470: 1. etc.

[0144] In some embodiments, the active agent or antigen is a HIV antigen. For example, in some embodiments, the active agent or antigen is an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gp!20, or VI V2 region of gpl20, or a nucleic acid encoding the same, such as a DNA or RNA molecule, e.g., a mRNA.

[0145] In some embodiments, the formulating step in the methods herein can comprise a step of absorbing a surfactant on the surface of the carbon nanotube, such as on the CNT of the carbon nanotube conjugate of Formula IV. Suitable surfactants are not limited and include any of those described herein. For example, in some embodiments, the surfactant comprises a polyethylene glycol chain, such as a polyethylene glycol chain of PEG100, PEG500, PEG1000, PEG2000, PEG3000, etc., which is optionally capped, e.g., with an alkoxy group, such as a Ci-io alkoxy group, preferably, a methoxy capping group. In some embodiments, the surfactant is a PEGlated lipid described herein. In some embodiments, the surfactant can be a phospholipid, such as a glycerol-based phospholipid containing the polyethylene glycol chain. In some embodiments, the surfactant can be a glycerol-based phospholipid, in which at least one of the glycerol hydroxy groups forms a fatty ether or fatty ester, and one of the glycerol hydroxy groups is linked with a polyethylene glycol chain through a phosphate containing group. For example, in one preferred embodiment, the surfactant is 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol) -2000]. The amount of the surfactant is also not particularly limited. In some embodiments, the surfactant can be present in an amount of about 0.01% to about 0.5% by weight of the carbon nanotube (prior to any functionalization with the one or more surface molecules), for example, about 0.025% to about 0.1% by weight of the carbon nanotube.

[0146] The present disclosure also contemplates all of the pharmaceutical compositions, intermediates, and conjugates that are produced by any of the methods described herein. For example, any of the vaccine compositions produced by any of the methods described herein are also novel compositions of the present disclosure. These compositions can be formulated for any route of administration, for example, for intramuscular or subcutaneous injection. In some embodiments, the compositions can be formulated for intranasal administration. In some embodiments, the composition can be formulated for intraperitoneal or intradermal administration. The forms of the compositions are also not limited, which can be any of those described herein, such as in the form of a lyophilized powder, a solution, a gel, or a suspension.

Exemplary Application and Use of the Technology

[0147] The development of a successful vaccine against the human immunodeficiency virus (HIV-1) is a major global challenge. Although many strategies have been investigated for development of HIV-1 vaccines, a tolerable and effective candidate is still unavailable. Luna Labs has therefore implemented its carbon nanotube-based antigen/mRNA delivery platform to address this critical global health challenge. Though work and preliminary data shown below is directly relevant to the HIV-1 vaccination effort, the general processing and surface chemistry of our vehicle could we widely adaptable to any situation where delivery of proteins, peptides, or nucleic acids is needed. In the HIV-1 vaccine effort, the vehicle was designed to possess HIV-1 particle-like morphology, with similar dimensions (length, diameter) and a surface protein coating density to mimic the HIV-1 virion. This protein coating consists of a heterotrimeric envelope glycoprotein that is the target of neutralizing antibodies (NAbs). The first and second variable region (V1V2) of the envelope glycoprotein as the delivery protein model is recognized as a strategic target for vaccine development. We shorten, sort, collect, and select multiwalled carbon nanotubes to have similar size to the HIV- 1 virion and conjugate an HIV-1 VI V2 scaffold immunogen to their surface to serve as the mucosal vaccine formulation. To maintain good biocompatibility while achieving a high antigen loading and excellent membrane permeability, short CNTs in the range of 100-200 nm arc preferred based on our preliminary HIV vaccine formulation results and analysis. Our short CNTs has a very high surface area (800-1000 m ² /g), which allow it to effectively interact with biomolecules, enabling the binding of 1-100’s of proteins per CNT, leading to significantly improved binding capacity and delivery efficiency. Our carbon nanotube delivery platform is experimentally demonstrated to be recognized and efficiently uptaken by antigen presenting cells (APCs) for the induction of a greater immune responses IgG and IgA than that obtained from antigen HIV-1 envelope glycoprotein using commercial adjuvant such as IFA in animal model.

[0148] CNT can also be used for nucleic acid delivery. In HIV vaccine research, the specifically designed and functional CNT-mRNA promoted rapid uptake within 5 minutes of exposure to antigen presenting cells (APCs), such as dendritic cells (DCs). We demonstrated that DCs took up 10 times higher amounts of CNT-mRNA complexes without compromising cellular integrity compared to mRNA alone. We also determined that the CNT-mRNA complex activated DCs to express high levels of CD83, the cell surface marker for mature human DCs. DCs showed much greater responses to the CNT-mRNA compared to mRNA alone, primarily due to the CNT shape and functionality. The CNT-mRNA was able to fully activate the DC cells to become mature, as demonstrated by CD83 expression and morphology, which indicated the DCs matured and were able to promote MHC class II (major histocompatibility complex) and CD86 expression, critical to T cell activation and immune response. Due to the higher efficiency of CNT-mRNA uptake into APCs for transfection, it will take significantly less time to complete the dosing, thus shortening the time to develop an effective antibody response and T cell responses.

[0149] The CNT platform herein was initially developed as an intranasal and intramuscular/subcutaneous administrative formulation. It demonstrated successful induction of mucosal responses in mice and rabbits, with generation of both IgG and IgA responses. These findings demonstrate the clinical potential of the CNT delivery platform for delivering a broad range of antigens. Due to the design of this delivery platform, it can be rapidly adopted for additional therapeutic or vaccine delivery of nucleic acids and proteins. Further Exemplary Uses

[0150] In some embodiments, the disclosure provides a method of inducing an immune response against one or more antigcn(s) in a subject that comprises administering an immunogenic amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) comprising the one or more antigens and/or nucleic acid(s) encoding the one or more antigens. In further embodiments, the one or more antigen(s) is a protein (e.g., a glycoprotein) or peptide. In some embodiments, the one or more antigens is derived from or corresponds to an antigen from an infectious agent or a cancer. In further embodiments, the one or more antigen(s) is a polypeptide(s) and/or a fragment(s) thereof, and/or a nucleic acid(s) and/or fragment(s) thereof that is derived from or corresponds to a protein or peptide of an infectious agent such as a virus, bacteria, fungus, protozoan, and/or a parasite. In some embodiments, the one or more antigen(s) is a polypeptide(s) and/or a fragment(s) thereof, and/or a nucleic acid(s) and/or fragment(s) thereof that is derived from or corresponds to a protein or peptide expressed by a cancer. In some embodiments, the subject is a human.

[0151] As used herein "subject" or "individual" or "animal" or "patient" or "mammal," refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; fclids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. In other embodiments, a subject is a human patient. In a particular embodiment, a subject is a human patient in need of treatment.

