Nanotube composition and method for promoting stem cell growth

Abstract

This application relates to compositions and methods useful for growing living cells such as stem cells. The compositions utilize a mixture of an extracellular matrix and discrete carbon nanotubes. The extracellular matrix may also contain components selected from a class of proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof. The discrete carbon nanotubes are typically present in less than about 1% by weight of the dry weight of the total composition.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Application No. 63 / 521,451, filed on June 16, 2023, which is incorporated herein by reference.

[0002] Field of the Disclosure The present disclosure generally relates to compositions and methods using an extracellular matrix (ECM) containing discrete carbon nanotubes less than about 1 wt% to promote the growth of live cells and / or increase the lifespan of live cells.

Background Art

[0003] There is a great need for the growth of live cells, such as stem cells, and / or an increase in the lifespan of live cells, among other things. The potential of pluripotent stem cells as a treatment for a wide variety of diseases and injuries has been established in recent years. Pluripotent stem cells can differentiate into local cell types, divide, and ultimately regenerate diseased or wounded tissues when transplanted into a tissue or organ system.

[0004] One problem faced by both the development and implementation of stem cells as a treatment is their relatively slow growth rate. In order for a treatment to be successful, stem cells must be grown in large numbers in order to regenerate tissue and integrate into existing organ systems. The amount of time required for primary stem cells to grow to a usable level is a major bottleneck in both stem cell treatment and research.

[0005] The extracellular matrix (ECM), used to grow living cells such as stem cells, consists of materials including, but is not limited to, fibrous proteins, macromolecules, sugars, lipids, and cell signaling molecules, and plays a major role in the health, differentiation, and proliferation of stem cells. The biochemical and biophysical properties of the ECM are carefully regulated by cells and tissues to yield favorable results in cell behavior and growth. The physical and chemical properties of the extracellular matrix, such as mechanical strength, stiffness, and conductivity, as well as the concentrations of a wide range of growth factors and signaling molecules, can all influence cell-to-cell and cell-to-matrix interactions.

[0006] The extracellular matrix (ECM) is a non-cellular component present in all living tissues and organs, providing not only an essential physical scaffold for cellular components but also initiating crucial biochemical and biomechanical signals necessary for tissue morphogenesis, differentiation, and homeostasis. The importance of the ECM is demonstrated by a wide range of syndromes arising from genetic abnormalities in ECM proteins, which can range from mild to severe. While fundamentally composed of water, proteins, and polysaccharides, each tissue possesses an ECM with a unique composition and topology, generated during histogenesis through dynamic and reciprocal interactions of biochemical and biophysical properties between various cellular components (e.g., epithelial, fibroblasts, adipocytes, endothelial elements) and the evolving cellular-protein microenvironment. Indeed, the physical, topological, and biochemical composition of the ECM is not only tissue-specific but also remarkably heterogeneous. Cell adhesion to the ECM is mediated by ECM receptors such as integrins, discoidin domain receptors, and syndecans. Adhesion mediates cytoskeletal coupling with the extracellular membrane (ECM) and is involved in cell migration through the ECM. Furthermore, the ECM is a highly dynamic structure that is constantly being remodeled enzymatically or non-enzymatically, and its molecular components undergo countless post-translational modifications. Through these physical and biochemical properties, the ECM generates the biochemical and mechanical properties of each organ, such as its tensile and compressive strengths, as well as elasticity, and mediates protection through buffering that maintains extracellular homeostasis and water retention. In addition, the ECM directs essential morphological organization and physiological functions by binding to growth factors (GFs) and interacting with cell surface receptors to induce signal transduction and regulate gene transcription. The biochemical and biomechanical properties, as well as protective and histological properties, of the ECM in a given tissue can vary significantly from tissue to tissue (e.g., lung vs. skin vs. bone), even within a single tissue, and from one physiological state to another (normal vs. cancerous).

[0007] Modifications to the endocrine cortex (ECM) may have beneficial effects on implanted stem cells. However, it is crucial that these modifications are precisely and carefully tailored, taking into account the sensitivity of stem cells to the properties of their local environment. Furthermore, modifications to the ECM would best be carried out by general-purpose additives or methods that can simultaneously alter multiple properties without affecting toxicity.

