WO2020018535A1

WO2020018535A1 - Dose and time-dependent intracelluar penetration of surface-modified nanotubes for delivery of molecular materials into cells

Info

Publication number: WO2020018535A1
Application number: PCT/US2019/042004
Authority: WO
Inventors: Aaron Wallace TASSET; Milos MARINKOVIC; Kevin CASTILLO; Kurt W. Swogger
Original assignee: Molecular Rebar Design, Llc
Priority date: 2018-07-16
Filing date: 2019-07-16
Publication date: 2020-01-23

Abstract

Discrete, individualized carbon nanotubes having targeted, or selective, oxidation levels or content and a functionalized surface coating. Such carbon nanotubes can have little to no inner tube surface oxidation, or differing amounts and/or types of oxidation between the tubes' inner and outer surfaces. These new discrete carbon nanotubes are useful for intracellular and extracellular delivery and controlled release of payload molecules including drugs, chemicals, compounds, genetic materials, various types of proteins, enzymes, gene-editing systems such as CRISPR, transcription activator-like effector nucleases, zinc finger nucleases and all combinations of associated proteins and RNAs/DNAs, and/or small molecules. The functionalized surface coating may be utilized to preferentially allow the nanotubes to associate with, penetrate or be removed from a cell membrane, cell wall, and/or nuclear membrane and deliver a payload molecule. The described nanotubes are useful for molecular delivery in mammalian, bacterial, fungal and plant cells, as well as tissues and cell-derived materials such as bacterial and fungal biofilms.

Description

DOSE AND TIME-DEPENDENT INTRACELLULAR PENETRATION OF SURFACE-MODIFIED NANOTUBES FOR DELIVERY OF MOLECULAR MATERIALS INTO

CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application No. 62/698,745, filed July 16, 2018; this application is related to United States Application Nos. 62/672,453 filed on May 15, 2018, 62/319,599 filed on April 7, 2016, and 62/424,606 filed November 21, 2016, the entire disclosures of which are incorporated herein by reference. This application is also related to United States Application Nos. 14/628,248 filed Feb. 21, 2015, as well as 13/164,456, filed June 20, 2011, and their progeny; and 13/140,029, filed August 9, 2011 and its progeny, the entire disclosures of which are incorporated herein by reference.

FIELD OF INVENTION

[0002] The present application is directed to novel carbon nanotube compositions with functional coatings that permit medical or industrial applications using discrete carbon nanotubes. The present application also encompasses the preparation of such carbon nanotubes.

BACKGROUND AND SUMMARY OF THE INVENTION

[0003] The transport of molecules across the cell wall or plasma membrane may be inefficient and is typically dependent on chemical and physical properties of the molecule. Techniques which enable transport of molecules into cells are often toxic, harmful to membrane integrity and can increase the likelihood of mutagenic processes which may lead to tumors. Due to its capacity to readily enter cells without causing damage, the disclosed nanotubes can be loaded with various types of inorganic and/or organic chemistries, ionic species, molecules and macromolecular assemblies, nanoparticles, genetic materials, nucleic acids, oligonucleotides, peptides, proteins, enzymes, viruses/vectors, antibodies, small molecules, cell-derived components various sorts of gene-editing complexes, for example, but not limited to, CRISPR-based systems, transcription activator-like effector nucleases, and zinc finger nucleases, other types of biologically derived molecules and/or complexes thereof in order to facilitate more efficient intracellular transport of these molecules and improve the effectiveness of their activity within cells and reduce toxicity. Detergents, lipid-based transfection agents and viral vectors are known in the art, however the disclosed nanotubes and compositions have the benefits of lowered toxicity, a more versatile mechanism for intracellular penetration such as, but not limited to, endocytosis and subsequent delivery of molecular cargo, preferential targeting to the nucleus and other organelles, controlled delivery of molecular cargo based on surface modification of the nanotubes, cellular imaging and/or sensors, molecular sequestration and/or extraction in cells and purification and enrichment of nanoparticle-containing cellular subpopulations. The lowered toxicity most likely results at least in part from the delivery mechanism. Cell membrane integrity is also not disrupted, or less disrupted, as compared to lipid or surfactant-based delivery systems, as well as relative to membrane-piercing transfection technologies such as gene guns or electroporation. The disclosed compositions have lower safety concerns relative to using viral vectors and higher efficacy as the disclosed nanotubes have been shown to have preferential delivery to the cytoplasm and/or nucleus of cells, as well as an capability for organelle-targeting The disclosed compositions also allow the ability to control delivery as a result of nanotube surface modifications.

[0004] Carbon nanotubes can be classified by the number of walls in the tube, single wall, double wall and multiwall. Carbon nanotubes are typically manufactured as agglomerated nanotube balls, bundles or forests attached to substrates. The use of carbon nanotubes as a delivery device for drugs and small molecules is an area in which carbon nanotubes are predicted to have significant utility. However, utilization of carbon nanotubes in these applications has been hampered due to the general inability to reliably produce individualized carbon nanotubes and the ability to disperse the individualized carbon nanotubes biological environments such as blood or tissue. Bosnyak et al., in various patent applications (e.g., US 2012-0183770 Al and US 2011-0294013 Al), have made discrete carbon nanotubes through judicious and substantially simultaneous use of oxidation and shear forces, thereby oxidizing both the inner and outer surface of the nanotubes, typically to approximately the same oxidation level on the inner and outer surfaces, resulting in individual or discrete tubes.

[0005] The present inventions can utilize the tubes of those earlier Bosnyak et al. applications and disclosures, but also uses new targeted oxidation discrete tubes. The present invention describes a composition of discrete, individualized carbon nanotubes having targeted, or selective, oxidation levels and/or oxygen content on the exterior and/or interior of the tube walls. Such novel carbon nanotubes can have little to no inner tube surface oxidation, or differing amounts and/or types of oxidation between the tubes’ inner and outer surfaces. In certain embodiments, drugs, chemicals, compounds, and/or small molecules may be loaded onto the exterior and/or into the interior of the nanotubes for delivery and/or controlled release.

[0006] One embodiment of the present invention is a composition comprising a plurality of discrete carbon nanotubes, wherein the discrete carbon nanotubes comprise an interior and exterior surface, each surface comprising an interior surface oxidized species content and an exterior surface oxidized species content, wherein the interior surface oxidized species content differs from the exterior surface oxidized species content by at least 20%, and as high as about 80% about 90%, about 95%, about 99%, about 99.5% or about 100%, preferably wherein the interior surface oxidized species content is less than the exterior surface oxidized species content.

[0007] The oxidized species in this case relates to those chemical entities containing an oxygen atom that are carboxylic, hydroxyl, lactone, lactol or ketone that are derived from oxidation of the carbon nanotubes for example, but not limited to, by strong oxidizing agents such as concentrated nitric acid.

[0008] The interior surface oxidized species content can be up to 3 weight percent relative to total carbon nanotube weight as determined by, but not limited to, thermogravimetric analysis of the carbon nanotubes, preferably from about 0.01 to about 3 weight percent relative to carbon nanotube weight, more preferably from about 0.01 to about 2, most preferably from about 0.01 to about 1. Especially preferred interior surface oxidized species content is from zero to about 0.01 weight percent relative to carbon nanotube weight.

[0009] The exterior surface oxidized species content can be from about 1 to about 6 weight percent relative to carbon nanotube weight, preferably from about 1 to about 4, more preferably from about 2 to about 3 weight percent relative to carbon nanotube weight. This is determined by comparing the exterior oxidized species content for a given plurality of nanotubes against the total weight of that plurality of nanotubes.

[0010] The interior and exterior surface oxidized species content totals can be from about 1 to about 9 weight percent relative to carbon nanotube weight.

[0011] For discrete carbon nanotubes where the average number of walls are known, the total oxidation content of the interior and exterior surfaces ranges from about 1% to a maximum % given by 35% multiplied by the number of walls oxidized divided by the total number of walls of the discrete carbon nanotubes.

[0012] The discrete carbon nanotubes of any composition embodiment herein may comprise a plurality of open-ended tubes. The discrete carbon nanotubes of any composition embodiment herein are preferred wherein the inner and outer surface oxidation difference is at least about 0.2 weight percent.

[0013] The surface chemistry of the disclosed nanotubes can be modified in order to control loading of diverse types of molecules, including genetic materials, oligonucleotides, peptides, proteins, viruses/vectors, small molecules and any sort of gene-editing complex, as well as to regulate the off-loading rate. Loading does not usually alter the payload molecule and does not typically impact the biological functionality. Various types of chemical surface properties, including specific ionic/non-ionic, hydrophobic/hydrophilic functional groups, combinations of functional groups and both lipid and non-lipid based polymeric surface coatings, including, but not limited to, PEG-based molecules, can be used to control on/off rate of molecular cargo, as well as the ability of the nanotubes to penetrate the cell. Controlling the rate of off-loading can directly impact the toxicity profile of a molecule by modulating the pharmacokinetics (PK).

[0014] The disclosed nanotubes may also have the ability to associate with or penetrate the cellular nucleus, releasing its molecular cargo near or within the nucleus. This creates significant potential for use in gene therapies and genetic modification modalities such as, but not limited to, CRISPR-based gene-editing and, transcription activator-like effector nucleases (TALEN) and zinc finger nucleases. Because the composition of the disclosed nanotubes can be selected to penetrate cells, they may be administered both in vitro (cell culture) and in vivo. In animals, nanotubes may be introduced by any therapeutic administration method, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, topical, oral or rectal. For unicellular or small multicellular organisms such as bacteria, yeasts, fungi, and algae, nanotubes may be introduced in the extracellular environment for uptake or injected directly into the cells. In the case of plants, nanotubes may be provided in water, soil or in the form of administered nutrients. In mammalian, fungal, bacterial, plant cell or tissue culture, as well as in biological reactors and fermentation vessels, carbon nanotubes may be administered directly to the culture media and incubated with cells and/or tissues.

[0015] The disclosed nanotubes may also protect molecular cargos from cellular metabolism or other forms of degradation, thus increasing the duration of time molecular cargos exert a biological activity, both in the intercellular or intracellular environment. This feature may be used to stabilize rapidly metabolized molecules. This shielding effect may be modulated by altering the surface characteristics of the carbon nanotube.

[0016] The compositions described herein can be used as an ion or chemical transport. Various species or classes of compounds, drugs, chemicals, and/or small molecules which demonstrate this ion transport effect can be used, including ionic, some non-ionic compounds, hydrophobic and/or hydrophilic compounds.

[0017] The compositions described herein can be used as storage for various organic or inorganic materials and their subsequent release.

[0018] The compositions of nanotubes described herein can be used as a molecular sequestration, extraction or enrichment system for proteins, oligonucleotides or other type of molecules found inside cells and/or in the extracellular environment. The compositions comprising the novel discrete oxidized carbon nanotubes may also be used as a component in, or as, an imaging, sensor or diagnostic tool.

[0019] The compositions disclosed herein can also be used as a component in, or as, molecular and/or drug delivery or controlled release formulations.

[0020] The compositions disclosed herein can also be used as a component in, or as, a molecular delivery system to cells, including delivery all types of small molecules, drugs, medicines, surfactants, polymers, composites, macromolecules, organic and inorganic nanoparticles, proteins, peptides, enzymes, nucleic acids, oligonucleotides such as but not limited to mRNA, siRNA, microRNA, carbohydrates, lipids, gly cos aminogly cans, proteoglycans, glycoproteins, steroids, antibodies, growth factors, viruses, viral vectors, viral- derived components, genetic materials, gene-editing complexes such as, but not limited to, CRISPR-based systems, TALENs and zinc finger nucleases, and associated oligonucleotides, micelles, other small or large molecular weight chemical entities, cell-derived components, other molecules derived from biological means and/or combinations thereof.

[0021] The compositions disclosed herein can also be used as a component in, or as, a biomolecular delivery system to the extracellular environment, trans-membrane transport, transport into the internal cellular environment (cytoplasm), and transport to the cellular nucleus and/or other cellular physiological targets and organelles, such as mitochondria.

[0022] The compositions disclosed herein can also be used as a component in, or as, a biomolecular delivery system for all types of eukaryotic and prokaryotic cells, including mammalian, fungal, plant and bacterial cells and tissues.

[0023] The compositions disclosed herein can also be used as a component in, or as, a biomolecular delivery system whereas the predominance of a delivery mechanism such as described above in, for example, paragraph [0014] may be controlled by selection of surface coatings and physical and chemical properties of the materials.

[0024] The compositions disclosed herein can also be used as a component in, or as, a biomolecular delivery system targeting specific cell-types within organs and tissues, by selection of surface coatings and physical and chemical properties of the carbon nanotubes.

[0025] The present inventions may also comprise carbon nanotubes with surface coatings. A surface coating may be functionalized, may be covalently linked to the nanotube surface and/or may be non-covalently bound through hydrophobic, hydrophilic, amphiphilic and/or electrostatic interactions. The present application discloses a novel manufacturing process for surface coated carbon nanotubes.