[0152] Any techniques known to one of ordinary skill in the ail may be used to determine if an immune response is elicited following administration of a vaccine provided herein. Successful immunization may further be determined in a number of additional ways known to the skilled person including, but not limited to, hemagglutination inhibition (HAI) and serum neutralization inhibition assays to detect functional antibodies; challenge studies, in which vaccinated subjects arc challenged with the associated pathogen to determine the efficacy of the vaccination; and the use of fluorescence activated cell sorting (FACS) to determine the population of cells that express a specific cell surface marker, e.g. in the identification of activated or memory lymphocytes. Also, vaccine efficacy in stimulating a humoral immune response can be assessed by ELISA detection of antigen- specific antibody levels in the serum of immunized subjects. A skilled person may also determine if immunization with a composition of the invention elicited a humoral (or antibody mediated) response using other known methods. See, for example, Current Protocols in Immunology Coligan et al., ed. (Wiley Interscience, 2007). Techniques known in the art can likewise routinely be applied to determine if an immune response to an antigen vaccine provided herein is of comparable magnitude to for example, another vaccine or in the case of a multiple vaccine antigen each antigen as a single antigen vaccine or another vaccine. For example, enzyme-linked immune absorbent spot (ELISPOT) (e.g., for secretion of IFNy) may determine the magnitude of the immune response. In some cases, the ELISPOT may detect rodent, non-human primate or human peptides.

[0153] In some embodiments, the disclosure provides a method of inducing an immune response to an infectious agent in a subject that comprises administering an immunogenic amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) that comprises one or more antigens that is derived from or corresponds to an antigen(s) from an infectious agent. In some embodiments, the infectious agent is a microorganism. In some embodiments, the infectious agent is a virus, bacteria, fungus, protozoan, and/or a parasite.

[0154] In some embodiments, the disclosure provides a method of inducing an immune response to a cancer in a subject that comprises administering an immunogenic amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) that comprises one or more antigens that is derived from or corresponds to an antigen(s) expressed by a cancer. In some embodiments, one or more of the antigens is expressed by a cancer in the subject.

[0155] In some embodiments, the provided pharmaceutical composition, vaccine composition, or carbon nanotube conjugate comprises and/or is administered in combination with a composition that is an adjuvant. As used herein, "adjuvant" means an agent that does not constitute a specific antigen, but modifies (Thl/Th2), boosts the strength and longevity of an immune response, and/or broadens the immune response to a concomitantly administered antigen.

[0156] In additional embodiments, the disclosure provides a method of vaccinating a subject against one or more antigens that comprises administering to the subject an effective amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) that comprise the one or more antigens. In some embodiments, the disclosure provides a method of vaccinating a subject against an infectious agent. In some embodiments, the disclosure provides a method of vaccinating a subject against a cancer.

[0157] In some embodiments, the disclosure provides a method of vaccinating a subject against an infectious agent that comprises administering to the subject an effective amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) that contains one or more different antigens derived from or corresponding to an infectious agent.

[0158] In some embodiments, the disclosure provides a method of vaccinating a subject against a viral infectious agent that comprises administering to the subject an effective amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) that contains one or more different antigens derived from or corresponding to the viral infectious agent.

[0159] Viruses, or parts thereof, useful as antigens and for which a corresponding vaccination can be accomplished according to the claimed methods include, without limitation, poxvirus, monkeypoxvirus, cowpoxvirus, vaccinia virus, pseudocowpox virus, human herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), cytomegalovirus, human adenovirus A-F, polyomavirus, human papillomavirus (HPV), parvovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, human immunodeficiency virus (HIV), orthoreovirus, rotavirus, ebola virus, parainfluenza virus, influenza virus (e.g. H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, rubella virus, pneumovirus, severe acute respiratory syndrome virus, human respiratory syncytial virus, rabies virus, California encephalitis virus, Japanese encephalitis virus, arboviral encephalitis virus, JC virus, echovirus, coxsackie virus, HTLV virus, molluscum virus, poliovirus, rabies virus, Hantaan virus, lymphocytic choriomeningitis virus, coronavirus, such as SARS-COV-2, enterovirus, rhinovirus, poliovirus, norovirus, flaviviruses, dengue virus, West Nile virus, yellow fever virus and varicella. In some specific embodiments, the virus is HIV. In some specific embodiments, the antigen is an HIV antigen, such as an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V 1V2 region of gpl20. In some specific embodiments, the antigen is an HIV antigen, such as a nucleic acid (e.g., mRNA) encoding an envelope glycoprotein antigen or partial or region of glycoprotein antigen, e.g., gpl20, or V1V2 region of gpl20.

[0160] In some embodiments, the disclosure provides a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate that has use as a cancer vaccine. A "cancer vaccine" is an immunogenic composition intended to elicit an immune response against one or more particular antigens in the subject to which the cancer vaccine is administered. A cancer vaccine typically contains a tumor antigen which is able to induce or stimulate an immune response against the tumor antigen. A "tumor antigen" is an antigen that is present on the surface of a target tumor. A tumor antigen may be a molecule which is not expressed by a non-tumor cell or may be, for example, a neoantigen or an altered version of a molecule expressed by a non-tumor cell (e.g., a protein that is misfolded, truncated, or otherwise mutated).

[0161] In some embodiments, the present disclosure provides a method of vaccinating a subject against a cancer that comprises administering to the subject an effective amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) that contains one or more different tumor antigen(s) derived from or corresponding to an antigen expressed by a cancer.

[0162] The terms "cancer," and "tumor" are used herein to refer to cells which exhibit autonomous, unregulated growth, such that the cells exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, and/or treatment in the context of the invention include cancer cells (e.g., cancer cells from an individual with cancer), malignant cancer cells, pre-metastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancers of virtually every tissue are known. Many types of cancers are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, and circulating cancers such as leukemias. Cancer includes any form of cancer, including but not limited to, solid tumor cancers (e.g., lung, prostate, breast, gastric, bladder, colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas, melanomas, and neuroendocrine) and liquid cancers (e.g., hematological cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including minimal residual disease, and including both primary and metastatic tumors.

[0163] In additional embodiments, the disclosure provides a method of treating or preventing a disease in a subject that comprises administering an effective amount of pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section) to a subject in need thereof. In some embodiments, the disease treated or prevented by the provided method is an infectious disease. In some embodiments, the disease treated or prevented by the provided method is cancer. In some embodiments, the disease treated or prevented by the provided method is a disorder of the immune system. In some embodiments, the subject is a human.

[0164] In some embodiments, the disclosure provides a method of treating or preventing an infection with a microorganism (e.g., virus), wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1-60 and 91-94 in the Brief Summary Section). Without limitation, the microorganism may include for example a virus, bacteria, fungus, protozoan, parasite or helminth.

[0165] Examples of viruses causing infections that are treatable by methods of the present disclosure include without limitation, poxvirus, monkeypoxvirus, cowpoxvirus, vaccinia virus, pseudocowpox virus, human herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), cytomegalovirus, human adenovirus A-F, polyomavirus, human papillomavirus (HPV), parvovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, human immunodeficiency virus (HIV), orthoreovirus, rotavirus, ebola virus, parainfluenza virus, influenza virus (e.g. H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, rubella virus, pneumovirus, severe acute respiratory syndrome virus, human respiratory syncytial virus, rabies virus, California encephalitis virus, Japanese encephalitis virus, arboviral encephalitis virus, JC virus, echovirus, coxsackie virus, HTLV virus, molluscum virus, poliovirus, rabies virus, Hantaan virus, lymphocytic choriomeningitis virus, coronavirus, such as SARS-COV-2, enterovirus, rhinovirus, poliovirus, norovirus, flaviviruses, dengue virus, West Nile virus, yellow fever virus and varicella. In some specific embodiments, the virus is an HIV virus.