[0008] Changes in the extracellular matrix (ECM) can support normal cellular behavior or induce cancer and other harmful conditions, thus potentially contributing to the health of implanted cells. External modifications of the ECM can induce positive effects such as eliminating disease conditions in surrounding cells and tissues or increasing beneficial parameters of a given microenvironment.

[0009] The discrete carbon nanotubes of the present invention can be incorporated into the extracellular matrix (ECM) to improve cell proliferation rates. Conversely, commercially available carbon nanotubes can present several problems in biological applications. Products such as CNano Flowtube carbon nanotubes are typically produced in bundles of 2–500 μm in size, where the carbon nanotubes are highly entangled. Single-walled carbon nanotubes are energetically favored to align and form ropes, which contain thousands of single-walled carbon nanotubes. These ropes can entangle with each other to form bundles. Finally, metal catalysts required for carbon nanotube synthesis can be contained within the bundles, potentially introducing elements that are toxic to cells and tissues.

[0010] U.S. Patent No. 10,414,655 by Bosnyak et al. discloses stem cell, bone, and nerve scaffolds containing discrete carbon nanotubes in a weight fraction exceeding 1% of the scaffold's weight. The discrete carbon nanotubes have targeted or selective oxidation levels and / or content both inside and outside the tube walls, as described in U.S. Patent No. 10,414,656, incorporated herein by reference. The invention disclosed herein differs from U.S. Patent No. 10,414,655 in that the amount of discrete carbon nanotubes used to enhance the ECM may be less than about 1% by weight, preferably less than about 0.1% by weight, of the extracellular matrix or scaffold, and yet it exhibits unexpectedly enhanced cell growth. At less than about 1% by weight of the extracellular matrix, the discrete carbon nanotubes can be dispersed such that individual carbon nanotubes do not come into contact with other discrete carbon nanotubes to form a support structure or scaffold.

[0011] In this embodiment, novel compositions and methods are provided that result in a substantial increase in cell growth using relatively small amounts of nanotubes. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent No. 10,414,655 [Patent Document 2] U.S. Patent No. 10,414,656 [Overview of the project]

[0013] A composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes is disclosed herein, wherein the extracellular matrix comprises components selected from the class of proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof, and the discrete carbon nanotubes are less than about 1% by weight of the dry weight of the total composition. The composition may further contain living cells, which may be stem cells or primary stem cells.

[0014] At least a portion of the extracellular matrix, or at least a portion of the discrete carbon nanotubes, or both, can be at least partially oriented to promote directed growth of living cells.

[0015] In the composition, at least a portion of the discrete carbon nanotubes may be physically attached to or chemically bonded to at least one component of the composition.

[0016] The composition can be subjected to static current, oscillating current, static voltage, oscillating voltage, or any combination thereof to stimulate the growth of living cells.

[0017] A composition comprising a mixture of extracellular matrix and discrete carbon nanotubes may contain discrete carbon nanotubes substantially separated from other discrete carbon nanotubes in the extracellular matrix.

[0018] A composition comprising a mixture of extracellular matrix and discrete carbon nanotubes further comprises a dispersant that substantially prevents the aggregation of the discrete carbon nanotubes. The dispersant may be physically or chemically bonded to the discrete carbon nanotubes and can be selected from the classes of anionic, cationic, nonionic, and zwitterionic molecules.

[0019] The composition may further comprise at least a portion of discrete carbon nanotubes that further comprise molecules having a different composition and structure from those of the extracellular matrix, where the molecules are at least partially bound to the nanotube surface and are selected from the class of molecules that function as antibacterial agents, growth factors, growth stimulants, nutrients, or combinations thereof.

[0020] Additional fillers such as, but not limited to, graphene, graphene oxide, carbon nanofibers, cellulose nanofibers, hydroxyapatite, silk, and mixtures thereof can be added to the composition. These additional fillers can serve to vary the stiffness or porosity of the ECM. The discrete carbon nanotubes can associate with these additional fillers.

[0021] The composition can be in the form of an open-cell foam, a closed-cell foam, a film, a strand, a particle, or a mixture thereof. A closed-cell foam structure is preferred to allow nutrients and air to reach the living cells.

[0022] The composition may further comprise water.

[0023] A method for growing living cells with the composition of the present invention is disclosed.

[0024] The composition is useful for the treatment of injuries, burns, wound care, sensors, bone, skin, muscle, nerve, blood, and organs for accelerating the repair of damage as compared to a similar composition of the extracellular matrix that does not contain discrete carbon nanotubes, but is not limited thereto. The composition can be in any suitable form for application and can be molded, injected, implanted, or fitted.