[0026] The present application also discloses a novel use for the carbon nanotubes in nanotube-mediated controlled delivery of drugs, chemicals, compounds, small molecules, organic or inorganic macromolecular complexes, nanoparticles, antibodies, adjuvants, lipids, micelles, viruses, viral-derived components oligonucleotides, peptide, proteins, enzymes, antibodies, other types of biologies and/or complexes thereof. In some embodiments, by covalent or non-covalent attachment a drug, small molecule and/or biologic to the surface of a nanotube or to a chemical modification of carbon nanotube surface or a surface coating, such as, for example, polyethylene glycol (“PEG”), which can regulate insertion of the nanotube into the cell through the plasma membrane and/or cell wall, adherence of the carbon nanotube to- or repulsion from- the surface of the plasma membrane and/or cell wall and removal of the nanotube from the cell by exiting through the plasma membrane and/or cell wall. Typical targets include human cell-types such as T lymphocytes, dendritic cells, macrophages, stem cells (e.g. embryonic, mesenchymal, iPSC), cancer cells, tissue-derived cells such as fibroblasts or adipocytes, as well as other non-human, mammalian cell-types, bacterial, fungal and/or plant cells.

[0027] In some embodiments, covalent or non-covalent attachment of a nanotube surface modification, surface coating and/or payload surface molecules consisting of: small molecules, organic or inorganic macromolecular complexes, nanoparticles, antibodies, adjuvants, lipids, micelles, viruses, viral-derived components oligonucleotides, peptide, proteins, enzymes, antibodies, other types of biologies and/or complexes thereof, may be useful for imaging, sensing and/or diagnostic applications, we all as modulating cellular biochemical processes such as gene delivery or sequestration or removal of cell-derived components or products such as proteins or nucleic acids.

[0028] In some applications, the biodistribution of carbon nanotubes can be controlled via selection and functionalization of the surface coating. In certain embodiments, the drug- loaded nanotubes may be used to transport covalently or non-covalently associated payloads into eukaryotic and prokaryotic cells grown in culture. In some embodiments, nanotubes may be preferentially directed to specific cells on the basis of intrinsic characteristics of the nanotube, such as: length, diameter, curvature and chemistry, as well as the function of covalently or non-covalently associated surface coatings and/or payloads, such as, but not limited to, oligonucleotides, peptides, antibodies, adjuvants, cytokines, cell- or viral-derived components, viral vectors and/or any other molecular or macromolecular component used to cell-targeting

[0029] Another aspect of the disclosed inventions is the controlled release of drugs, chemicals, compounds, nanoparticles, and/or small molecules which may be bound to a nanotube surface coating. Nanotube surface coatings may include molecules which undergo chemical and/or physical changes in response to changes in environmental conditions, including but not limited to temperature, ionic concentration, and/or pH. Such molecules may be incorporated, either directly through covalent or ionic, hydrophilic, hydrophobic and electrostatic interactions onto the surface of nanotubes in order to further regulate delivery. As one of many examples, a pH-sensitive polymer which decomposes at acidic pH below 7.4 would allow for selective delivery of drugs to acidic environments such as tumor-like environments.

[0030] Nanotubes may include molecular surface coatings or payloads whose uptake and/or release from the nanotube may be controlled by competitive association of ionic or biomolecular species with the external and/or internal surfaces of the nanotube or the molecular surface coatings of payloads themselves. As one of many examples, differences in calcium concentration gradients may be used as means of controlling release of biomolecular payloads in systems where calcium ions above a threshold concentration compete with biomolecular surface coatings or payloads for occupying the nanotube surface, causing release of the biomolecular surface coating or payload.

[0031] Nanotubes may feature targeting agents and/or adjuvants, covalently or non- covalently associated with the external surfaces, internal surfaces and/or payload molecules associated with either, which alter the accumulation and/or clearance of the nanotubes within a particular populations and/or targeted subpopulations of cells. These targeting agents may be both inorganic and/or organic in nature, of components such as nanoparticles, small molecules, oligonucleotides, peptide, proteins, enzymes, antibodies, other types of biologies and/or complexes thereof.

[0032] Discrete nanotubes, covalently or non-covalently associated with molecular surface coatings and/or payloads, which enable accumulation and/or clearance from cells in culture and/or targeted populations thereof, with approximately normal distributions of average lengths ranging from 800nm to lOnm may be preferred for greater (by mass) and more rapid uptake and/or clearance. Molecular surface coatings and/or payloads can consist of: ionic and non-ionic species, inorganic and organic nanoparticles, small molecules, polysaccharides, oligonucleotides, peptide, proteins, enzymes, antibodies, other types of biologies and/or complexes thereof. For example, a collection of discrete nanotubes with an approximately normal size distribution averaging 700nm is demonstrated to provide approximately 500% to 1000% increase in distribution to cultured mesenchymal stem cells relative to a population of discrete nanotubes with an approximately normal size distribution averaging 850nm.

[0033] It is envisioned nanotubes with different inherent physical characteristics (such as: length, diameter, curvature, surface roughness and/or chemistry), as well as different covalently or non-covalently associated surface coatings and/or payloads such as, but not limited to, oligonucleotides, peptides, antibodies, adjuvants, cytokines, cell- or viral-derived components, viral vectors, other biologies and/or any or combinations thereof, may be preferred for internalization and/or clearance for distinct physiological cell-types of cells of a distinct immunophenotype, or bridging a particular spatial or temporal span. In another example, a preferential distribution of nanotube length may be used in an application where distinct biological payloads on each of a nanotube are used to associate, bind to or other affect two distinct surface receptors on cells, such as a therapeutic, imaging or sensing system in which a nanotube complexed with two different antibodies binds distinct membrane proteins. [0034] The inherent physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads referenced in, for example, paragraph [0032] above, impact the mechanism and rate of nanotube internalization, clearance and/or association with membranes, organelles and walls in mammalian, bacterial, fungal and/or plant cells. For example, nanotubes with PEO-based surface coatings, characterized by an approximately normal length distribution averaging 850nm, are known to traffic into mammalian cell-types such as mesenchymal stem cells via a vesicular transport mechanism. In the case of cell-types featuring polysaccharide walls, such as plants or bacteria, for nanotubes with the same PEO-based surface coating, a nanotube subpopulation with a smaller average length is preferred for internalization, while a longer average length nanotube subpopulation is preferred for association and/or payload delivery to the external cellular surface.

[0035] In certain embodiments, delivery of chemical, molecular, macromolecular and/or biological payloads using nanotubes or delivery of surface-modified nanotubes to cells would preferably proceed by association of the nanotube with the surfaces of mammalian, bacterial, fungal and/or plant cells, tissue, biofilm or other material, as opposed to direct internalization of the nanotube. In these applications, inherent physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads which regulate the affinity of nanotubes for association with the surfaces of mammalian, bacterial, fungal and/or plant cells, tissue or other material are preferred over those what facilitate internalization. Similarly, physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads which confer targeting of nanotubes for accumulation, enrichment and/or deplition of particular physiologic cells-types, cellular immunotypes, cellular phenotypes, or the expression of certain biomolecular components, such as surface markers or membrane receptors, or any other distinguishing biological or non-biological component of cells, tissues and/or materials such as, but not limited to, bacterial or fungal biofilms.

[0036] In certain embodiments, inherent physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads which confer the ability to identify, quantity, isolate, purify, enrich, retrieve and/or remove nanotubes from a gaseous, liquid, gel or solid media and/or from within cells, tissues and materials, may be preferable. For example, nanotubes loaded with molecular fluorophores such as DAPI (4',6- diamidino-2-phenylindole) can be useful for quantifying rate and/or quantity of intracellular trafficking and accumulation in mesenchymal stem cells and T lymphocyte-derived cell lines.

[0037] In further embodiments, inherent physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads which confer the ability to identify, quantity, isolate, purify, enrich, retrieve and/or remove cells, tissues, bacterial or fungal biofilms and/or other materials which have internalized nanotubes may be preferable. The referenced physical characteristics of nanotubes, surface coatings and/or payloads of nanotubes may serve an ancillary function to technologies for cell identification, phenotyping and/or sorting, such as flow cytometry or fluorescence-activated cell sorting (FACS).

[0038] In certain embodiments of the invention, inherent physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads which reduce cellular doubling-time, extend the number of population doublings and/or reduce phenotypic drift.

[0039] In certain embodiments of the invention, inherent physical characteristics of nanotubes, as well as different covalently or non-covalently associated surface coatings and/or payloads which confer targeted accumulation in lymphocytes, neutrophils and monocyte/macrophage immune cells, for example, but not limited to, dendritic cells, macrophages, T cells, B cells and NK cells and/or well as adjuvants to increase the immunological response to an antigen in in vitro, ex vivo and in vivo administration.

[0040] In another aspect the invention is a composition comprising a plurality of functionalized discrete single-wall, double-wall, or multi-wall carbon nanotubes having an innermost wall and an outermost wall, the inner-most wall defining an interior cavity, and at least one type of payload molecule; wherein the functionalized discrete carbon nanotubes are open on at least one end; and wherein greater than about 30 weight percent of the at least one type of payload molecule is within the interior cavity of the discrete single-wall, double-wall or multi-wall carbon nanotubes.

[0041] The functionalizing groups and/or the surface coating are not limited to, but can be selected from, the group consisting of ionic/non-ionic, hydrophobic/hydrophilic functional groups, bio-compatible surfactants, ionic and zwitterionic moieties. Preferred bio-compatible surfactants include, but are not limited to, PLA (poly lactic acid), PVOH (polyvinyl alcohol), PEO (polyethylene oxide), PGLA (polyglycolic acid), CMC (carboxymethyl cellulose), PVP polyvinylpyrrolidone, PAA polyacrylic acid, aminoacids, peptides, polysaccharides, carboxy betaine-based systems, phosphoryl choline-based systems, nucleic acids for example, but not limited o, DNA) and proteins for example, but not limited to albumin. Zwitterions may employed to provide a physical and energetic barrier against protein adsorption. Open ended multi-wall discrete carbon nanotubes preferably comprise at least one end having attached thereto a bio-compatible polymer, amino acid, protein or peptide.

[0042] The attachment may be via covalent bonding, ionic bonding, hydrogen bonding or pi-pi bonding in nature. That is,“attached” as used herein may, depending on the context, include covalent bonding, ionic bonding, hydrogen bonding, pi-pi bonding, or other adherence, as well as, combinations thereof. The functionalized discrete carbon nanotubes can include at least one cell, organelle or tissue-targeting moiety. Use of cell, organelle or tissue-targeting moieties is known in the art to facilitate association of nanoparticles or molecules to a particular cell-type, organelle, tissue or other biological target. The compositions may also be directed to certain cellular receptors, such as through receptor ligands attached to the functionalized carbon nanotube. In some cell-types, such as but not limited to stem cells, immunological cells or cancer cells certain cellular receptors can be overexpressed, unexpressed or in a high-affinity or low-affinity binding state. In this example, direction of the compositions herein to cellular receptors advantageously provides a means of targeting a particular cell-type or cellular immunotype and/or phenotype. The at least one tissue- targeting moiety is selected from a group including, but not limited to, aptamers, nucleic acids, antibodies, antibody fragments, polysaccharides, peptides, proteins, hormones, receptor ligands, synthetic derivatives thereof, adjuvants and combinations thereof. Various extracellular or intracellular recognition sites exist for these moieties, allowing for directed tissue targeting of the compositions.

[0043] At least one type of payload molecule is preferably at least partially released from the open-ended multi-wall discrete carbon nanotubes by a mechanism comprising diffusion, electromagnetic radiation exposure (e.g., MRI (Magnetic Resonance Imaging)), local pH changes, electrolyte balance, chemical concentration gradients, chemical and/or conformational changes caused by interaction with an organic or inorganic chemical entity in the extracellular or intracellular environment, dissolution, chemical and/or conformational changes caused by interactions with biological structures (e.g., binding to surface receptors on a plasma membrane) or biological (e.g., enzymatic) digestion of the biopolymer coat.

[0044] Due to the relevance of both covalent and non-covalent attachment for combination of various chemistries with carbon nanotubes, both as surface coatings and payloads, any referenced to attachment in this patent referes to both covalent and non-covalent linkages unless otherwise stated. [0045] The plurality of functionalized discrete open-ended multi-wall carbon nanotubes preferably comprises nanotubes of varying lengths. The tube length distribution may be monomodal, bimodal or multimodal. For tube length distributions comprising at least 2 groups of lengths, preferred is wherein each group’s tube length varies on average by at least about 10% from the other group’s average tube length to control drug release rates. Different length distributions may contain different payload molecules, different targeting moieties, and/or different surface coatings.