[0166] In additional embodiments, the disclosure provides a method of treating or preventing cancer in a subject that comprises administering to the subject an effective amount of a pharmaceutical composition, vaccine composition, or carbon nanotube conjugate provided herein (e.g., any of those described in Embodiments 1 -60 and 91 -94 in the Brief Summary Section) that contains one or more different tumor antigcn(s) derived from or corresponding to an antigen expressed by a cancer.

[0167] In embodiments, the disclosure provides a method of stabilizing an active agent by mixing the active agent with the provided surface functionalized carbon nanotube herein (e.g., any of Embodiments 1-32 described in the Brief Summary Section). The mixing is not particularly limited, which can include forming a covalent link between the active agent and the carbon nanotube and/or immobilizing the active agent with the carbon nanotube through non-covalent interactions as described herein. For example, in some embodiments, the disclosure provides a method of stabilizing a nucleic acid composition, the method comprising mixing the nucleic acid (e.g., any of those described herein, such as a RNA, for example, a mRNA, siRNA, antisense RNA, etc.) with the provided surface functionalized carbon nanotubc herein (e.g., any of Embodiments 1-32 described in the Brief Summary Section). In some embodiments, the nucleic acid that is mixed (such as immobilized or bound to) with the provided surface functionalized carbon nanotube herein is more stable (chemically and/or physically) compared to the nucleic acid in a control formulation without the provided surface functionalized carbon nanotube. Determination of stability of nucleic acids is well known in the art.

[0168] In some embodiments, the disclosure also provides a method of stabilizing a small molecule, the method comprising mixing the small molecule with the provided surface functionalized carbon nanotube herein (e.g., any of Embodiments 1-32 described in the Brief Summary Section). In some embodiments, the small molecule that is mixed (such as immobilized or bound to) with the provided surface functionalized carbon nanotube herein is more stable (chemically and/or physically) compared to the small molecule in a control formulation without the provided surface functionalized carbon nanotube. Determination of stability of small molecules is well known in the art.

[0169] In some embodiments, the disclosure also provides a method of stabilizing a biologies such as a protein, peptide, enzyme, etc., the method comprising mixing the biologies (e.g., any of those described herein) with the provided surface functionalized carbon nanotube herein (e.g., any of Embodiments 1-32 described in the Brief Summary Section). In some embodiments, the biologies that is mixed (such as immobilized or bound to) with the provided surface functionalized carbon nanotubc herein is more stable (chemically and/or physically) compared to the small molecule in a control formulation without the provided surface functionalized carbon nanotube. Determination of stability of biologies is well known in the art.

[0170] In some embodiments, the disclosure also provides a method of delivering an active agent (e.g., any of those described herein) into a cell, the method comprising (1) formulating the active agent (e.g., any of those described herein) with the provided surface functionalized carbon nanotube herein (e.g., any of Embodiments 1-32 described in the Brief Summary Section) to provide the active agent loaded carbon nanotube; and (2) contacting the active agent loaded carbon nanotube with the cell. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the cell can be an antigen presenting cell, such as a dendritic cell. Formulating the active agent with the surface functionalized carbon nanotube can include any of the methods described herein. For example, in some embodiments, the active agent can be covalently conjugated to the carbon nanotube. In some embodiments, the active agent can be bound to the carbon nanotube through noncovalent interactions. In some embodiments, the active agent can be an antigen described herein, such as a protein, peptide, or nucleic acid, e.g., a DNA or RNA molecule. The efficiency of delivering the active agent into the cell can be readily determined by any suitable methods, such as those exemplified herein.

[0171] In embodiments, the administering for the methods provided herein is not particular limited. For example, in some embodiments, the administering is an injection, such as an intramuscular injection or subcutaneous injection. In some embodiments, the administering for the methods provided herein is an intranasal administration. In some embodiments, the administering for the methods provided herein is an intraperitoneal or intradermal administration.

Definitions

[0172] As used herein, the singular form “a”, “an”, and “the”, includes plural references unless it is expressly stated or is unambiguously clear from the context that such is not intended.

[0173] As used herein, the term “about” modifying an amount related to the invention refers to variation in the numerical quantity that can occur, for example, through routine testing and handling; through inadvertent error in such testing and handling; through differences in the manufacture, source, or purity of ingredients employed in the invention; and the like. As used herein, “about” a specific value also includes the specific value, for example, about 10% includes 10%. As used herein, when "about" is used to modify a range, both the lower limit and higher limit should be understood as preceding with the term "about", and the lower limit and higher limit should have the same unit unless otherwise specified. For example, about 1-5 mM should be understood as about 1 mM to about 5 mM. Whether or not modified by the term “about”, the claims include equivalents of the recited quantities. In one embodiment, the term “about” means within 20% of the reported numerical value.

[0174] The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0175] Where, features or embodiments of the disclosure are described in terms of a Markush group, it is intended that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0176] The use of "including," "comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0177] As used herein, the terms "treatment", "treat" and "treating," refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, "treating" cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

[0178] As used herein, the terms "prevent", "preventing" and "prevention" refer to prophylactic and preventative measures, wherein the object is to reduce the chances that a subject will develop the pathologic condition or disorder over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the pathologic condition or disorder in the subject The term "prophylactic" refers to a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. [0179] As used herein, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

[0180] Headings and subheadings are used for convenience and/or formal compliance only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology. Features described under one heading or one subheading of the subject disclosure may be combined, in various embodiments, with features described under other headings or subheadings. Further it is not necessarily the case that all features under a single heading or a single subheading are used together in embodiments.

EXAMPLES

Example 1. Preparation of carbon nanotubes

[0181] In this example, precisely engineered carbon nanotubes were prepared to mimic the size/shape of HIV.

[0182] The HIV-1 virion maintains a roughly spherical shape with a diameter of approximately 120 nm. It is decorated with several envelope spikes, which consist of a cap made of three gpl20 molecules, and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope. [9] The antigen carrier CNTs were properly synthesized and chemically modified for the best mimicking of the size of HIV virion. CNTs are typically synthesized (for most of the players in this field) with polydisperse micrometer lengths with poor quality controls. To acquire short individual nanotubes with diameters in the range of the HIV average size, CNTs were properly cut and sorted. Three different types of starting CNTs were used: single-walled carbon nanotubes (SWCNTs) and two types of multi-walled carbon nanotubes (MWCNTs). The SWCNTs have average diameter of 1-2 nm and the length of 5- 30 pm. Two types of MWCNTs: one has an average diameter of 30-50 nm with the length of 10-20 pm; and the other one has an average diameter of 60-100 nm with length of 1-2 pm. [0183] The carbon nanotube delivery platform can be specifically designed to have variable lengths, different diameters and tunable density and orientation of antigen loaded on the surface, as shown in the schematic FIG. 2. As a result, we modified these three types of CNTs to have obtained 12 tunable sizes of candidates for antigen carrier and then further narrowed down to select 2 CNTs as the representative CNTs to demonstrate HIV glycoprotein loading. The highly stable and monodispersed CNT solutions were shown in FIG. 3. HIV particles have average sizes around 120 nm and also it was reported that the CNTs shorter than 500 nm in lengths are more ideal for in vivo applications including adequate tissue distribution and eventual elimination from the body [10], so the SCNT with the length range from 100-200 nm (Figure 3-1) was selected as one of the HIV antigen loading carrier. This was followed by conjugation of specific HIV-1 antigens to the surface, as described in the next section. A similar process as this can be performed to mimic other vaccine sizes or optimize delivery of other antigens.