[0025] The composition can comprise discrete carbon nanotubes (dCNT) that are single-walled (dSWCNT), double-walled (dDWCNT), or multi-walled (dMWCNT), which may or may not be oxidized or functionalized.

[0026] To facilitate a more complete understanding of the present invention, reference is now made to the accompanying drawings, which are not to be construed as limiting the invention, but are intended only to illustrate different aspects and embodiments of the invention.

Brief Description of the Drawings

[0027] [Figure 1] Shows the improvement of cell proliferation experienced by stem cells when incubated in a 3D ECM-like matrix containing dMWCNT. One sample of mesenchymal stem cells was grown in Vitrogel, and the other sample was grown in Vitrogel incubated with dMWCNT at a concentration of 0.001 mg / ml. Each condition was seeded in a 24-well plate at a cell concentration of 5.0x10^5 per well and then incubated in a 37°C, 5% CO2 incubator for 10 days. Subsequently, viable cells were extracted and counted using a bioactive colorimetric assay (WST-8). This assay uses a water-soluble tetrazolium salt to quantify the number of live cells by generating an orange formazan dye through bioreduction in the presence of an electron carrier, which is a marker of metabolic activity.

[0028] [Figure 2] Shows how the health and growth rate of stem cells increase when incubated in a 3D ECM-like scaffold injected with dMWCNT. In this example, the dMWCNT is dispersed in DSPE-PEG(NH2) at a CNT:PEG ratio of 1:0.55. For comparison, stem cells incubated in a 3D ECM-like scaffold containing only DSPE-PEG(NH2) without dMWCNT are shown. The scale bar is 100 μm, and all conditions were incubated for 9 days before imaging.

[0029] [Figure 3] Shows bright-field microscopy images of each concentration of dMWCNT in Vitrogel.

[0030] [Figure 4]This shows single-walled carbon nanotubes non-covalently dispersed in DSPE-PEG(NH2). [Modes for carrying out the invention]

[0031] Next, exemplary embodiments of the present invention will be described to illustrate various features of the present invention. The embodiments described herein are not intended to limit the scope of the present invention, but rather to provide examples of the components, uses, and operations of the present invention.

[0032] Furthermore, the features, advantages, and characteristics described in the embodiments can be combined in any suitable manner. Those skilled in the art will recognize that embodiments may be implemented without one or more of the specific features or advantages of the embodiments. In other examples, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. This embodiment generally relates to a mixture of an extracellular matrix containing components selected from proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof, and discrete carbon nanotubes, wherein the discrete carbon nanotubes constitute less than about 1% of the dry weight of the total composition, preferably less than about 0.05% of the dry weight of the total composition, more preferably less than about 0.01% of the dry weight of the total composition, and most preferably less than about 0.001% of the dry weight of the total composition.

[0033] The discrete carbon nanotubes (dCNTs) used in the present invention, which function as additive components of the extracellular matrix, may consist of single-walled, double-walled, or multi-walled graphene shells, or mixtures thereof.

[0034] Discrete carbon nanotubes (dCNTs) refer to carbon nanotubes that have a clearly defined and measurable length and can be made unbound or unentangled so as to be substantially separated from each other along the length of the carbon nanotube. Discrete carbon nanotubes can be individually dispersed in a given medium by selecting the thermodynamic interactions of the tube surface, the medium, and the tube concentration in the medium.

[0035] During the process of producing discrete or exfoliated carbon nanotubes (which may be single-walled, double-walled, and multi-walled configurations, or mixtures thereof) from bundles or entangled clumps of carbon nanotubes, the carbon nanotubes may be cut into segments having at least one open end, and residual catalyst particles on the outside and / or inside of the carbon nanotubes may be removed by an acid such as nitric acid. Cutting of carbon nanotubes can be carried out by high-energy mechanical and / or chemical means. Discrete carbon nanotubes with smaller aspect ratios are easier to exfoliate than those with larger aspect ratios. Exfoliation here means that the discrete carbon nanotubes can be separated as individual carbon nanotubes. Appropriate selection of the carbon nanotube feedstock, related to the type and distribution of catalyst particles within the carbon nanotubes, allows for greater control over the resulting individual tube lengths and the overall tube length distribution. A preferred choice is when the internal catalyst sites are evenly spaced and the catalyst is most efficient. A further preferred choice is when Stone-Wales defects are present along the walls or multiple walls of the carbon nanotubes. Individual discrete carbon nanotubes can have aspect ratios of about 5 to about 10,000, preferably about 25 to about 2,000, more preferably about 25 to about 500, and most preferably about 50 to about 200. The selection of discrete carbon nanotubes for the extracellular matrix can be evaluated using electron microscopy, as well as the determination of the length and length distribution of discrete or detached carbon nanotubes.