[0046] The plurality of functionalized discrete open-ended multi-wall carbon nanotubes comprises an average aspect ratio (Length/Diameter) of from about 25 to about 500, preferably 25-250 and most preferably 40-120.

[0047] The plurality of functionalized discrete open-ended multi-wall carbon nanotubes can comprise 0.01 to 99.9% by weight of the composition, preferably 0.1 to 99%, more preferably 0.25 to 95% by weight of the composition.

[0048] Based on the desired rate of payload delivery 10% by weight or less of the discrete carbon nanotubes of the composition can comprise L/D of about 100 to 200 and about 30% or more of the discrete carbon nanotubes (known and referred to herein as Molecular Rebar (“MR”) of the composition can comprise L/D of 40 to 80. The L/D of the discrete carbon nanotubes can be a unimodal distribution, or a multimodal distribution (such as a bimodal distribution). The multimodal distributions can have evenly distributed ranges of aspect ratios (such as 50% of one L/D range and about 50% of another L/D range). The distributions can also be asymmetrical - meaning that a relatively small percent of discrete nanotubes can have a specific L/D while a greater amount can comprise another aspect ratio distribution.

[0049] Some embodiments of the disclosed nanotubes are referred to as MGMR. MGMR nanotubes are discrete nanotubes and may be in a stable dispersion. They have at least about 99.8% purity, are generally sterile, have an average length of about 800nm to about 900nm, and an average diameter of about l2nm to about l4nm. MGMR nanotubes are generally used in medical applications. Figure 2 shows one particular embodiment of MGMR nanotubes. It will be appreciated that many of the disclosed embodiments are not limited to the specific features identified above.

[0050] The payload molecule is not limited to, but can be selected from, the group consisting of small molecular weight chemical entities, large molecular weight chemical entitites, inorganic and/or organic chemistries, ionic species, molecules and macromolecular assemblies, nanoparticles, drug molecules, radiotracer molecules, radiotherapy molecules, diagnostic imaging molecules, fluorescent tracer molecules, proteins, peptides, enzymes, nucleic acids, oligonucleotides, lipid vesicles, polymer vesicles, genetic materials, anti-bodies, adjuvants, cell-derived components, various sorts of gene-editing complexes (e.g., CRISPR- based systems, transcription activator-like effector nucleases and zinc finger nucleases), other types of biologies and/or combinations thereof. Exemplary types of payload molecules that may be covalently or non-covalently associated with the discrete functionalized carbon nanotubes disclosed herein may include, but are not limited to, proton pump inhibitors, H2- receptor antagonists, cytoprotectants, prostaglandin analogues, beta blockers, calcium channel blockers, diuretics, cardiac glycosides, antiarrhythmics, antianginals, vasoconstrictors, vasodilators, ACE inhibitors, angiotensin receptor blockers, alpha blockers, anticoagulants, antiplatelet drugs, fibrinolytics, hypolipidemic agents, statins, hypnotics, antipsychotics, antidepressants, monoamine oxidase inhibitors, selective serotonin reuptake inhibitors, antiemetics, anticonvulsants, anxiolytic, barbiturates, stimulants, amphetamines, benzodiazepines, dopamine antagonists, antihistamines, cholinergics, anticholinergics, emetics, cannabinoids, 5-HT antagonists, NSAIDs, opioids, bronchodilator, antiallergics, mucolytics, corticosteroids, beta-receptor antagonists, anticholinergics, steroids, androgens, antiandrogens, growth hormones, thyroid hormones, anti-thyroid drugs, vasopressin analogues, antibiotics, antifungals, antituberculous drugs, antimalarials, antiviral drugs, antiprotozoal drugs, radioprotectants, chemotherapy drugs, cytostatic drugs, cytotoxic drugs such as paclitaxel, and biologies, including proteins, such as antibodies and antibody fragments, molecular technologies for gene-editing such as, but not limited to, CRISPR and/or combinations of associated proteins, transcription activator-like effector nucleases, zinc finger nucleases enzymes and gRNAs as well as nucleic acids, oligonucleotides, gene expression vectors, siRNAs, vaccines, and the like.

[0051] In another aspect of the invention, a payload or drug molecule delivery system composition comprising discrete carbon nanotubes is disclosed, wherein at least a portion of discrete nanotubes have a ratio of number average value of ((tube contour length (TCL)):(tube end to end length (TEE))) of from about 1.1 to about 3, preferably from about 1.1 to about 2.8, more preferably from about 1.1 to about 2.4, most preferably from about 1.1 to about 2 and especially from about 1.2 to about 2.

[0052] Another aspect of the inventions is a payload or drug delivery system composition comprising a plurality of discrete carbon nanotubes wherein at least a portion of discrete nanotubes have a number average tube contour length (TCL) of at least 10% greater than, and up to about 300% of, a number average tube end to end length (TEE), wherein the number average TCL and TEE are obtained from the same batch of discrete carbon nanotubes.

[0053] In another aspect, in a payload or drug delivery system composition comprising a plurality of discrete carbon nanotubes having an average actual aspect ratio, of at least about 5% (volume) of the discrete carbon nanotubes having an apparent aspect ratio from about 50% to about 99% of the average actual aspect ratio of the discrete carbon nanotubes. The apparent aspect ratio can be from a low of about 60% or 70%, to as high as about 80%, 90%, about 99% of the actual aspect ratio.

[0054] The composition can have at least about 10% (volume), preferably 20%, more preferably 50%, most preferably 75%, and especially 95%, of the discrete carbon nanotubes that have an apparent aspect ratio from about 50% to about 99% of the actual aspect ratio of the discrete carbon nanotubes.

[0055] Another aspect of the invention is in a payload or drug delivery system composition comprising a plurality of discrete carbon nanotubes having a number average contour length TCL, the improvement comprising at least about 5% (volume) of the discrete carbon nanotubes have a number average end-to-end tube length TEE low value from about 50%, 60%, or 70% to a high value of about 80%, 90%, or 99% of the number average TCL.

[0056] The composition can have at least about 10% (volume), preferably 20%, more preferably 50%, most preferably 75%, and especially 95%, of the discrete carbon nanotubes have a number average TEE from about 50% to about 99% of the number average TCL of the discrete carbon nanotubes.

[0057] The at least a portion of discrete nanotubes can have a number average value (the ratio of discrete TCL to TEE) of about 1.1 to as high as about 3, is greater than 5% by number, preferably greater than 20% by number and most preferably greater than 50% by number of tubes.

[0058] In applications where carbon nanotubes are exploited for internationalization into cells or association with their external surface and the cells are selected from the group of cells having polysaccharide walls, discrete carbon nanotubes with an average length in the range of about less than 1000 nanometers, preferably less than about 850 nanometers and most preferably less than about 500 nanometers, are deemed to be the most effective. Conversely, in applications where internalization of the carbon nanotubes in these cells is not desirable, biocompatible surface modifications of the carbon nanotubes which prevent cellular association may be used.

[0059] In applications where carbon nanotubes are exploited for internationalization into cells or association with their external surface and the cells are selected from the group of mammalian, bacterial protoplasts, fungal protoplasts, plant protoplasts wherein the discrete carbon nanotubes have an average length greater than that required for cellular penetration, preferably greater than 500 nm, more preferably greater than about 750nm, are deemed to be the most effective. Conversely, in applications where internalization of the carbon nanotubes in these cells or protoplasts is not desirable, biocompatible surface modifications of the carbon nanotubes which prevent cellular association may be used.

[0060] Processes to make the discrete carbon nanotubes are also described herein. These additional (and optional) steps can be selected from adding the discrete carbon nanotubes to a material to react with the oxidized discrete carbon nanotubes, adding surfactants, and adding other molecules including drugs, chemicals, compounds, and/or small molecules.

[0061] For a more complete understanding of the present disclosure, and the advantages thereof, reference is now made to the following descriptions describing specific embodiments of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0062] Figure 1 shows the relative scale of one potential nanotube compared to various biological features.

[0063] Figure 2 depicts an electron micrograph (35,000x magnification) of the disclosed carbon nanotubes.

[0064] Figure 3 describes the effect of carbon nanotubes with different types of surface coatings on the proliferation of human bone marrow-derived mesenchymal stem cells within a 72-hour period.

[0065] Figure 4 depicts internalization kinetics of several particular embodiments of the disclosed nanotubes in human bone marrow-derived mesenchymal stem cells, measured by intracellular fluorescence of DAPI. [0066] Figures 5a-5h feature fluorescence and brightfield micrographs (4x magnification) depicting human bone marrow-derived mesenchymal stem cells incubated for 96 hours with several particular embodiments of the disclosed nanotubes, loaded with DAPI or treated for the same length of time with DAPI alone.

[0067] Figure 6 features a transmission electron micrograph of human mesenchymal stem cells, depicting internalization of a particular embodiment of the described nanotubes following 72-hour incubation.

[0068] Figure 7 features a transmission electron micrograph of human mesenchymal stem cells incubated with nanotubes for 72 hours and depicts the stages of the intracellular trafficking mechanism of a particular embodiment of the described nanotubes.

[0069] Figures 8a-8h outline the membrane penetration and localization of a particular embodiment of the disclosed nanotubes in cultured PTK2 kidney epithelium cells.

[0070] Figure 9 outlines the genotoxicity testing of a particular embodiment of the disclosed nanotubes in 3T3L1 murine pre-adipocyte fibroblasts.

[0071] Figure 10 outlines the carcinogenic testing of a particular embodiment of the disclosed nanotubes in 3T3L1 murine pre-adipocyte fibroblasts.

[0072] Figures l la-l lb depict micrographs of LNCaP cells treated with nanotubes without a peptide payload (Fig. 1 la) and nanotubes with a peptides payload (Fig. 1 lb).

[0073] Figure 12 describes the transportation of a peptide payload molecule using a particular embodiment of the disclosed nanotubes in LNCaP cells.

[0074] Figure 13 describes the transportation of a genetic molecular payload (siRNA oligonucleotides), using a particular embodiment of the disclosed nanotubes, in HeLa cancer cells.

[0075] Figure 14 describes the transportation of a genetic molecular payload (mRNA oligonucleotides), using a particular embodiment of the disclosed nanotubes, in T- lymphocytes.

[0076] Figure 15 describes the transportation of a genetic molecular payload (mRNA oligonucleotides), using a particular embodiment of the disclosed nanotubes, in macrophages.

[0077] Figure 16 outlines the bacterial toxicity testing of a particular embodiment of the disclosed nanotubes in Escherichia coli.

[0078] Figures l7a-l7c depict the fluorescence micrographs of Escherichia coli incubated with nanotubes surface coated with an Alexa 488 fluorescent tag.

[0079] Figures l8a-l8c outline tunable affinity of particular embodiments of the disclosed nanotubes for the bacterial cell wall of Escherichia coli.

DETAILED DESCRIPTION

[0080] In the following description, certain details are set forth such as specific quantities, sizes, etc., so as to provide a thorough understanding of the present embodiments disclosed herein. However, it will be evident to those of ordinary skill in the art that the present disclosure may be practiced without such specific details. In many cases, details concerning such considerations and the like have been omitted inasmuch as such details are not necessary to obtain a complete understanding of the present disclosure and are within the skills of persons of ordinary skill in the relevant art.

[0081] While most of the terms used herein will be recognizable to those of ordinary skill in the art, it should be understood, however, that when not explicitly defined, terms should be interpreted as adopting a meaning presently accepted by those of ordinary skill in the art. In cases where the construction of a term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition, 2009. Definitions and/or interpretations should not be incorporated from other patent applications, patents, or publications, related or not.

[0082] Functionalized carbon nanotubes of the present disclosure generally refer to the chemical modification of any of the carbon nanotube types described hereinabove. Such modifications can involve the nanotube ends, sidewalls inside and/or outside, or both. Chemical modifications may include, but are not limited to covalent bonding, ionic bonding, chemisorption, intercalation, surfactant interactions, polymer wrapping, cutting, solvation, and combinations thereof. In some embodiments, the carbon nanotubes may be functionalized before, during and after being individualized or exfoliated.