[0184] CNTs are typically polydisperse with lengths measured in micrometers. To acquire short individual nanotubes with lengths in the range of the HIV-1 virion, we precisely cut, ^[13] sorted, and purified commercially available multiwall carbon nanotubes (MWCNTs) (SES research, 900-1280). The MWCNTs were obtained with original diameter of 60-100 nm and starting lengths of 1-2 pm prior to processing and surface modifications. First, we used the oxidation shortening reaction which is necessary to obtain different lengths of CNTs. We used concentrated H2SO4/HNO3 mixtures (3: 1 volume ratio) to functionalize/oxidize and cut the long CNTs using bath sonication. This resulted in the generation of short, open-ended, and non-cntanglcd carbon nanotubes as shown in the Figure 1. During the reaction, carboxylic groups were also generated on the CNT surface. We used a titration method which was reported previously the quantify the COOH functionalities [11], After functionalization, obtain 4-12% of carboxyl group on CNT surfaces. Purification and separation steps followed as shown in FIG. 4, and the final product purity and length distribution were determined using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), Dynamic Light Scattering (DLS), and Scanning Electron Microscopy (SEM). During purification of the carbon nanotubes after the acid treatment, we first removed small impurities known as carboxylate carbonaceous fragments that were produced during the oxidation reaction and subsequently physiosorbed to the CNT scaffolds. The treated CNTs were processed through several filtration-purification steps, and we then obtained CNTs of various size ranges. The relatively long CNTs were first removed using an 0.8 pm pore size filter membrane and then the shorter CNTs were collected in the filtrate using 0.4 pm or 0.1 pm size filters (Polycarbonate membrane, 90mm; Sterlitech Corporation). After three sequential filtration and purification runs, we obtained purified CNTs with a narrow size distribution that was confirmed using SEM and DLS. We can disperse sorted CNT in DI water (Figure ID) for these CNT. The final short carbon nanotubes have size range at 112.8±69.7nm. (See FIGs. 4B and 4C for DLS analysis and Zeta potential analysis of the short carbon nanotubes.) For comparison, longer CNT obtained prior to the last 0.1 um filter filtration, were 215.2+92.3 nm. The two ranges of CNTs were then be used for V1V2 immunogen conjugation. This size range of CNT products were subsequently selected as the delivery platform, as their sizes are most close to the HIV-1 virion.

[0185] To obtain high-quality short carbon nanotubes for preclinical studies, we evaluated the purity of SCNT using ICP-OES to monitor trace metals. Results indicated a very low level (ppm) of metal contaminants were found in the purified CNT sample, including cobalt (0.009 ppm), copper (0.02 ppm) and nickel (1.1 ppm); all of which could originate from the catalysts used to synthesize the CNTs. The nickel specifically (1.1 ppm) could precipitate inside the carbon nanotube channels, making it difficult to remove during the purification process. We also determined that sodium (3.4 ppm) was present at higher amounts than the other metals. Since the sodium found at 3.4 ppm primarily originated from the purification reagents, we hypothesized that it could be removed after subsequent washes and purification steps. Results indicated that when CNTs were purified twice to remove the excess sodium and other metals. The sodium content was reduced by almost half (3.4 to 1.8 ppm), but the nickel content remained constant after second purification, which indicates that nickel is likely encapsulated inside the CNTs originating from catalytic growth. However, nickel is widely found in food and water and is safe in accordance with FDA 184.1(b)(1). From these results, the produced CNTs were determined to be sufficiently pure for in vitro and in vivo use. Example 2. Conjugation of HIV antigens to carbon nanotubes

[0186] In this example, HIV antigens were conjugated to the surface of biocompatible PEGylated CNTs with proper density.

[0187] The density of surface carboxylic groups on CNTs is critical factor for controlling the loading and spatial orientation of the immobilized antigens or mRNA. In this example, CNTs with 100-200 nm in length (SCNT) and CNTs with 200-400 nm (LCNT) were selected as the synthetic vector to deliver HIV antigens. The surface functionality and the density of the functionality are very important to control the loading and the spatial orientation of the envelope glycoprotein (Env) antigen, thus modulating antibody-mediated immune response. Evidence also supports the idea that surface epitope density is a driving factor for controlling antibody function, thus modulating antibody-mediated immune response [12], As a result, different ratios, and chemistry of functionality on CNT were prepared to find out the optimized condition for antigen loading.

[0188] During the immunogen conjugation process, PEG2k (polyethylene glycol, NH2- PEG k-COOH) linkers were first introduced to the CNT surface by conjugation through EDC reaction. This step is crucial to enhance CNT solubility, stability, and biocompatibility as well as improve tissue distribution and elimination, as has been previously demonstrated [13]. Protein conjugation was then performed by attaching the immunogen to the distal end of PEG2k linkers. The UV-Vis spectra and thermal gravimetric analysis (TGA) were used for the CNT protein conjugates to confirm the conjugation of protein on the surface of CNT.

[0189] Two candidate HIV-1 antigens gpl20 A244A11 and gpl20 B6240A11 were used here as an example. Different surface density (loading) of these two antigens conjugated on LCNT and SCNT were successfully prepared from molar ratio of 27: 1 to 470: 1 as shown in the Table 2. The protein content and ratios to CNT were determined by BCA protein assay. Table 2. Representative CNT-antigen conjugates and their parameters

Example 3. Uptake of antigen modified carbon nanotubes to Antigen Presenting Cells

[0190] This example shows that modified CNT enhanced the uptake of the antigen to APCs by 10-fold. This example also demonstrated enhanced stimulation of the immune cell responses by CNT-antigen conjugates in vitro.

[0191] Mature human dendritic cells (mDCs) are the most powerful Antigen Presenting Cells (APCs) known today, having the unique ability to induce primary immune responses. To evaluate the uptake of CNT-antigen and compare the uptake efficiency between CNT-antigen and antigen by itself, human dendritic cells were chosen as the APCs. To confirm that the CNT as a vector to effectively bring antigen into the APC cells, the red iFluor dye labelled antigens with and without conjugating to CNTs were visualized under the fluorescence microscope CNT as the antigen vector improved the efficiency of antigen uptake into the APCs Figure 5. The fluorescence intensity was more than 10 times greater compared with the antigen alone. And the DCs could internalize large amount of CNTs without compromising cellular integrity. The internalization of CNT-antigen conjugates into the DCs was observed as dose and time dependent. The CNT-antigen was observed taken up into cells within 5 minutes of incubation as seen in Figure 6. The uptake pattern is punctuated, presenting numerous vesicles (blue arrows) within the first 5-15 minutes. With longer time and higher dose of CNT exposure, the CNT began to form aggregates insides cells, suggesting increasingly more CNT-antigen conjugates were internalized. The rapid and vesicular uptake is consistent with the micropinocytosis, known to be an important mechanism of macromolecular antigen uptake in dendritic cells. [0192] Maturation is the critical step for DCs to become potent APCs and to be able to activate naive T cells. The bright view images (Figure 6) also demonstrate the morphology changes of the DCs after exposure to the antigen, CNT-antigcn after 15 min and 5 days. For 15 min, we found DC cells already started interacting with foreign CNT-antigen and stimulated the maturation process. The morphology of DC after 5-day exposure to the antigen alone has fewer changes compared with CNT-antigen ones. The cells exposed to short CNT delivered antigen demonstrated full maturation which was similar to the positive control. It indicated that APCs have stimulated by SCNT.