[0036] Discrete oxidized carbon nanotubes are obtained from as-fabricated bundled or entangled carbon nanotubes by oxidation-inducing methods, including but not limited to the use of concentrated nitric acid, concentrated sulfuric acid and a combination of concentrated nitric acid, or peroxides. Bundled or entangled carbon nanotubes can be prepared by any known means, such as chemical vapor deposition, laser ablation, and high-pressure carbon monoxide synthesis. Carbon nanotubes are preferably prepared via a non-toxic catalyst, such as iron, rather than, for example, cobalt. Bundled carbon nanotubes can exist in various forms, including, for example, soot, powder, fibers, and buckypaper. Furthermore, bundled carbon nanotubes can be of any length, diameter, or chirality. Carbon nanotubes can be metallic, metalloid, semiconductor, or nonmetallic, based on their chirality and the number of walls. Examples of discrete carbon nanotubes include single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes, and combinations thereof. Those skilled in the art will recognize that many of the specific embodiments of the present invention illustrated using certain types of carbon nanotubes can be similarly implemented within the spirit and scope of this disclosure using other types of carbon nanotubes. Other types of carbon nanotubes include, for example, boron.

[0037] A preferred choice of carbon nanotube in this invention is the incorporation of a portion of a structure called Stone-Wales defects, which is a rearrangement of the six-membered rings of graphene into heptagonal-pentagonal pairs that fit into the hexagonal lattice of condensed benzene rings constituting the wall of the carbon nanotube. These Stone-Wales defects are useful in creating sites with higher bond strain energy for easier reactions such as oxidation of the graphene or carbon nanotube wall. These defects and other types of condensed ring structures can also facilitate bending or curling along the length of the carbon nanotube, which is advantageous in maintaining the fluidity of a mixture of discrete carbon nanotubes.

[0038] Stone-Wales defects are thought to be more common at the end caps where the curvature of the carbon nanotube walls is greater. During oxidation, the ends of the carbon nanotube can be open and may undergo a higher degree of oxidation than along the walls. The higher degree of oxidation, and therefore higher polarity or hydrogen bonding at the ends of the tube, is thought to help reduce the ratio of the average tube contour length to the distance between tube ends when the tube is in water and other polar media.

[0039] According to a preferred embodiment, discrete carbon nanotubes are further functionalized. Functional groups linked to dCNTs include, but are not limited to, hydroxyl groups, thiol groups, amide groups, amine groups, and carboxyl groups. Functionalized dCNTs enable reduced aggregation between dCNT molecules in the culture medium. Functionalization enables interaction with organic molecules, including, but not limited to, inorganic metals, inorganic salts, ether groups, imine groups, amide groups, or ester groups, as well as with organisms, including, but not limited to, DNA, RNA, peptides, proteins, and enzymes. Discrete carbon nanotubes can associate with or bond with polymers such as poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethylacrylamide) (PDMA), and poly(N-acryloylmorpholine) (PAcM). Particularly useful polymer molecules for functionalizing discrete carbon nanotubes are, but are not limited to, modified polyethylene oxide copolymers such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol) and DSPE-PEG-NH2.

[0040] Other functionalized carbon nanotubes in this disclosure generally refer to any type of modification of carbon nanotubes described above. Such modifications may include the ends, sidewalls, or both of the nanotubes. Modifications may include, but are not limited to, covalent bonding, ionic bonding, chemisorption, intercalation, surfactant interactions, polymer wrapping, cleavage, solvation, and combinations thereof.

[0041] At least a portion of the identified discrete carbon nanotubes preferably have a number mean ratio of tube contour length (TCL) to tube end-to-end distance (TEE) of about 1.1 to about 3, preferably about 1.1 to about 2.8, more preferably about 1.1 to about 2.4, most preferably about 1.1 to about 2, and particularly about 1.2 to about 2. The TCL and TEE values ​​can be measured by scanning electron microscopy.