[0083] Any of the aspects disclosed in this application with discrete carbon nanotubes may also be modified within the spirit and scope of the disclosure to substitute other tubular and non-tubular nanostructures, including, for example, organic, inorganic, or mineral nanotubes, planar nanostructures, and/or other nanostructures. Inorganic or mineral nanotubes include, for example, silicon nanotubes, boron nitride nanotubes and carbon nanotubes having heteroatom substitution in the nanotube structure. The nanotubes may include or be associated with organic or inorganic elements such as, for example, carbon, silicon, boron and nitrogen. Association may be on the interior or exterior of the inorganic or mineral nanotubes via Van der Waals, ionic or covalent bonding to the nanotube surfaces, all of these forces are individually and collectively referred to by the non-limiting term “attached”. Planar nanostructures include substantially planar carbon compounds such as graphene and similar structures composed of or including silicon, boron nitride, and carbon structures having heteroatom substitution in the nanostructure. These planar nanostructure may include or be associated with organic or inorganic elements such as, for example, carbon, silicon, boron and nitrogen. Association may be on either or both surfaces of the planar nanostructure via Van der Waals, ionic or covalent bonding to the planar nanostructure surfaces. Other nanostructures include three dimensional carbon structures such as fullerenes and similar structures composed of or including silicon, boron nitride, and carbon structures having heteroatom substitution in the nanostructure. These other nanostructures may also include or be associated with organic or inorganic elements such as, for example, carbon, silicon, boron and nitrogen. Association may be on the interior or exterior of the nanostructure via Van der Waals, ionic or covalent bonding to the nanotube surfaces

[0084] In various embodiments, a plurality of carbon nanotubes is disclosed comprising single wall, double wall or multi wall carbon nanotubes having an aspect ratio of from about 10 to about 500, preferably from about 40 to about 200 and more preferably from about 45 to about 120 and an overall (total) oxidation level of from about 1 weight percent to about 15 weight percent, preferably from about 1 weight percent to about 10 weight percent, more preferably from about 1 weight percent to 5 weight percent, more preferably from about 1 weight percent to 3 weight percent. The oxidation level is defined as the amount by weight of oxygenated species covalently bound to the carbon nanotube. The thermogravimetric method for the determination of the percent weight of oxygenated species on the carbon nanotube involves taking about 7-15 mg of the dried oxidized carbon nanotube and heating at 5 °C/minute from 100 degrees centigrade to 700 degrees centigrade in a dry nitrogen atmosphere. The percentage weight loss from 200 to 600 degrees centigrade is taken as the percent weight loss of oxygenated species. The oxygenated species can also be quantified using Fourier transform infra-red spectroscopy, FTIR, particularly in the wavelength range 1730-1680 cm-l

[0085] The carbon nanotubes can have oxidation species comprising carboxylic acid or derivative carbonyl containing species and are essentially discrete individual nanotubes, not entangled as a mass. Typically, the amount of discrete carbon nanotubes after completing the process of oxidation and shear is by a far a majority (that is, a plurality) and can be as high as 70, 80, 90 or even 99 percent of discrete carbon nanotubes, with the remainder of the tubes still partially entangled in some form. Complete conversion (i.e., 100 percent) of the nanotubes to discrete individualized tubes is most preferred. The derivative carbonyl species can include phenols, ketones, quaternary amines, amides, esters, acyl halogens, monovalent metal salts and the like, and can vary between the inner and outer surfaces of the tubes.

[0086] For example, one type of acid can be used to oxidize the tubes exterior surfaces, followed by water washing and the induced shear, thereby breaking and separating the tubes. If desired, the formed discrete tubes, having essentially no (or zero) interior tube wall oxidation can be further oxidized with a different oxidizing agent, or even the same oxidizing agent as that used for the tubes' exterior wall surfaces at a different concentration, resulting in differing amounts - and/or differing types - of interior and surface oxidation.

[0087] The discrete carbon nanotubes have oxidized species on the surface, also known herein as functionalized groups. In this disclosure, the amount of oxidation can be from about 1 to about 15% by weight of the dried carbon nanotubes. Oxidized species include but not limited to carboxylates, hydroxyls, lactones, and combinations thereof. The oxidized species can react advantageously with species such as, but not limited to, an acylchloride, epoxy, isocyanate, hydroxyl, or amine group. The Molecular Rebar may further comprise a biocompatible dispersing agent or surfactant, adhesively, ionically or covalently bonded to the Molecular Rebar surface. The biocompatible dispersing or surfactant molecule can be chosen such that the size of the surfactant molecule in the liquid media prevents it from entering within the discrete carbon nanotube. The selection of the minimum size of the surfactant molecule that cannot enter into a tube opening is related to the diameter of the tube opening and the hydrodynamic radius of the molecule in the liquid media.

[0088] Hydrodynamic radius, RH, of polymer molecules in liquid media has been well- studied in the scientific literature, for example M.S. Ahmed, M.S. El-Aassar and J. W. Vanderhoff, ACS Symp. Series 240:77 (1983). Techniques to measure the radius of gyration commonly include viscometry and photon correlation spectroscopy. In the studies by Ahmed et al, the values of RH of polyvinyl alcohol adsorbed onto polystyrene particles of diameter l90nm in water were found to follow an equation RH = 0.03 Mw° ⁵³⁸. [0089] The size of the surfactant molecule that can disperse the discrete carbon nanotube in aqueous media and is not expected to be able to enter within the cavity of the open- ended carbon nanotube is preferably greater than about 30,000 Daltons, more preferably greater than about 60,000 Daltons and most preferably greater than about 100,000 Daltons. An example of a biocompatible polymer that is of size that does not fit within carbon nanotubes with an internal diameter opening of 5nm is polyvinyl alcohol of molecular weight about 61,000 Daltons, available as Mowiol 10-98, supplied by Kuraray.

TABLE 1

The Hydrodynamic Radius of Various Molecules in Water for Various Molecules

[0090] The hydrodynamic radius, RH of single amino acids, small di- and tripeptides as well as denatured proteins fit an equation RH = 0.027M^{0 5}nm. (J. Danielson. PhD. Thesis Stockholm University 2007). For PVOH this has been found to be RH = 0.03M^{0 538}nm. It is recognized that the value of the hydrodynamic radius is also dependent on the solvent quality, i.e., RH will decrease for insulin in acid conditions versus neutral conditions. Likewise a change in temperature can also cause a change in values of RH. This change in hydrodynamic radius may be conducive to fit molecules within the interior cavity of the discrete carbon nanotube; then to change the liquid media environment and force expansion of the molecules’ hydrodynamic radius and cause expulsion of the drug molecule from the interior cavity, one needs to change the environment, such as for example, by changing liquid media environment temperature, pH or both.

[0091] Single-wall and double-wall carbon nanotubes typically have internal diameters of about 0.9 to about 1.2 nm. Multi-wall carbon nanotubes typically have internal diameters from about 1.8 to about 50nm. Molecules are considered unlikely to enter into open-ended carbon nanotubes if their hydrodynamic radius is about 10% larger than that of the carbon nanotube opening. This means, for example looking at Table 1 (above), that insulin, with a hydrodynamic radius of 2. lnm would not be able to enter inside an opened single wall or double wall carbon nanotube. Likewise, bovine serum albumin with a hydrodynamic radius of 7 nm would not enter into an open-ended multiwall carbon nanotube of internal diameter 5.5nm. This means that the selection of the innermost wall diameter of the discrete carbon nanotube plays a key role in selecting the maximum size of molecule that can enter into the carbon nanotube.

[0092] The biocompatible dispersing or surfactant molecule can also be chosen to help solubilize a drug or other molecule in an aqueous media such that the drug and surfactant conjugate can enter into an open-ended nanotube, followed by the nanotube and contents being encapsulated with a larger biomolecule that cannot enter into the tube. The size of the surfactant molecule being able to enter into the nanotube is preferably less than about 10,000 Daltons, more preferably less than about 5,000 Daltons and most preferably less than about 2,000 Daltons. An example of this type of surfactant being able to enter into a multiwall nanotube is polyoxyethene sorbitan monostearate of molecular weight about 1,309 Daltons and is commercially available as Tween-60 (Tween is a registered Trademark of Croda International PLC). As a result of the aforementioned, discrete carbon nanotubes may result in advantageous drug transport properties.

[0093] The internal tube diameter of the open-ended carbon nanotube can be selected to allow a maximum size of the molecule to enter within the tube. This can be useful to select a certain size molecule from a mixture of molecules of different sizes. Open-ended carbon nanotubes of different internal diameter tubes and/or different lengths can be used to control the rate of drug delivery, or combinations of drug types or sizes. Discrete open-ended carbon nanotubes of differing functionality can also be used to control the rate of release of the drug to the treatment site.

[0094] The discrete oxidized carbon nanotubes alternatively termed exfoliated carbon nanotubes, of the present disclosure can take advantage of properties such as electrical, thermal, physical and drug transport, offered by individual carbon nanotubes that are not apparent when the carbon nanotubes are aggregated into bundles. An example of properties offered by individual carbon nanotubes rather than bundled or associated carbon nanotubes would be to deliver drug concentrations more accurately and for individual carbon nanotubes to be preferentially oriented alongside cell walls or to enter within cells.

[0095] Discrete oxidized carbon nanotubes, alternatively termed exfoliated carbon nanotubes, are obtained from as-made bundled carbon nanotubes by methods disclosed in USSN 13/164,456 and USSN 13/140,029, the disclosures of which are incorporated herein by reference, are particularly useful in producing the discrete carbon nanotubes used in this invention. The bundled carbon nanotubes can be made from any known means such as, for example, chemical vapor deposition, laser ablation, and high pressure carbon monoxide synthesis. The bundled carbon nanotubes can be present in a variety of forms including, for example, soot, powder, fibers, and bucky paper. Furthermore, the bundled carbon nanotubes may be of any length, diameter, or chirality. Carbon nanotubes may be metallic, semi-metallic, semi-conducting, or non-metallic based on their chirality and number of walls. The discrete oxidized carbon nanotubes may include, for example, single-wall, double-wall carbon nanotubes, or multi-wall carbon nanotubes and combinations thereof. One of ordinary skill in the art will recognize that some of the specific aspects of this invention illustrated utilizing a particular type of carbon nanotube may be practiced equivalently within the spirit and scope of the disclosure utilizing other types of carbon nanotubes. However, for control of the desired structures of a plurality of discrete carbon nanotubes requires a specific control of chemistry, thermal and mechanical energy which varies according to the starting structure of the carbon nanotubes.

[0096] In particular for forming carbon nanotubes of this invention is the incorporation of a portion of structures called Stone-Wales defects which are the rearrangement of the six- membered rings of graphene into heptagon-pentagon pairs that fit within the hexagonal lattice of fused benzene rings constituting a wall of the carbon nanotubes. These Stone-Wales defects are useful to create sites of higher bond-strain energy for more facile oxidation of the graphene or carbon nanotube wall. These defects and other types of fused ring structures may also facilitate bending or curling along the length of the carbon nanotubes.

[0097] Stone-Wales defects are thought to be more prevalent at the end caps that allow higher degrees of curvature of the walls of carbon nanotubes. During oxidation the ends of the carbon nanotubes can be opened and also result in higher degrees of oxidation at the opened ends than along the walls. The higher degree of oxidation and hence higher polarity or hydrogen bonding at the ends of the tubes are thought useful to help increase the average contour length to end to end ratio where the tubes are present in less polar media such as oil. The ratio of the contour length to end to end distance can be advantageously controlled by the degree of thermodynamic interaction between the tubes and the medium. Surfactants and electrolytes can be usefully employed also to modify the thermodynamic interactions between the tubes and the medium of choice. Alternate means to influence the ratio of contour length to end to end ratio include the use of inorganic or ionic salts and organic containing functional groups that can be attached to or contacted with the tube surfaces. [0098] Bundled Carbon Nanotubes

[0099] As manufactured carbon nanotubes are obtainable in the form of bundles or entangled agglomerates and can be obtained from different sources, such as CNano Technology, Nanocyl, Arkema, and Kumho Petrochemical, to make discrete carbon nanotubes. An acid solution, preferably nitric acid solution at greater than about 60 weight % concentration, more preferably above 65% nitric acid concentration, can be used to prepare the carbon nanotubes for later shear to make the discrete tubes. Mixed acid systems (e. g. nitric and sulfuric acid) as disclosed in US 2012-0183770 Al and US 2011-0294013 Al, the disclosures of which are incorporated herein by reference, can be used to produce discrete, oxidized carbon nanotubes from as- made bundled or entangled carbon nanotubes. The carbon nanotubes may be used consistent with the methods described in U.S. Patent No. 7,992,640; U.S. Application No. 2015/0368541; and U.S. Application No. 2014/0014586, all of which are incorporated herein by reference.

[00100] As-made carbon nanotubes using metal catalysts such as iron, aluminum or cobalt can retain a significant amount of the catalyst associated or entrapped within the carbon nanotube, as much as five weight percent or more. These residual metals can be deleterious in such applications as drug delivery, treatment, imaging, and/or diagnostics because of such residual metals may not be biocompatible. Furthermore, these divalent or multivalent metal ions can associate with carboxylic acid groups on the carbon nanotube and interfere with the discretization of the carbon nanotubes in subsequent dispersion processes. In other embodiments, the oxidized carbon nanotubes comprise a residual metal concentration of less than about 25,000 parts per million, ppm, and preferably less than about 5,000 parts per million. The metals composition and concentration can be conveniently determined using energy dispersive X-ray spectroscopy or thermogravimetric methods.