Example 4. Surface functionalization of carbon nanotubes with cationic functionalities

[0193] This example shows that preparation of carbon nanotubes by immobilizing cationic functionalities on the surface for high efficiency transfection.

[0194] Depending on the loading entity on the CNT surface, the surface charge, linker length, and density were considered during functionalization. Luna Labs focused on functionalization of CNT surface such as immobilizing cationic functionalities and optimizing the surface charges on the CNT surface to obtain stable CNT-mRNA formulation and enhancement of cellular transfection. Different cationic functionalities including polyethyeneimine (PEI, branched), multivalent cationic lipid (MVL), and polyamidoamine dendrimer (PAMAM) that was tested to be localized on the CNT surfaces. Different ratios of CNTs to these functionalities and mRNA were tested in order to achieve the optimal interaction between RNA and delivery vectors as shown in Table 3 and summary Table 1. Our data shows that optimized combinations of a multivalent lipid (named “Lipid” in Figure 7) and PEI on CNT surface can improve transfection efficiency as shown in Figure 7. Table 3. CNT Formulations for in-vitro transfection test using APC THP-1 cells

*Transfection data: “means negative, “+” means positive.

[0195] Among the tested formulations, two (Formulation 1: SCNT-PEI+MVL and Formulation 2: SCNT-MVL both at 1: 1 weight ratio) were found to result in high efficiency in vitro transfection as shown in Figure 7. The surface charge of CNT was significantly increased to a positive charge after polymer coating for both CNT samples. They both have relatively higher surface charges >40mV after centrifugation purification to remove unbounded polymer from the solution. At this high value of surface charge, the suspension was shown very stable and able to be stored at refrigerated conditions (0-4 °C) for long term (> 3 months). The zeta potential was consistent during storage, and we didn’t visualize any precipitation during the 3-month storage. For the finalized complex formulation of CNT- mRNA, a ratio of 10:1 demonstrated the best transfection (as shown in the Table 3) in vitro using reporter gene (GFP) which present as “+++”.

Example 5. Polyethyleneimine conjugated carbon nanotubes

[0196] This example shows that preparation of carbon nanotubes by immobilizing cationic functionalities on the surface for high efficiency transfection.

[0197] Polyethyleneimine (PEI) as the one of the functionalization candidates has shown improved transfection of the vehicle according to above results. To further refine the formulation, four MW (25k, 10k, 2k, and 600) branched and two MW (2.5k and 25k) of linear PEI were used for the functionalization of either short CNT or long CNT which were referenced as CNT (s) and CNT (1), respectively, which is shown in Table 4. First, we needed to activate the CNT surface. 10 mg of CNT was dispersed in 10 mL of MES (BupH MES Buffered Saline, Thermo Scientific, product No. 28390) buffer (Img/mL). To this CNT solution, 1 mg of EDC (MW = 191.7) was added, resulting in a 5-fold molar excess of EDC to CNT. Subsequently, 2.75 mg of Sulfo-NHS was added into the reaction and mixed by vortexing. The reaction was kept at room temperature for 15 minutes. After reaction, the activated CNT was purified by centrifugal filtration (molecular weight cutoff 300 KDa) with DI water for three times to remove excess EDC and Sulfo-NHS. After purification, the activated CNT was dispersed in 10 mL of PBS (Thermo Scientific, product no. 28372). Second, 10 mg of PEI in solution was added into the activated CNT solution, which was well mixed by vortexing. The reaction was allowed to proceed for 2 hours at room temperature. After conjugation, the CNT-PEI conjugates were purified by centrifugal filtration (molecular weight cutoff 300 KDa) with DI water for three times to remove excess unreacted PEI. All the formulations were resuspended in the PBS buffer in order to obtain stable solution. Before PEI was coated on CNT surfaces, the CNT had a negatively charged surface since carboxylic groups were present on the CNT. Once the PEI was successfully conjugated, this caused the surface charge to increase to 33 and 50 mV for short CNT and long CNT Table 4. The zeta potential changes reflect the successful surface modification of MWCNTs, suggesting that the surface potentials of MWCNTs can be manipulated through PEI-mediated reactions.

Table 4. Characteristics of Zeta potentials for CNT-PEI conjugations as the formulations 8] Another method to formulate the positively charged CNT-PEI complexes is non- covalent physical absorption method using above PEIs. In general, 1 mL of Img/ml long or short CNT solution was mixed with 100 pl of 10 mg/ml PEI solutions by strong sonication. The reaction was sonicated using bath sonication for 1 hour. After reaction, zeta potentials were measured before and after centrifugation for each sample (data not shown, but all samples before centrifugation had a range of 10 mV-17mV lower than final products). After centrifuged for 30 minutes at 14,000 rpm, supernatant was decanted, and the pellets were resuspended in 1ml DI water. Complex colloid stability is very important for effective mRNA delivery, and zeta potential is one of the indicators of colloidal stability. Electrostatic repulsion between complexes tends to increase stability, and the higher the zeta potential the more stable the complexes are expected to be. All of complexes had positive zeta potentials (Table 5) in solution (>30 mV). This can be attributed to the incorporation of PEI at CNT surfaces. Zeta potentials results were shown in Table 5.

Table 5. Characteristics of Zeta potentials for CNT+PEI complexes as the formulations

[0199] Furthermore, in vitro transfection assay was used to validate the CNT-mRNA formulations. Luna has investigated different cationic lipid functionalities including multivalent cationic lipid (MVL5: Nl-[2-((lS)-l-[(3- aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzam ide), and also EPC (1- palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine chloride salt) that can be localized on the CNT surfaces but would synergistically enhance the mRNA transfection. Multiple ratios of CNTs to these functionalities and mRNA were tested to achieve the optimal interaction between RNA and delivery vectors (Table 1). Our data shows that optimized combinations of an EPC and PEI on CNT surface (Figure 8) can even improve the transfection efficiency and stabilize the formulation from precipitation. Possible conditions that can be modified on CNT surface was concluded in the Table.