[00101] General Process to Produce Discrete Carbon Nanotubes Having Targeted Oxidation

[00102] A mixture of 0.5% to 5% carbon nanotubes, preferably 3%, by weight is prepared with CNano grade Flotube 9000 carbon nanotubes and 65% nitric acid. While stirring, the acid and carbon nanotube mixture is heated to 70 to 90 degrees C for 2 to 4 hours. The formed oxidized carbon nanotubes are then isolated from the acid mixture. Several methods can be used to isolate the oxidized carbon nanotubes, including but not limited to centrifugation, filtration, mechanical expression, decanting and other solid - liquid separation techniques. The residual acid is then removed by washing the oxidized carbon nanotubes with an aqueous medium such as water, preferably deionized water, to a pH of 3 to 4. The carbon nanotubes are then suspended in water at a concentration of 0.5% to 4%, preferably 1.5% by weight. The solution is subjected to intensely disruptive forces generated by shear (turbulent) and/or cavitation with process equipment capable of producing energy densities of 106 to 108 Joules/m³. Equipment that meet this specification includes but is not limited to ultrasonicators, cavitators, mechanical homogenizers, pressure homogenizers and microfluidizers (Table 2). One such homogenizer is shown in U.S. Patent 756,953, the disclosure of which is incorporated herein by reference. After shear processing, the oxidized carbon nanotubes are discrete and individualized carbon nanotubes. Typically, based on a given starting amount of entangled as- received and as-made carbon nanotubes, a plurality of discrete oxidized carbon nanotubes results from this process, preferably at least about 60%, more preferably at least about 75%, most preferably at least about 95% and as high as 100%, with the minority of the tubes, usually the vast minority of the tubes remaining entangled, or not fully individualized.

[00103] Another illustrative process for producing discrete carbon nanotubes follows: A mixture of 0. 5% to 5% carbon nanotubes, preferably 3%, by weight is prepared with CNano Flotube 9000 grade carbon nanotubes and an acid mixture that consists of 3 parts by weight of sulfuric acid (97% sulfuric acid and 3% water) and 1 part by weight of nitric acid (65-70 percent nitric acid). The mixture is held at room temperature while stirring for 3- 4 hours. The formed oxidized carbon nanotubes are then isolated from the acid mixture. Several methods can be used to isolate the oxidized carbon nanotubes, including but not limited to centrifugation, filtration, mechanical expression, decanting and other solid - liquid separation techniques. The acid is then removed by washing the oxidized carbon nanotubes with an aqueous medium, such as water, preferably deionized water, to a pH of 3 to 4. The oxidized carbon nanotubes are then suspended in water at a concentration of 0.5% to 4%, preferably 1.5% by weight. The solution is subjected to intensely disruptive forces generated by shear (turbulent) and/or cavitation with process equipment capable of producing energy densities of 106 to 108 Joules/m³. Equipment that meet this specification includes but is not limited to ultrasonicators, cavitators mechanical homogenizers, pressure homogenizers and microfluidizers (Table 2). After shear and/or cavitation processing, the oxidized carbon nanotubes become oxidized, discrete carbon nanotubes. Typically, based on a given starting amount of entangled as-received and as-made carbon nanotubes, a plurality of discrete oxidized carbon nanotubes results from this process, preferably at least about 60%, more preferably at least about 75%, most preferably at least about 95% and as high as 100%, with the minority of the tubes, usually the vast minority of the tubes remaining entangled, or not fully individualized.

[00104] Example 1 : ENTANGLED OXIDIZED AS MWCNT - 3 Hour (oMWCNT-31

[00105] One hundred milliliters of>64% nitric acid is heated to 85 degrees C. To the acid, 3 grams of as-received, multi-walled carbon nanotubes (C9000, CNano Technology) are added. The as-received tubes have the morphology of entangled balls of wool. The mixture of acid and carbon nanotubes are mixed while the solution is kept at 85 degrees C for 3 hours and is labeled "oMWCNT-3". At the end of the reaction period, the oMWCNT-3 are filtered to remove the acid and washed with reverse osmosis (RO) water to pH of 3-4. After acid treatment, the carbon nanotubes are still entangled balls. The tubes are dried at 60°C to constant weight.

[00106] Example 2: ENTANGLED OXIDIZED AS MWCNT- 6 Hour

(oMWCNT-6)

[00107] One hundred milliliters of >64% nitric acid is heated to 85 degrees C. To the acid, 3 grams of as-received, multi-walled carbon nanotubes (C9000, CNano Technology) are added. The as-received tubes have the morphology of entangled balls of wool. The mixture of acid and carbon nanotubes are mixed while the solution is kept at 85 degrees for 6 hours and is labeled "oMWCNT-6". At the end of the reaction period, the oMWCNT-6 are filtered to remove the acid and washed with reverse osmosis (RO) water to pH of 3-4. After acid treatment, the carbon nanotubes are still entangled balls. The tubes are dried at 60°C to constant weight.

[00108] Example 3: DISCRETE CARBON NANOTUBE - OXIDIZE

OUTERMOST WALL ( out-dMWCNT)

[00109] In a vessel, 922 kilograms of 64% nitric acid is heated to 83 °C. To the acid,

20 kilograms of as received, multi-walled carbon nanotubes (C9000, CNano Technology) is added.

The mixture is mixed and kept at 83 °C for 3 hours. After the 3 hours, the acid is removed by filtration and the carbon nanotubes washed with RO water to pH of 3-4. After acid treatment, the carbon nanotubes are still entangled balls with few open ends. While the outside of the tube is oxidized forming a variety of oxidized species, the inside of the nanotubes have little exposure to acid and therefore little oxidization. The oxidized carbon nanotubes are then suspended in RO water at a concentration of 1.5% by weight. The RO water and oxidized tangled nanotubes solution is subjected to intensely disruptive forces generated by shear (turbulent) and/or cavitation with process equipment capable of producing energy densities of 106 to 108 Joules/m³ .The resulting sample is labeled "out-dMWCNT" which represents outer wall oxidized and "d" as discrete.

Equipment that meet this shear includes but is not limited to ultrasonicators, cavitators, mechanical homogenizers, pressure homogenizers, and micro fluidizers (Table 2 ). It is believed that the shear and/or cavitation processing detangles and discretizes the oxidized carbon nanotubes through mechanical means that result in tube breaking and opening of the ends due to breakage particularly at defects in the CNT structure which is normally a 6 member carbon rings. Defects happen at places in the tube which are not 6 member carbon rings. As this is done in water, no oxidation occurs in the interior surface of the discrete carbon nanotubes.

[00110] Example 4: DISCRETE CARBON NANOTUBE - OXIDIZED OUTER AND INNER WALL ( out/m-dMWCNT)

[00111] To oxidize the interior of the discrete carbon nanotubes, 3 grams of the out-dMWCNT is added to 64% nitric acid heated to 85°C. The solution is mixed and kept at temperature for 3 hours. During this time, the nitric acid oxidizes the interior surface of the carbon nanotubes. At the end of 3 hours, the tubes are filtered to remove the acid and then washed to pH of 3-4 with RO water. This sample is labeled "out/in-dMWCNT" representing both outer and inner wall oxidation and "d" as discrete.

[00112] Oxidation of the samples of carbon nanotubes is determined using a thermogravimetric analysis method. In this example, a TA Instruments Q50 Thermogravimetric Analyzer (TGA) is used. Samples of dried carbon nanotubes are ground using a vibration ball mill. Into a tared platinum pan of the TGA, 7-15 mg of ground carbon nanotubes are added. The measurement protocol is as follows. In a nitrogen environment, the temperature is ramped from room temperature up to l00°C at a rate of l0°C per minute and held at this temperature for 45 minutes to allow for the removal of residual water. Next the temperature is increased to 700°C at a rate of 5°C per minute. During this process the weight percent change is recorded as a function of temperature and time. All values are normalized for any change associated with residual water removal during the l00°C isotherm. The percent of oxygen by weight of carbon nanotubes (%Ox) is determined by subtracting the percent weight change at 600°C from the percent weight change at 200 °C.

[00113] A comparative table (Table 3 below) shows the levels of oxidation of different batches of carbon nanotubes that have been oxidized either just on the outside (Batch 1, Batch 2, and Batch 3), or on both the outside and inside (Batch 4). Batch 1 (oMWCNT-3 as made in Example 1 above) is a batch of entangled carbon nanotubes that are oxidized on the outside only when the batch is still in an entangled form (Table 3, first column). Batch 2 ( oMWCNT -6 as made in Example 2 above) is also a batch of entangled carbon nanotubes that are oxidized on the outside only when the batch is still in an entangled form (Table 3, second column). The average percent oxidation of Batch 1 (2.04% Ox) and Batch 2 (2.06% Ox) are essentially the same. Since the difference between Batch 1 (three hour exposure to acid) and Batch 2 (six hour exposure to acid) is that the carbon nanotubes were exposed to acid for twice as long a time in Batch 2, this indicates that additional exposure to acid does not increase the amount of oxidation on the surface of the carbon nanotubes.

[00114] Batch 3 (Out-dMWCNT as made in Example 3 above) is a batch of entangled carbon nanotubes that were oxidized on the outside only when the batch was still in an entangled form (Table 3, third column). Batch 3 was then been made into a discrete batch of carbon nanotubes without any further oxidation. Batch 3 serves as a control sample for the effects on oxidation of rendering entangled carbon nanotubes into discrete nanotubes. Batch 3 shows essentially the same average oxidation level (1.99% Ox) as Batch 1 and Batch 2. Therefore, Batch 3 shows that detangling the carbon nanotubes and making them discrete in water opens the ends of the tubes without oxidizing the interior.

[00115] Finally, Batch 4 (Out/In-dMWCNT as made in this Example 4 herein) is a batch of entangled carbon nanotubes that are oxidized on the outside when the batch is still in an entangled form, and then oxidized again after the batch has then been made into a discrete batch of carbon nanotubes (Table 3, fourth column). Because the discrete carbon nanotubes are open-ended, in Batch 4 acid enters the interior of the tubes and oxidizes the inner surface. Batch 4 shows a significantly elevated level of average oxidation (2.39% Ox) compared to Batch 1, Batch 2 and Batch 3. The significant elevation in the average oxidation level in Batch 4 represents the additional oxidation of the carbon nanotubes on their inner surface. Thus, the average oxidation level for Batch 4 (2.39 % Ox) is about 20 % higher than the average oxidation levels of Batch 3 (1.99% Ox). In Table 3 below, the average value of the oxidation is shown in replicate for the four batches of tubes. The percent oxidation is within the standard deviation for Batch 1, Batch 2 and Batch 3.

[00116] Disclosed embodiments may also relate to a composition useful for targeted delivery of drugs, chemicals, compounds, and/or small molecules. Embodiments may also relate to directing the controlled release or adjusting the breakdown or clearance of drugs, chemicals, compounds, and/or small molecules including but not limited to genetic materials, oligonucleotides, peptides, proteins, viruses/vectors, small molecules and any sort of gene editing complex.

[00117] Disclosed embodiments may comprise a plurality of discrete carbon nanotubes, wherein the discrete carbon nanotubes comprise an interior and exterior surface. Each surface may comprise an interior surface oxidized species content and/or an exterior surface oxidized species content. Embodiments may also comprise at least one biologically or chemically active molecule that is attached on either the interior or the exterior surface of the plurality of discrete carbon nanotubes. Such embodiments may be used in order to deliver known biologically and/or chemically active molecules to a desired location within the body and/or to maintain such biologically and/or chemically active molecules at a desired location once delivered.

[00118] Cellular Non-toxicity of The Disclosed Nanotubes

[00119] Figure 1 features a scaled depiction of 850nm length nanotubes relative to relevant biological structures, while Figure 2 depicts typical physical characteristics of a particular embodiment of the disclosed nanotubes. Figure 3 indicates that some embodiments of the disclosed nanotubes increase cellular proliferation. Figure 3 depicts cultured human bone marrow-derived mesenchymal stem cells, incubated for 72 hours, with 0.002 mg/mL of three particular embodiment of the disclosed nanotubes, featuring three distinct surface coatings, in 15% FBS -containing aMEM media. Figure 3 also demonstrates the benign cytotoxicity profile of particular embodiments of nanotubes.

[00120] In certain embodiments, the disclosed nanotubes may be used to treat murine fibroblast-like preadipocyte cells. In certain embodiments, the disclosed nanotubes may be used to treat human mesenchymal stem cells, prostate cancer cells, myeloma cells, multiple myeloma cells, T lymphocytes, natural killer cells, dendritic cells, macrophages and any other type of eukaryotic or prokaryotic cells, including but not limited to mammalian, fungal, plant and bacterial cells. Figure 16 depicts in bacterial cell culture, the growth kinetics of Escherichia coli colonies are not impacted by 24-hour incubation to a 0.02mg/mL concentrations of particular embodiment of nanotubes. This data indicates that particular embodiments of discrete nanotubes are benign for not solely mammalian cells, as the cells depicted in Figure 16 are bacterial.

[00121] Figure 9 shows that certain embodiments of the disclosed nanotubes are not genotoxic in vitro. 3T3L1 murine pre-adipocyte fibroblast cells were seeded into lOOmm dishes and allowed to rest for 48 hours before treatment. Cells were then treated with 0.05, 0.1 and 0.5mg/mL of discrete, biocompatibilized nanotubes for 120 hours (continuous treatment). 24 hours before harvesting the cells, one dish was treated with O.lug/mL of mitomycin C to be used as a positive control. Cocelmid was added at 0. lug/mL for 30 minutes to each dish before experiment was harvested to arrest cells in metaphase. Following cocelmid incubation, culture medium was collected into a l5mL conical to ensure no loss of loosely adherent mitotic cells. Cell were washed with lx PBS and trypsinized off of the plate and spun at 1,000 rpm for 5 minutes. Cells were resuspended and incubated in 10ML of 0.075M KCL hypotonic solution for 10 minutes before a 3: 1 methanol: glacial acetic acid fixative mixture was added to cells (lmL). Cells were spun down at 1,000 rpm for 5 minutes before being resuspended. lOmL of fixative was added and incubated for 20 minutes. This was repeated an additional two times. Slides were made, stained with gemsa, and cover slipped with cytoseal for analysis. For analysis, 100 metaphases were scored per concentration under a lOOx oil objective for chromosomal damage (breaks, gaps, rings, dicentrics, acentric, centromere spreading and exchanges). Chromosomal aberration testing showed no significant genotoxicity over a wide range of nanotube concentrations including about 0.05mg/ml, about O. lmg/ml, and about 0.5 mg/ml.

[00122] Figure 10 shows that certain embodiments of the disclosed nanotubes are not carcinogenic in vitro. HOS cells were seeded into 100 mm clear tissue culture treated plates at 100,000 cells per dish. Treatment of cells included: no treatment, 0.01 mg/mL of discrete, biocompatible nanotubes, 0.1 mg/mL discrete biocompatible nanotubes, 1 mg/mL discrete, biocompatible nanotubes and O. lug/mL 3-methylcholanthrene. Cells were treated continuously for 120 hours. Following treatment, cells were trypsinized and seeded at 20,000 cells per 60 mm dishes and there were five dishes per treatment group. Morphological cellular transformation was assessed by the presence of focus formation daily. If a focus was found, it was isolated by ring cloning and seeded into a 12 well plate for sub culturing. Dishes were stained with crystal violet after focus isolation and assess for foci not noticed under the microscope. Cells were observed for five passages which translated into sub culturing once a week for five weeks. Foci isolated were cultured into cell lines and used for testing anchorage independence in soft agar using Cytoselect cell transformation assay. Experimental results show that these nanotubes do not significantly transform cells to soft-agar proliferating colonies, which is indicative of tumorigenicity.

[00123] Intracellular and Extracellular Trafficking of The Disclosed Nanotubes

[00124] Various physical and chemical properties of the described nanotubes, as well as those of their surface coatings and/or payloads can influence the rate, duration, localization and concentration of trafficking inside, outside or to the surface of: cells, cell- derived materials (such as biofilms), tissues and biomaterials. Figure 4 shows the intracellular trafficking kinetics of certain embodiments of a particular embodiments of the disclosed nanotubes are influenced by the length and oxidation, wherein a subpopulation of nanotubes with an average length of 700nm and oxidation of 2.2% w/w showed significantly more rapid and concentrated internalization in human bone marrow-derived stem cells. Cells were seeded into a 96-well plate at a density of 7.0c10^L3 per well in MEM-Alpha medium (15% FBS, 1% anti-anti) and were incubated for 24-hrs before experimental treatments (day -1). At Day 0, day -1 medium was aspirated from wells and cells were then treated with fresh medium containing CNT complexes and incubated for 96 hrs. CNT complexes were formed by mixing various formulations of CNTs (1, 2, 3 and s) at two different weight ratios of DAPI to CNT (.2: 1 and .4: 1). Complexes were sonicated for 1 hr in waterbath sonicator before adding DSPE-PEG- NH2 at weight ratio of .55: 1 DSPE-PEG-NH2:CNT and another hour of sonication in waterbath sonicator. Solutions were centrifuged at 3.5k xg for 15 min and supernatants were used for experimentation. Cells were taken out every 24 hours (day 1, day 2, day 3, day 4) to acquire images under an inverted microscope at a total magnification of lOOx. Using a plate reader, the cells’ fluorescent excitation (380) and emission (455) was read every 24 hours and quantitated using [insert software] RFU readings were averaged (n=5). Figure 5 depicts fluorescence and brightfield micrographs from aforementioned study, depicting the increased fluorescent staining of nuclei evident for cells incubated with a particular embodiment of DAPI-loaded nanotubes with an average length of 700nm and 2.2% oxidation relative to those incubated with DAPI only, or DAPI-loaded embodiments of nanotubes with different average lengths and oxidations.

[00125] Figures 6 and 7 depict transmission electron micrographs of human bone marrow-derived mesenchymal stem cells incubated with a particular embodiments of the disclosed nanotubes for 72 hours. Cells were cultured 15% FBS -containing aMEM media supplemented with O. lmg/mL concentration nanotubes. Figure 6 shows that nanotubes are internalized, via endocytotic transport, within vesicular intracellular structures which traffic into the cytosol. Figure 7 provides an overview of the mechanism of intracellular transport of the same particular embodiment of disclosed nanotubes. Area 1 shows an early endosomal vesicle containing nanotubes, newly-formed near the surface of the plasma membrane. Area 2 shows a more mature vesicle, also containing nanotubes, which has deeper in the intracellular space. Area 3 shows a late-stage vesicle, in the process of releasing nanotubes as it breaks- down.

[00126] The disclosed nanotubes are useful for penetrating the cell membrane and/or transporting cargo molecules across the membrane. In some embodiments, the disclosed nanotubes localize within the nucleus, nucleoli, and/or actin cytoskeleton of a cell. This feature makes the disclosed nanotubes suitable for the controlled delivery of genetic materials or cell impermeable cargo molecules. Figure 8 shows the accumulation of nanotubes within PTK2 cells over time. Discrete biocompatible nanotube was labeled with a small peptide (QSYQAKANNYC) with a cysteine on the C terminus. Briefly 10 mg of discrete biocompatible CNT was coupled with 10 mg of peptide with 30 mg EDC for 2 hours at RT. The peptide-CNT complex was then treated with 1 mM DTT for 10 minutes to reduce the cysteines and then the peptide-CNT complex, following rinsing to remove the excess DTT, was labeled with 5 mg of tetramethyl rhodamine iodoacetamide (TMRIA) overnight and then rinsed 25x until there was nominal fluorescence in the supernatant. The resulting labeled product is further referred to as Rho-CNT. Human epithelial cells (PTK2 CCL— 56) were obtained from ATCC. The cells were grown in DME medium (ThermoFisher) supplemented with 10% calf serum (Sigma, St. Louis, MO), 50 pg/ml streptomycin and 50 U/ml penicillin. Cells were plated on glass coverslips 12—24 hours before the time-lapse studies. Just before experimentation the glass coverslips were sealed to 3D printed culture slides with clear silicone. Following addition of fresh media, the culture slide was sealed with a second glass coverslip on top.

[00127] Fluorescence microscopy was performed on two different inverted microscopes (configuration 1 : Lecia DM IRE 2 Fluorescent microscope with Leica DCFX Color CCD camera, Configuration 2: Keyence BZ-X700 fluorescent microscope with a 2/3inch, 2.83 million pixel monochrome CCD with 63x or lOOx oil objectives). Based on static time points of the endocytosis of Rho-CNT paraformaldehyde fixed cells, we determined the optimal time course for the video microscopy. Briefly, cells were incubated with Rho-CNT at time points 5, 15, 30, and 120 minutes. The Rho-CNT labeled cells were then rinsed with Tris Buffered Saline with 0.1% Tween 20 (TBST) or 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCh, pH 6.9; (PHEM buffer, Schliwa and van Blerkom, 1981) and then fixed with 3.3 % paraformaldehyde (Polysciences Inc., Warrington, PA) diluted with TBST for 5 min. Cells were imaged as described above. Preliminary evidence suggested that we should use Rho - CNT at a starting concentration of 10 pg/ml. Based on the optimal time course of endocytosis determined from the static fixed cells, we performed time- -lapse on a microscope stage. Images were taken at a low light level to reduce photo bleaching, F12K media was used reduce the auto fluorescence caused by phenol red in the DMEM. The images were stacked to provide a time-lapse video of the Rho-CNT endocytosis.

[00128] Figure 17 depicts Escherichia coli incubated with a particular embodiment of disclosed nanotubes loaded with Alexa647 fluorophore (ThermoFisher), at a concentration of 10mg/mL for 1 hour. Following incubation, cells were washed with PBS to remove nanotubes other than those bound to the bacterial cell wall and imaged by fluorescence microscopy. The punctate structures visible on the surface of the washed bacterial cells are indicative of a high affinity of the particular embodiment of disclosed nanotubes for cell walls of Escherichia coli.

[00129] Figure 18 depicts the incubation of Escherichia coli cells with three different particular embodiments of disclosed nanotubes, each with a distinct surface chemistry. The bacterial cells were incubated for 2 hours with lOpg/mL of each of the three types of nanotubes. These different nanotube surface chemistries resulted in a range of affinities for the bacterial cell wall, as indicated by the incorporation of nanotubes in the bacterial pellets following centrifugation. Nanotubes surface chemistries with high affinity of the surface of Escherichia coli were substantially enriched in the pellet and depleted from the supernatant. Conversely, nanotube surface chemistries with low affinity for the bacterial cell wall partitioned poorly to bacterial pellet following centrifugation, while the majority of nanotubes remained distributed in the supernatant. [00130] Figures 17 and 18 demonstrate the ability to use certain embodiments of the nanotubes as payload delivery systems targeted to specific types of cells and organisms, such as bacteria. This offers the possibility of using bacteria-targeted nanotubes as a selective delivery system for a payload in a heterogenous cell culture, such as a bioreactor containing gram-negative bacteria and another type of cell, such as gram-positive bacteria, or another type of organism, such as fungus.

[00131] Addition of Payload Molecules

[00132] Aqueous solubility of drug substances or other molecules is an important parameter in pre-formulation studies of a drug product. Several drugs are sparingly water- soluble and pose challenges for formulation and dose administration. Organic solvents or oils and additional surfactants to create dispersions can be used. If the payload molecule is easily dissolved or dispersed in an aqueous media, the filter cake need not be dried. If the payload molecule is not easily dissolved or dispersed in aqueous media, the filter cake is first dried at 80 °C in vacuo to constant weight. The payload molecule in the liquid media at the desired concentration is added to the discrete carbon nanotubes and allowed several hours to equilibrate within the tube cavity. The mixture is then filtered to form a cake, less than about 1 mm thickness, then the bulk of the payload solution not residing within the tubes are removed by high flow rate filtration. The rate of filtration is selected so that little time is allowed for the payload molecules to diffuse from the tube cavity. The filter cake plus payload drug is then subjected to an additional treatment if desired to attach a large molecule such an aqueous solution of a biopolymer, nucleic acid, oligonucleotide, amino acid, protein, peptide, enzyme, and/or combination thereof. Table 4 outlines anon-exhaustive list of payload molecules loaded on a particular embodiment of the disclosed nanotubes. The disclosed nanotubes may be loaded with a wide array of payload molecules. Table 4 shows the loading capabilities and customizability of a particular embodiment of the disclosed nanotubes. [00133]

Table 4

[00134] Nanotubes may serve as intracellular transport vehicles for molecular cargos involved in genetic engineering and modification of gene expression in all types of cells and organisms, including but not limited to mammalian cells, bacteria, algae, yeasts and plant cells. The physical and chemical properties may be modified to load and deliver diverse types of molecular cargos involved in genome engineering and expression modification including DNA sequences such as CRISPR, associated proteins/enzymes such as Cas9, Cpfl and C2cl/2/3, gRNAs and any other molecular machinery (protein, enzyme, nucleic acid, small molecule or polymer thereof) which interacts with cellular genetic material. In these applications, nanotubes may be provided to cultured cells, or delivered by any means of pharmacological administration to organisms, including watering, spraying, and soil deposition in the case of plants.

[00135] In some embodiments, surface modification of the nanotubes may be altered to improve loading depending on the class of molecular cargo being utilized. For example purposes only, Cas-9, Cpfl, C2cl/2/3, and/or CRISPR complex delivery and/or efficacy may benefit from covalent attachment to the nanotube surface or end of a PEG chain attached to the nanotube surface rather than relying on surface-surface interactions.