Example 6. Evaluation of the antigenicity of CNT-antigen conjugate formulations

[0200] HIV-1 antigens gpI20 A244A11 and gpl20 B6240A11 was successfully conjugated on the LCNT and SCNT to generate four different candidates for the ELISA antigenicity test. Two broadly neutralizing antibodies (bNAbs) PGT128, GP9 and mAb 830A were used for the evaluation, which were selected according to the binding site differences as shown in Figure 9. These antibodies appear to interact directly with the HIV glycan coat with high binding affinity. The ELISA results indicated that CNT by itself has not shown responses to these selected antibodies, however, the CNT enhanced the antigen responses to these three antibodies. The short carbon nanotube as the antigen delivery vector has the highest responses for the antibodies PG9 and PGT 128, however, for the longer carbon nanotube, the highest responses was present on 830A antibody which were shown in the ELISA curves (Figure 10). The broadly neutralizing antibodies PG9 is targeting V1/V2 and PGT 128 is targeting V3. The 830A is the conformation dependent V2 mAbs and the epitope is at the distal end of the VI V2 domain on the opposite side from the PG9 epitope [14]. The ELISA results indicated that modified CNT can enhance the conjugated antigens’ responses to the antibodies selectively. In a certain context, the site-specific conjugation technique can control the orientation of antigens to preserve the specific epitope which is ready for the effective binding.

[0201] Binding epitope exposure by PEG-Lipid decoration on CNT-V1 V2 demonstrated the highest antigenicity.

[0202] An ELISA was used to determine the epitope specificity of the CNT-V 1 V2 formulation and to evaluate if the formulation altered the antibody recognizing epitopes for the well-characterized Env-specific mAbs. First, we selected three VI V2 specific antibodies PG9, CH58 and 830A for the test. The broadly neutralizing antibody PG9 recognizes the strand conformation of the C-strand region of V1V2 while CH58 recognizes its helical formation. The 83OA is the conformation dependent VI V2 mAb, and its epitope is located at the distal end of the VI V2 domain on the opposite side from the PG9 epitope. Thus, these three mAbs can detect the major conformations of V 1 V2 and are often utilized for characterizing the antigenicity of V1V2 immunogens. From the results of these three antibodies, PG9 and 83OA have shown the best responses to VI V2(ZM53)-2F5K (Figure 10). As the result, we chose the broadly neutralizing antibody PG9 for the antigenicity test for CNT-V1V2 formulations.

[0203] First, we evaluated the V1V2 immunogen immobilized on CNT surfaces alone without adding PEG2k linker and the result did not show much PG9 binding (data not shown). As a result, the linker PEG2k were added in order to keep the flexibility of VI V2 immunogen (named CNT-V1V2) to allow the binding site open which increased the PG9 antibody binding opportunities as shown in Figure 11 compared with VI V2 alone. Furthermore, in order to enhance the final formulation bioavailability, permeability and retention, we selected 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethyleneglycol)-2000] (PEG-Lipid) as the CNT surface functionality (Figure 12). It was absorbed on the surface of CNTs by 7t-7t interactions at any free location that was not already conjugated by the V1V2 immunogen. After localizing these lipids, we found it further enhanced the PG9 binding (Figure 11). At the same time, the dispersability and stability of CNTs in aqueous solution was also improved. Multiple concentration ranges (0.025-0. lwt.% of CNTs: low, medium and high) of PEG-Lipid were tested for ELISA in order to evaluate the effect of PEG-Lipid density on results of antigenicity. The PEG-Lipid concentration at 0.025% of CNT was found that was demonstrated the highest response in ELISA results. As a result, we assume that the binding site of the epitope is preferably oriented or the propensity for the strand conformation was increased by adding PEG-Lipid in between immunogen molecules on the CNT, so that it maintains epitope availability for effective binding. The PEG-Lipid performed as a potential protein “conformation protector” in this delivery system.

Example 7. In vitro mucosal penetration study

[0204] In this example, CNT formulations demonstrated mucosal penetration in vitro.

[0205] The nasal route of administration offers several advantages for vaccination, including ease of self-administration and induction of mucosal as well as systemic immunity. To develop an intranasal formulation of immunogen-loaded carbon nanotubes, we evaluated the in vitro mucosal penetration efficiency. Calu-3 cells, derived from a human bronchial adenocarcinoma, are well documented as a model for examining the response of proximal airway epithelial cells to pharmacological compounds [15]. Therefore, we established a polarized, liquid-covered culture of Calu-3 cells for a mucosal permeability test. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by monitoring the passive equilibration of dye (known as a flux assay) between the apical and basolateral compartments. Once TEER measurements plateaued at or above 800 Qxcm2 (approximately 8 days), the Calu-3 liquid covered culture was ready for use to examine the penetration efficiency. All the samples were prepared in culture media (EMEM, 10% FBS). After replacing the basal chamber of each well with ImL of fresh media, 500 pL of each prepared sample solution along with media controls were added to the apical chamber of each insert in triplicate. The results indicated a drop of the TEER values, demonstrating that the protein can pass through the tight junctions. As shown in Figure 13A, the CNT-VIV2+PEG- Lipid sample demonstrated a continuously decreasing TEER, and the end point TEER value was around 300 Q after 24 hours. TEER serves as a surrogate marker for epithelial permeability, a widely accepted quantitative technique to measure and represent the integrity of intracellular junction dynamics in vitro.

[0206] A flux assay was performed in parallel and confirmed these results, as shown in Figure 13B. The addition of PEG-Lipid to the CNT-V1V2 surface, as discussed previously, was confirmed to have demonstrated to enhance permeation as compared to CNT-V1V2 alone. The permeability from apical to basal layers was demonstrated to increase up to 5.8 times for this formulation. This assay confirms that the short CNT, in combination with PEG- Lipid, showed the highest permeability through the cell membrane. The relationship between TEER and intracellular junctions is supported by the correlation between TEER and rates of the V1V2 immunogen flux.

Example 8. Intranasal delivery of CNT formulations in mouse model

[0207] This example shows that CNT-VlV2+PEG-Lipid formulation successfully penetrated the murine mucosal barrier for delivery of V 1 V2 immunogen.

[0208] We used a mouse model to investigate antibody production efficiency following intranasal delivery of the VI V2 immunogen immobilized on CNTs. Due to the limitations of administration volume when applied by intranasal instillation to the small mouse nasal cavity, the CNT-V1V2+ PEG-Lipid antigen solution-based vaccine formulation was concentrated to 3 mg/mL in PBS for administration, and each animal had a total of 50 pg of the V 1 V2 protein administrated. In addition, the same formulation was lyophilized into a powdered form (Group 1, Figure 14) to compare with the solution-based formulation (Group 2, Figure 14B) in this mucosal immunogenicity test. There were three dosing time points (2-week interval between each dose) for each group of animals (N=5) during the study. As shown in Figure 14, the lyophilized powdered form of the vaccine formulation demonstrated a higher IgG response when compared with the solution-based formulation. Further, HIV-1 Env- specific plasma IgA responses was also evaluated via ELISA. IgA responses were observed for all CNT-VlV2+PEG-Lipid formulations administrated to the mice. The lyophilized formulation (Group 1) had similar peak responses as Group 2 in the six-week serum sample, but less of a response at the fourth week. These results demonstrate that mucosal delivery of both powdered (lyophilized) and solution-based CNT-VlV2+PEG-Lipid formulations to mice successfully induced immune responses with the generation of both IgG and plasma IgA. In order to demonstrate the mucosal site protection using vaccine candidates, subsequent rabbit immunogenicity studies evaluated IgA production in urine and vaginal wash samples.