[00136] Biodegradable linkages may additionally or alternatively be utilized to improve loading or control the release of molecular cargo following successful cellular uptake of loaded discrete nanotubes. As one of many examples, an enzymatic degradable linkage between a peptide and discrete nanotubes or a nanotube-PEG complex that degrades following cellular uptake in the endosome or transition to lysosome may be used to control the release of the peptide or other molecular cargo.

[00137] Example 5

[00138] A calibration curve for the UV absorption of niacin as a function of the concentration of niacin in water was determined. A solution was prepared by mixing 0.0578 grams of discrete functionalized carbon nanotubes of this invention with 0.0134 grams of niacin in 25 ml of water [0.231 grams niacin/gram of carbon nanotube]. The tubes were allowed to settle and an aliquot of the fluid above the tubes removed hourly. The UV -vis absorption of this aliquot was measured and the resulting amount of niacin in the solution recorded. The amount of niacin in solution stabilized after 6 hours. A final sample was taken 20 hours after mixing. The difference between the amounts of niacin remaining in the solution and the original amount was determined to be the amount of niacin associated with the discrete functionalized carbon nanotubes. It was found that 0.0746 grams of niacin associated with each gram of carbon nanotubes. The total amount of niacin absorbed by the carbon nanotubes was 0.0043 grams. Assuming an average carbon nanotube length of 1,000 nm, external diameter of 12 nm and internal diameter of 5 nm, the available volume within the tube is 0.093 cm³ per gram of carbon nanotubes. Since the density of niacin is 1.473 g/cm³, then the maximum amount of niacin that can fit in the tubes is 0.137 grams. Therefore, the measured absorption of 0.0746 g niacin / g CNT amount could be confined to the interior of the tube.

[00139] Example 6

[00140] A poly (vinyl alcohol), PVOH, is sufficiently large (30 kDa-70 kDa) that it cannot be absorbed internally in a carbon nanotube. PVOH is used as a surfactant for carbon nanotubes because it associates and wraps the exterior of the carbon nanotube. In this experiment, PVOH was added to a mixture of 0.0535 g of carbon nanotubes and 0.0139 g niacin (0.26 grams niacin to 1 gram carbon nanotubes) in 25 ml water. This was allowed to rest overnight. Using the UV-vis technique of Example 5, the amount of niacin associated with the carbon nanotubes was determined to be 0.0561 grams niacin per gram of carbon nanotubes, less than the 0.0746 grams in Example 5. The total amount of niacin absorbed was 0.003 grams.

[00141] Calculations were made assuming carbon nanotube length of 1,000 nm, external diameter of 12 nm and internal diameter of 5 nm. Given the density of PVOH is 1.1 g/cm³ and the ratio of PVOH to carbon nanotubes was 0.23 to 1, the average layer thickness of PVOH on the carbon nanotube is 0.6 nm. Therefore there is sufficient PVOH to encapsulate the carbon nanotube and displace any niacin on the surface of the tube and the measured amount of 0.0561 grams of niacin per gram of carbon nanotubes is in the interior of the carbon nanotube.

[00142] Delivery of Biomolecules to Cells Ex Vivo

[00143] The apoptosis inducing peptide KLA or KLAKLAK is known to disrupt mitochondrial membranes but has a poor cell penetrating potential. Figures 11 and 12 shows the results of an experiment in which DAPI stained cells were treated with various agents and compared to an untreated control group (CON). The agents included KLA peptide only, nanotubes only, and a composition of discrete nanotubes loaded with KLA. The micrograph in Figure 11 shows that cells treated with nanotubes not loaded with KLA remain in-tact and healthy, as indicated by their attached morphology, membrane integrity and culture density (left panel), while cells treated with KLA-loaded nanotubes are lysed and dead (right panel). As can be seen in Figure 12, the number of cells remaining after treatment with either KLA or discrete nanotubes individually was only slightly different from the untreated control group. In fact, cells treated with nanotubes only showed improve growth rates. This shows that neither KLA nor discrete nanotubes exhibits significant cytotoxicity in isolation. Figure 12 also shows that there were no viable cells remaining after treatment with KLA loaded discrete nanotubes. KLA loaded discrete nanotubes caused complete apoptosis of the cells within 72 hours. Discrete biocompatible nanotube was loaded with KLAKLAK by mixing of 1 mg/ml of discrete biocompatible nanotube + 30uM of lmg/ml KLAKLAK (0.230 mL) in 4.23mL of pH 10 25mM bicarbonate-carbonate buffer on a rocker overnight at room temperature.

[00144] Seeded 30,000 LNCaP cells per 96 well in tissue culture treated plate. Let cells sit for 24-48 hours. Following seeding, LNCaP cells were treated with the following groups:

[00145] a) Negative control (no treatment, just LNCaP cells alone)

[00146] b) KLAKLAK only treatment (no CNTs)

[00147] c) discrete biocompatible CNT only (no KLAKLAK)

[00148] d) KLAKLAK + discrete biocompatible CNT (loaded)

[00149] Resuspended discrete biocompatible CNT + KLAKLAK in 2 mLs of RPMI 1640 medium and transferred 0.2 mL to 10 wells containing LNCaP cells. Mixed 0.537 mL of lmg/ml of discrete biocompatible nanotube with no KLAKLAK into 2 mLs of RPMI 1640 medium and treated 10 wells containing with 0.2 mL each. Mixed 0.23 mL of 1 mg/ml KLAKLAK to 4.7 mL of RPMI 1640 media and treated 10 wells with 0.2 mL each. Cells were then incubated for 72 hours at 37C, 5% CCh and then harvested and analyzed for apoptosis through DAPI/Phalloidin 488 staining. Representative photos were taken for each well and cells that were positive for DAPI staining were counted using ImageJ software. [00150] This procedure shows the massive improvement in the efficacy of KLA once it is transported into the cell using discrete nanotubes. Figure 11 shows an image of healthy cells after treatment with discrete nanotubes only and an image of cells following complete apoptosis after treatment with KLA loaded discrete nanotubes.

[00151] Figure 13 depicts a similar experiment, human embryonic kidney (HEK) cells engineered to express the GFP gene for green fluorescent protein were seeded in l2-well plates and proliferated for 24 hours. Experimental groups included: no treatment (control), siRNA only (50nM), discrete biocompatible CNT only (unloaded), siRNA (50nM) + discrete biocompatible CNT, siRNA (50 nM) + transfection reagent. siRNA for GFP was loaded onto discrete biocompatible CNT at a weight ratio of 0.05 siRNA: 1 discrete biocompatible CNT, and treated with a sufficient mass of loaded discrete biocompatible CNT to deliver 50nM concentration of siRNA. Cells in the“discrete biocompatible CNT only” group were treated with an equivalent mass of unloaded discrete biocompatible CNT used to deliver siRNA in“siRNA+ discrete biocompatible CNT” group. Once cells atained 85% confluency, each group was maintained in OptiMEM media for 72 hours. Following the transfection period, each experimental group was imaged at lOx by fluorescent microscopy in order to visualize GFP expression. Cellular mRNA was extracted for qPCR analysis of GFP expression, using the following primer to detect GFP.

[00152] GFP gene expression was evaluated relative to the expression of beta actin, ACTB gene. Figure 13 shows GFP expression relative to ACTB confirming that discrete nanotubes facilitate intracellular delivery of siRNA, thereby reducing transcription level expression of a targeted gene.

[00153] Figures 14 and 15 demonstrate dose-dependent intracellular delivery of mRNAto human immune cells, T-lymphocyte and monocyte-like cells, respectively. Cell lines used in the experiment were Jurkat E6-1 clone (P6) from ATCC (TIB-152) and U937 monocyte-like cells also sourced from (ATCC). Cells were seeded into a l2-well plate at a density of 2.0c10^L5 cells per well in RPMI-1640 medium (w/ HEPES, 10% FBS, 1% Anti- Anti) and were incubated for 24-hrs before experimental treatments (day -1). At Day 0, cells were treated with CNT complexes. CNT complexes were formed by sonicating a mixture of 2: 1 weight ratio of eGFP mRNA with CNT for one hour in waterbath sonicator. The eGFP mRNA treatment concentration was 500ng/mL at day 0 for control and lx groups. Concentration was increased with 2.5x and 5x groups while maintaining 2: 1 weight ratio of mRNA: CNT.

[00154] After 72 hours, cells were aspirated and spun down at lOOOg for 5 minutes using a microcentrifuge. Supernatant was then discarded. Cell pellets were then washed using PBS and were then resuspended in 250ul of Trizol Reagent. 30-50ul of Chloroform was then added to homogenate. Homogenate was then spun for 15 minutes at l2,000g. Subsequent aqueous phase was then removed and placed into new microcentrifuge tube. Aqueous phrase was mixed with equal volume of isopropanol and frozen in -80c for 20 minutes to precipitate RNA. Precipitate was then spun down for 15 minutes at l2,000g. Precipitate was then was twice in ethanol for 15 minutes at l2,000g. RNA pellet was resuspended in DEP-C water and analyzed with NanoDrop for concentration analysis. Reverse Transcription was performed using High-Capacity cDNA Reverse Transcription Kit (applied biosystems # 4374966). Gene-expression analysis by qPCR was performed using Taqman multipled master mix and ThermoFisher’s eGFP and GAPDH verified primers. In both cases, MGMR loaded with mRNA provided the highest-efficiency transport of the oligonucleotides into the T cells and macrophages, respectively.

[00155] Modulating Biodistribution and Controlled Release of Loaded Discrete Nanotubes

[00156] In some embodiments, the nanotubes described herein have altered surface chemistry which allows for control of the biodistribution of nanotubes and the controlled penetration and delivery of payload molecules.

[00157] Nanotube surface chemistry may be altered by functionalizing the surface and/or coating the nanotubes. In some embodiments, PEG is used to functionalize the nanotube surface. The density and type of functionalization used, including the type of terminal group and/or terminal charge of any species attached to a nanotube influences aspects of the biodistribution, drug delivery: drug-loading, cell membrane penetration efficiency, cell- specific uptake and drug off-loading of the disclosed nanotubes and payload molecules.

[00158] In certain embodiments, PEG is covalently linked to the surface of the nanotubes. In particular embodiments, PEG is covalently attached to the surface of nanotubes using a thionyl chloride addition between the hydroxyl group of the PEG polymer and the carboxylic acid groups of an oxidized nanotube. In some embodiments, controlling the degree and location of nanotube oxidation allows for controlling of the degree and location of surface coating. In other embodiments, altering the surface chemistry of the nanotubes may be accomplished using a bio-compatible polymer, surface coating, and/or functionalizing agent other than PEG. In some embodiments, it is possible to attach various types of ligands or other molecules, including but not limited to carbohydrates, that interact preferentially with the cell, cell membrane, or cell surface to improve uptake of discrete nanotubes into the cell and/or cell nucleus.

[00159] In other embodiments, amphiphilic Poly(ethylene glycol) surfactant (DSPE-PEG) may, additionally or alternatively, be non-covalently attached to the nanotube surface through hydrophobic interactions between a phospholipid chain and hydrophobic pockets found on the surface of the nanotubes which are void of oxidation. Changing the surfactant ratio or terminal functional group of the PEG has been shown to cause changes in the biodistribution drug-loading and cell-specific uptake of the nanotubes. Such changes may also alter intracellular targets of the PEG-Nanotube complex. Merely as one example, changing from a methyl terminated PEG to a primary amine terminated PEG has been shown to cause a large change in biodistribution. As can be seen in Figures 18 and 19, when this change was tested, liver retention decreased and bone accumulation increased nearly 4 times. In Figures 18 and 19, the change from 2 to 3+ involved the addition of a primary amine, instead of a methyl group, to the DSPE-PEG surfactant which was used to disperse and functionalize the discrete nanotubes.

[00160] Specific ranges of PEG density on the nanotube surface are useful for biocompatibilization and may assist in maintaining nanotubes in a discrete form in the blood and tissues during delivery in vivo or in vitro. In some embodiments, the w/w range of PEG:Nanotube of covalently linked PEG may be as low as about 1%, about 3%, about 5%, about 7%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%. The w/w range of PEG:Nanotubes of covalently linked PEG may be as high as about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 13%, about 15%, about 17%, or about 19%. The range of covalently linked PEGmanotubes is preferably about 9-11% w/w.

[00161] In some embodiments, the weight ratios of PEG surfactant to discrete nanotubes of non-covalently attached PEG surfactant may be as low as about 0.01: 1, about 0.05: 1, about 0.1: 1, about 0. l5: l, about 0.2: l, about 0.25: l, about 0.3: l or about 0.4: 1. In some embodiments, the weight ratios of PEG surfactant to discrete nanotubes of non-covalently attached PEG surfactant may be as high as about 0.5: 1, about 0.6: 1, about 0.7: 1, about 0.75: 1, about 0.8: 1, about 0.85: 1, about 0.9: 1, or about 1 : 1. The weight ratios of PEG surfactant to discrete nanotubes of non-covalently attached PEG surfactant is preferably between about 0.2: 1 and 0.8: 1 and more preferably is about 0.5: 1.