Example 9. Immunogenicity studies in rabbits

F0209] This example shows that CNT-VlV2+PEG-Lipid demonstrated acceleration of immune responses compared to the V1V2 immunogen alone.

[0210] We investigated whether conjugation of the V1V2 immunogen onto the carbon nanotube delivery vehicle enhanced their immunogenicity and elicited specific immune responses in vivo using larger animal rabbits. Three boosts (100 pg of V1V2 content per dose) were administrated by intramuscular- injection to evaluate the immunogenicity in rabbits. A VI V2 immunogen-only group utilized Incomplete Freund's Adjuvant (IFA) for the delivery (no CNT-bascd delivery vehicle for this control). Starting from the third bleed, the CNT conjugated VI V2 immunogen group demonstrated a two-week acceleration of the immune response as compared to the VI V2 immunogen alone, as shown Figure 15. In these intramuscular studies, the CNT formulation accelerated the immune response with higher IgG titers as compared to the immunogen alone.

[0211] We next investigated intranasal administration to demonstrate that when CNT was used as a delivery platform for mucosal immunization, it would induce a more efficient mucosal antibody response than the common intramuscular administration route. In this study, the CNT formulation was compared with control VI V2 antigens in a rabbit model. There were two test groups of rabbits with five animals per group. These two groups included CNT- VlV2+PEG-Eipid conjugates, as well as VI V2 antigen mixed with PEG-Lipid, both for intranasal administration. The animals received three protein boosts of the CNT-V1V2 conjugates or antigen alone. A total of 100 pg of the boosting immunogen was administered via intranasal injection. Serum samples were collected prior to immunization, two weeks after the third DNA prime, and after each protein boost respectively in order to analyze the kinetics of the antibody responses during the immunization. For the intranasal administration test, the combination of five rabbit serum samples and vaginal washes were evaluated.

[0212] According to the results shown Figure 16, it was demonstrated that CNT delivery of the antigens accelerated the generation of systemic immune response by the second dose, which was two weeks earlier (Figure 16-A2) than treatment with the VI V2 immunogen alone (in the absence of CNT immobilization). Further, the CNT group doubled the titer of mucosal antibody IgA response in vaginal washes (Figure 16-B2) as compared to V1V2 immunogen alone after intranasal administration. The data from IgM production ELISA tests showed that the CNT-VlV2+PEG-Lipid also stimulated higher immediate IgM response in animal scrum as compared to the antigen alone.

[0213] To gain additional understanding of the conformational specificities of the antibody responses induced by CNT-VlV2+PEG-Lipid in rabbits, we performed an antibody competition assay for the rabbit immune sera with PG9. This competition ELISA was used to determine epitope specificity, i.e., whether sera from vaccinated animals contained antibodies recognizing epitopes similar to the well-characterized broadly neutralizing antibody PG9. The sera from the rabbit group immunized by the intranasal route using CNT as the delivery vector demonstrated pronounced competition against PG9 for the binding of VI V2 (ZM53)-2F5K, suggesting that the formulations induced production of antibodies whose epitopes overlap and are potentially conformationally similar to that of PG9.

Example 10. Immunogenicity studies using CNT-mRNA formulation

[0214] For testing the immunogenicity of CNT-mRNA candidates, HIS-A2/DR4 mice were used for the study. In brief, HIS mice have been made by infusing human hematopoietic stem cells (HSCs) to highly immunodeficient mice, NSG or NOG mice, which are made by backcrossing NOD/SCID mice to IL-2Ry-deficient mice. They have chosen AAV serotype 9 (AAV9) for transducing a high degree of transgenes into the host cells but is also capable of infecting a wide range of different tissues in animals as seen in the following reference [16] and Figure 17. Three vaccine formulation groups were tested in the HIS studies, including CNT-mRNA (intramuscular. Group 1), CNT-mRNA (intranasal, Group 2) and codelivery formulation CNT-VlV2+mRNA (intranasal, Group 3). Two doses were administrated at a 4- week interval. Group 4 is the control naive mice. Two weeks after boosting, all sera samples were collected from immunized and naive mice, and the titers (1/20 to 1/2500 dilutions) of mouse IgG against HIV glycoprotein VI V2 were determined by ELISA. Spleens were also harvested and splenocytes were isolated, followed by IFN-y ELISpot assay to determine the relative degree of HIV glycoprotein- specific human T-cell response. Figure 17 shows the ELISA and ELISpot results, respectively. When humoral response against HIV V1/V2 protein was measured by ELISA, all three vaccine groups of HIS-A2/DR4 mice generated humoral responses that resulted in significant human IgG titers against HIV VI V2. In particular, Group 1 mice immunized by CNT-mRNA (IM) all induced more than 1/500 or 1/2500 IgG titers against HIV VI V2, consistently. In the ELISpot assay, Group 1 formulation CNT-mRNA IM mice demonstrated significant T cell response. Since these HIS-A2/DR4 mice should have HLADR4- restricted human CD4+ T cells, these T cells likely helped human B cells to produce class-switched human IgG upon receiving vaccines that express HIV VI V2. Also, in the group 3, the CNT codelivery formulation administrated intranasally, one HIS mouse (#13) of group 3 showed a very high (highest) level of human T-cell response and the highest human IgG responses against V1/V2 in ELISpot and ELISA, respectively. However, a moderate level of human IgG against V1/V2 but no significant T-cell response were found in Group 2 mice. We hypothesize that this is because PEG-Lipid used in the group 3 played an important role in inducing intranasal absorption and T-cell responses.

[0215] CNT-mRNA also demonstrated immunogenicity in rabbit model. The female New Zealand White rabbits 6-8 weeks old (~2kg) each received three boosters. A total of 5 Female New Zealand White rabbits in each group were administrated with 50 pg of CNT-mRNA. The rabbits were immunized by CNT-mRNA at weeks 0, 2, and 4 with blood collected at weeks -2 (pre-bleed), 4, and 8 weeks (the end point of the experiment). Serum samples were collected to analyze the kinetics of the antibody response. Serum was separated from whole blood and stored at -20°C until assays were performed. Body fluids (virginal washes) were collected post-mortem. These analyses were specifically developed to compare both the breadth and potency of the antibody responses induced by mRNA and encoding protein. At the end of the study, animals were euthanized by non-inhaled barbiturate injection under anesthesia. For ELISA assay, the 96 flat-bottom wells of plates were coated overnight at 4°C with a HIV-1 V1V2 (ZM53)- 2F5K immunogen at Ipg/ml. The plates were washed with PBS containing 0.05% Tween 20 (PBST), pH 7.4. Non-specific binding sites were blocked with PBS containing 3% BSA for 1 hour. The plates were then washed once with PBST and subsequently 100 pL of rabbit serum was added and incubated for 1 hour. Serum samples were diluted at the ratio of 1 : 10 for antibody detection. After incubation, the plates were washed with PBST. The bound antibodies were detected by incubating with goat anti-rabbit IgG conjugated with alkaline phosphatase diluted at a ratio of 1:2000 in PBS. Finally, the plates were washed with PBST, and the bound alkaline phosphatase enzyme activity was revealed by adding 100 pL/well of substrate solution. Absorbances at 405 nm in each well were measured using a microplate reader. From the results shown in Figure 18, we did not see significant immune response during the earlier time points. However, after 8 weeks and the third booster of CNT-mRNA vaccine candidate, we observed a very high immune responses against HIV-1 VI V2 (ZM53)-2F5K target antigen. When we compared it with administration of CNT-V1V2 protein as the control, it demonstrated even higher and stronger Figure 18. ELISA sera evaluation of IgG responses for CNT-mRNA immunized rabbits against HIV-1 VI V2 (ZM53)-2F5K antigen. As a result, CNT demonstrated it is an effective adjuvant/delivery system for HIV-1 mRNA delivery.

Example 11. Safety Studies

[0216] This example shows that CNT formulation can be safely administrated by the intranasal route and intramuscular route.

[0217] Raw carbon nanotubes have highly hydrophobic surfaces and are not soluble in aqueous solutions. For biomedical applications, surface chemistry or functionalization is required to solubilize CNTs, and to render biocompatibility and low toxicity. Demonstration of biodistribution and safe dosing levels of the specific short carbon nanotubes we are utilizing will be critical for potential human uses or trials. First step, we worked on the animal biosafety studies for SCNTs. In order to monitor intranasal administration of carbon nanotubes, we implemented a previously reported method of labelling recombinant thermostable Luciola cruciate luciferase (LcL) [19] for in vivo imaging of short carbon nanotubes in male BALB/c mice using an in vivo imaging system (IVIS Spectrum In Vivo Imaging System, PerkinElmer). After conjugating LcL to SCNTs, it remained biologically active for the catalysis of D-luciferin in the presence of ATP to release a detectable number of photons for in vivo imaging. The biolumincsccncc of LcL allowed imaging of CNTs at high resolution with no need for an excitation source.

[0218] As shown in Figure 19A, the CNTs alone demonstrated no bioluminescence, but after LcL conjugation on their surface, the distribution of CNTs was easily visualized using whole mouse imaging following administration via the intranasal route. Four hours after intranasal delivery of CNT-LcL, a significantly elevated bioluminescence signal was observed in both the whole-body region of interest ROI (excluding the snout) and the snout ROI (Figure 19B). Elevated bioluminescent signal in the snout four hours post-dose indicated the ability to detect the presence of the luciferase labeled short CNT at or near the dosing site.

[0219] In order to characterize organ-specific distribution of the short carbon nanotubes, tissues were processed and analyzed. The process of quantifying SCNTs extracted from each organ involved a custom carbon nanotube quantification assay. Briefly, the fluorescent tag (LcL) serves as an indicator that is quenched in the presence of single stranded DNA (ssDNA), which interacts strongly with carbon nanotubes. ssDNA forms stable complexes with individual SCNTs, wrapping around them by way of 71-71 stacking interactions between the nucleotide bases and the SCNT sidewalls. The fluorescence quenching efficiency FO/F, where F0 and F are the fluorescence intensities of the fluorescent tag in the absence and presence of SCNTs, is used for the evaluation and calibration of the SCNT concentration in the samples. Low concentration calibration curves were generated as shown in SL3 and were applied for measurement of trace SCNTs in the samples. Five different organs including liver, lung, spleen, brain, and kidney were evaluated using this method. Results in Figure 20A indicated that the majority of SCNTs accumulated in the lung. In the spleen, liver, kidney, or brain, there was lower accumulation two days after intranasal administration. We also determined that the accumulation of SCNTs in the lung and liver decreased over two days. Further, we observed that four hours after administration, the SCNTs were able to penetrate the brain membrane, but after two days, the amount of SCNT in brain tissue was significantly decreased and undetectable (Figure 20A). These results indicate that SCNTs could be cleared and are potentially safe for intranasal administration. [0220] Finally, we evaluated the dose tolerability of SCNTs after intranasal administration to male BALB/c mice. SCNTs at 5, 10, and 30 mg/kg (Iml/kg dose volume) were administered intranasally as a solution (PBS)-bascd formulation and the mice were then monitored out to 24 hours post-dose. Clinical observations performed 2-, 4-, and 24-hours post-dose indicated adverse reactions in animals administered 10 and 30 mg/kg, and consisted mainly of increases in respiratory rate, hunched posture, and a scruffy appearance. Orbital tightening was also observed in animals that received the highest dose (30 mg/kg). None of these adverse reactions were observed in the 5 mg/kg test groups. Animals administered the 30 mg/kg SCNT dose demonstrated a mean weight loss of 8.02 ± 3.62% (p=0.0042) over approximately 24 hours (Figure 20B). Mean weight loss was dose-dependent, however, and was not significant at the 5 or 10 mg/kg dosing levels. Three animals in the 30 mg/kg dose group were euthanized at 24 hours post-dose specifically because their weight loss over 24 hours exceeded 10%. No statistically significant changes were noted in any parameters compared to vehicle that were measured 24 hours post-dose. The 30 mg/kg dose group also had elevated blood urea nitrogen (BUN) levels 72 hours post-dose (3.65x mean increase; p=0.0347), a potential indicator of dehydration which corresponds with weight loss in the high-dose group. Additionally, Group (30 mg/kg) had at least one animal that presented with elevated levels of cholesterol, CK, ALT, AST, ALP, amylase, potassium, and osmolarity that were outside the vehicle Group. Overall, treated groups approximately 72h post-dose, but did not result in statistically significant changes from the mean. More evaluations of the adverse effect on the function of the lung and respiratory tract would be considered for further investigation. So far, as a result of these clinic observations and adverse reactions at higher dosing levels, less than 10 mg/kg dose was selected as safer for dosing and used in immunogenicity animal studies.

[0221] CNT also demonstrated safety profile by intramuscular injection in rats. There was one or three doses of CNT at a dose level of 0- 30 mg/kg administrated for rats on Day 1, 14 and 28. Clinical observations were conducted daily. Body weight was collected approximately weekly and at the time of necropsy. Group 1-4 were euthanized under isoflurane on Day 2, approximately 24 hours post dose. Group 5-8 were euthanized on Day 42. Terminal blood was collected and processed for hematology and clinical chemistry as described in below. All these blood samples containing K3EDTA as an anticoagulant were analyzed. Heart, lungs, kidneys, liver, and spleen were collected, weighed, and fixed in 10% neutral-buffered formalin. Brain was removed, weighed, and discarded. Slides of fixed tissue will be prepared, stained with standard hematoxylin & Eosin, and reviewed by a qualified veterinary pathologist. From the results shown in Figure 21, there were no drug-related clinical observations or adverse effects as compared to vehicle controls for body weights or organ weights from day 0 to day 42 days for any of the doses tested. In all, we did not observe any concerns across the dosing range of 0-30 mg/kg.

[0222] References:

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[0223] The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0224] The present invention has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0225] With respect to aspects of the invention described as a genus, all individual species are individually considered separate aspects of the invention. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of’ or "consisting essentially of’ the feature.

[0226] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the ordinary skill of the ait, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications arc intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the ordinarily skilled artisan in light of the teachings and guidance.

[0227] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments.

[0228] All of the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0229] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.