[00162] The process of forming discrete nanotube/PEG dispersions may also be modified to control the type of drug-loading. Adding a desired drug and PEG to a discrete nanotubes sample in a single step or forming drug-encapsulating micelles prior to loading allows for the formation of drug-loaded micelles which may associate with the discrete nanotube surface in some embodiments. This type of drug-loading may produce different off loading characteristics and release kinetics relative to drug loaded on the discrete nanotubes following treatment with PEG.

[00163] Both lipid and non-lipid based polymeric surface coatings, including but not limited to PEG-based molecules, may be used to control the rate and quantity of cellular uptake of discrete nanotubes, or the loading and/or unloading of payload molecules. Additionally, chemical modifications to the discrete nanotube surface can increase or decrease the ability of the disclosed compositions to associate with or penetrate the cellular nucleus.

[00164] Molecules which undergo chemical or physical changes in response to changes in physiological conditions (temperature, ionic concentration, pH) may be incorporated on discrete nanotube surface in order to further regulate delivery. For example, a pH-sensitive polymer which decomposes at acidic pH (below 7.4) may allow for selective delivery of drugs to acidic or tumor like environments.

[00165] In some embodiments, zwitterionic molecules, including but not limited to carboxybetaine phosphoryl choline, and/or polymers therefrom, may be employed to regulate drug delivery: drug-loading, biodistribution, tissue/organ targeting, drug off-loading and/or clearance.

[00166] In some embodiments, chemicals, compounds, and/or small molecules, useful for scanning, imaging and/or diagnostics may be loaded onto discrete nanotubes alternatively or in addition to drugs, chemicals, compounds, and/or small molecules. These embodiments may be useful for monitoring, confirming, and/or quantizing the delivery of drug to a particular target.

[00167] Intracellular Delivery for Agriculture

[00168] As shown in Figures 12, 13, 14, 15 and 17 the disclosed nanotubes may be used to transport nutrients, genetic material, and gene-editing complexes for biological processes used in therapeutic, bio-industrial and agricultural settings. In some embodiments, the disclosed nanotubes may be administered to com, wheat, soybeans, rice, beans, algae, switch grass, hemp, linseed, bamboo, cotton, papyrus, sisal, borage, cannabis, Echinacea, Artemisia, tobacco, maize, potatoes, coffee, tea, cocoa, coconut palms, plantains, yams, sorghum, sweet potatoes, and cassava. The disclosed tubes may also be used to deliver pesticides to insects, bacteria, yeasts, or any other organism which feed on or otherwise associate with the plant.

[00169] Intracellular Delivery in Bio-manufacturing and other Industrial Processes

[00170] Figures 4,5, 8, 12, 13, 14, 15, 17 and 18 depict an example of how carbon nanotubes may be used for molecular delivery to cells involved in bio-manufacturing or other industrial processes wherein cells produce an industrial or diagnostic output. These cells maybe prokaryotic or eukaryotic, derived from mammalian, fungal, bacteria, plant or other types of organisms.

[00171] Embodiments

[00172] Embodiments disclosed in this application include at least: A composition comprising a plurality of discrete carbon nanotubes, wherein the discrete carbon nanotubes comprise an interior and exterior surface, the interior surface comprising an interior surface oxidized species content and the exterior surface comprising an exterior surface oxidized species content, wherein the interior surface oxidized species content comprises from about 0.01 to less than about 4 percent relative to carbon nanotube weight and the exterior surface oxidized species content comprises more than about 1 to about 10 percent relative to carbon nanotube weight, wherein a biocompatible and/or bioactive surface coating is attached and/or attracted to at least a portion of at least one surface of the discrete carbon nanotubes. The composition of embodiment 1, wherein the biocompatible surface coating is selected from the group consisting of PEG (polyethylene glycol), PLA (polylactic acid), PVOH (polyvinyl alcohol), PEO (polyethylene oxide), PGLA (poly glycolic acid), CMC (carboxymethyl cellulose), PVP (polyvinylpyrrolidone), PAA (polyacrylic acid), zwitterionic polymers, lipids, phospholipids, nucleic acids, amino acids, peptides, polysaccharides and proteins. The composition of embodiment 1, wherein the biocompatible surface coating is a polyethylene glycol. The composition of embodiment 3, wherein the terminal group of the polyethylene glycol is a methyl group. The composition of embodiment 3, wherein the terminal group of the polyethylene glycol is a primary amine. The composition of embodiment 3, wherein the polyethylene glycol is covalently linked to the exterior surface. The composition of embodiment 3, wherein the polyethylene glycol is a surfactant and is non-covalently linked to the exterior surface. The composition of embodiment 6, wherein the w/w range of polyethylene glycol to discrete carbon nanotubes is between about 7% to about 13%, preferably between about 9% to about 11%. The composition of embodiment 7, wherein the weight ratio of polyethylene glycol surfactant to discrete carbon nanotubes is between about 0.05:1 to about 1 : 1, preferably between about 0.2: 1 to about 0.8: 1. The composition of embodiment 1, further comprising at least one type of payload molecule. The composition of embodiment 10, wherein the payload molecule is at least partially atached to at least one surface, exterior and/or interior, of the discrete carbon nanotubes. The composition of embodiment 10, wherein the payload molecule is selected from the group consisting of PEG (polyethylene glycol), PLA (polylactic acid), PVOH (polyvinyl alcohol), PEO (polyethylene oxide), PGLA (polyglycolic acid), CMC (carboxymethyl cellulose), PVP (polyvinylpyrrolidone), PAA (polyacrylic acid), aminoacids, peptides, polysaccharides and proteins. The composition of embodiment 10, wherein the payload molecule is selected from the group consisting of small molecules, drugs, medicines, surfactants, composites, organic nanoparticles, inorganic nanoparticles, fluorescent tracers, radiotracers, radiotherapy molecules, diagnostic imaging molecules, amino acids, proteins, peptides, polysaccharides, nucleic acids, carbohydrates, lipids, glycosaminoglycans, proteoglycans, glycoproteins, steroids, antibodies, antibody fragments, growth factors, viral vectors, genetic materials, gene-editing complexes, micelles, liposomes, vesicles, cell-derived membranes, extracellular matrix components, and combinations thereof. The composition of embodiment 10, wherein the payload molecule has a molecular weight of less than about 10,000 Daltons. The composition of embodiment 1, further comprising a pH sensitive polymer attached to at least one surface of the discrete carbon nanotubes. The composition of embodiment 1, further comprising electromagnetic species such as iron or its oxides attached to at least one surface of the discrete carbon nanotubes. The composition of embodiment 1, wherein the biocompatible surface coating has a molecular weight greater than about 30,000 Daltons. The composition of embodiment 1, wherein the nanotubes are sterile. The composition of embodiment 1, wherein the nanotubes have an average length of between about 800nm and about 900nm and an average diameter of between about l2nm to about l4nm. The composition of embodiment 1, wherein the nanotubes are at least about 99.8 percent pure by weight. The composition of embodiment 1, wherein the biocompatible surface coating is derived from a polyethylene glycol precursor. The composition of embodiment 1, wherein the biocompatible surface coating is derived from CRISPR-based gene editing technology and any combination of associated proteins/enzymes such as Cas9, Cpfl and C2c 1/2/3, and/or gRNAs. The composition of embodiment 1, wherein the biocompatible surface coating is derived from a carboxy betaine precursor. The composition of embodiment 1, wherein the biocompatible surface coating is derived from a phosphoryl choline precursor. The composition of embodiment 1, wherein the biocompatible surface coating is derived from a zwitterionic moiety. A payload molecule delivery system composition comprising discrete oxidized carbon nanotubes, at least one payload molecule, and at least one type of biocompatible surface coating, wherein the distribution of aspect ratios of the discrete carbon nanotubes is bimodal. The composition of any embodiment above wherein the average length of the discrete carbon nanotube is in the range 10 to 1200 nanometers, preferably 50 to 900 nanometers and most preferably between 150 and 800 nanometers.

Claims

CLAIMS What is claimed is:

1. A composition comprising a plurality of discrete carbon nanotubes, wherein the discrete carbon nanotubes comprise interior and exterior surfaces, the interior surface comprising an interior surface oxidized species content and the exterior surface comprising an exterior surface oxidized species content, wherein the interior surface oxidized species content comprises from about 0.01 to less than about 1 percent relative to carbon nanotube total weight and the exterior surface oxidized species content comprises more than about 1 to about 10 percent relative to carbon nanotube total weight, wherein a biocompatible surface coating is attached to at least a portion of at least one surface of the discrete carbon nanotubes, and at least one type of payload molecule is attached to at least one surface of the nanotubes.

2. The composition of claim 1 wherein the average length of the discrete carbon nanotube is in the range of about 10 to about 1200 nanometers.

3. The composition of claim 1, wherein the biocompatible surface coating is derived from a precursor selected from PEG (polyethylene glycol) or derivatives, PLA (polylactic acid) or derivatives, PVOH (polyvinyl alcohol) or derivatives, PEO (polyethylene oxide) or derivatives, PGLA (polyglycolic acid) or derivatives, CMC (carboxymethyl cellulose) or derivatives, PVP (polyvinylpyrrolidone) or derivatives, PAA (polyacrylic acid) or derivatives, carboxy betaine precursor or derivative, phosphoryl choline precursor or derivative, zwitterionic moieties, amino acids, peptides, proteins, enzymes, polysaccharides, nucleic acids, oligonucleotides, antibodies, adjuvants, nanoparticles, lipids, vesicles, liposomes, micelles, viral-derived components, cellular plasma membrane-derived components, extracellular matrix derived-components, other cell- derived components and combinations thereof.

4. The composition of claim 3, wherein the polyethylene glycol or derivative precursor is covalently attached to the exterior surface.

5. The composition of claim 3, wherein the polyethylene glycol or derivative precursor is non-covalently attached to the exterior surface.

6. The composition of claim 3, wherein the weight ratio of polyethylene glycol to discrete carbon nanotubes is between about 7% to about 13%.

7. The composition of claim 3, wherein the weight ratio of polyethylene glycol surfactant to discrete carbon nanotubes is between about 0.05: 1 to about 1 : 1.

8. The composition of claim 1 wherein the payload molecule is selected from the group of small molecules less than about 1000 daltons, metal oxides, surfactants, polymers, composites, organic and inorganic nanoparticles, fluorescent tracers, radiotracers, peptides, proteins, enzymes, nucleic acids, oligonucleotides, carbohydrates, lipids, glycosaminoglycans, proteoglycans, glycoproteins, steroids, antibodies, growth factors, viral components, viral vectors, genetic materials, CRISPR-based gene editing complexes, transcription activator-like effector nucleases, zinc finger nucleases, adjuvants, micelles, liposomes, vesicles, cell-derived components, recombinant proteins, tissues, genes, allergens, blood components, molecules derived from biological materials, vaccines, adjuvants, biologic drugs, tracer entities and combinations thereof.

9. A payload molecule delivery system composition comprising discrete oxidized carbon nanotubes, at least one type of payload molecule, and at least one type of biocompatible surface coating, wherein the at least one type of biocompatible surface coating is attached to at least a portion of at least one surface of the discrete carbon nanotubes and wherein the at least one type of payload molecule is attached to the biocompatible surface coating.

10. The payload molecule delivery system composition of claim 9 wherein a distribution of aspect ratios of the discrete carbon nanotubes is bimodal.

11. The payload molecular delivery system composition of claim 9 wherein a biocompatible surface coating is attached to at least a portion of at least one surface of the discrete carbon nanotubes, and at least one type of payload molecule attached to at least one surface of the nanotubes further comprising cells wherein the cell type is selected from the group of mammalian, bacterial, fungal, plant cells and protoplasts, wherein the cell type has a doubling time in the range of about one minute to about seventy two hours.

12. The payload molecular delivery system composition of claim 11 wherein the cell doubling time of the cells is increased or decreased by at least 10% compared to about the same composition without a plurality of discrete carbon nanotubes.

13. The payload molecular delivery system composition of claim 9 further comprising cells wherein the cell type is selected from the group of mammalian, bacterial, fungal, plant cells and protoplasts wherein the biocompatible surface coating of the discrete carbon nanotubes is selected from the group of oligonucleotides, cholesterol derived molecules, lipid-derived molecules, glucosaminoglycans, glycoproteins, peptides, proteins, enzymes, antibodies, adjuvants, cytokines, cell-derived components, viral-derived components and viral vectors to target a molecular targets on the cell surface selected from the group of lipids, proteins, enzymes, carbohydrates, glycoproteins, glycolipids, nucleic acids, oligonucleotides, RNA, DNA, cholesterols and cholesterol-derived molecules.