[0042] The TCL to TEE ratio can be advantageously controlled by the degree of thermodynamic interaction between the tube surface and the medium. Surfactants can also be usefully used to modify the thermodynamic interaction between the tube and the selected medium. Alternative means of influencing the ratio of the contour length to the end distance of discrete carbon nanotubes include the use of inorganic or ionic salts, such as sodium chloride, and organic functional groups having oxygen or nitrogen functional moieties.

[0043] Another method for controlling the TCL to TEE ratio is by orientation. Discrete carbon nanotubes can be oriented by such means, but not limited to, extrusion through a circular or slit die, drawing fibers, or foaming or blown film, injection or tentering. Orientation is facilitated by the presence of a polymer in a fluid. One example of this is the use of the discrete carbon nanotubes of the present invention in the presence of polyvinyl alcohol and water, so that polyvinyl alcohol-oriented discrete carbon nanotube fibers can be obtained by electrospinning or through an orifice in the wall of a spinning centrifuge.

[0044] The carbon nanotubes used in the present invention as described herein do not need to contain 100% discrete carbon nanotubes. That is, some carbon nanotube bundles may still exist as entangled, non-discrete tubes. However, the carbon nanotubes used in this ECM invention, based on the entire carbon nanotube composition, contain at least 70% by weight of discrete carbon nanotubes, more preferably more than 80% by weight, most preferably more than 95% by weight, and particularly 99% or more of discrete carbon nanotubes.

[0045] Compositions containing discrete carbon nanotubes may further contain polymers selected from a variety of natural, synthetic, and biosynthetic polymers that are biocompatible or biodegradable. Polymers based on a CC backbone tend to resist degradation, while heteroatom-containing polymer backbones confer biodegradability. Therefore, biodegradability can be designed into polymers by the appropriate addition of chemical bonds such as anhydride, ester, or amide bonds. The usual mechanism of degradation is by hydrolysis or enzymatic cleavage of unstable heteroatom bonds, resulting in cleavage of the polymer backbone. Macroorganisms can ingest polymers, sometimes digest them, and initiate mechanical, chemical, or enzymatic aging. Biodegradable polymers with hydrolyzable chemical bonds are widely studied for biomedical, pharmaceutical, agricultural, and packaging applications. For use in medical devices and controlled drug release applications, biodegradable polymers must be biocompatible, processable as biomaterials, sterilizable, and meet other criteria to be certified as capable of controlled stability or degradation in response to biological conditions. Often, the chemical properties of the degradation products, rather than the chemical properties of the polymer itself, have a significant impact on biocompatibility. Polylactides (PLA), polyglycolides (PGA), polycaprolactones (PCL), and poly(esters) based on their copolymers are widely used as biomaterials. The degradation of these materials yields corresponding hydroxy acids, which make them safe for in vivo use. Other biodegradable and environmentally degradable polymers include polyvinyl alcohol, poly(hydroxyalkanoates) such as the polyhydroxybutyrate-polyhydroxyvalerate class, additional poly(esters), and natural polymers, particularly modified poly(saccharides), such as starch, cellulose, and chitosan.

[0046] The stiffness of an ECM containing discrete carbon nanotubes can be selected based on the choice of polymer, the ratio of polymer to discrete carbon nanotubes, and the density of the scaffold. The stiffness of the scaffold can determine the type of cells that stem cells differentiate into.

[0047] ECM containing discrete carbon nanotubes may further include other additives that help direct stem cell differentiation, such as other inorganic types like graphite, graphene, and silicate structures, polymer additives, such as, but not limited to, classes of polymers like polyolefins, as well as, but not limited to, natural products like silk or DNA strands.

[0048] The stem cells to which this invention applies are not limited and include any stem cells having unique characteristics such as undifferentiated, unlimited proliferation, and the ability to differentiate into specific cell types. Preferred stem cells used in this invention are classified into two groups: pluripotent stem cells, such as embryonic stem cells and embryonic germ cells, and multipotent stem cells. Embryonic stem cells originate from the inner cell mass of the blastocyst, and embryonic germ cells originate from primordial germ cells present in the gonad at 5-10 weeks of age. Multipotent stem cells are found in embryonic tissue, fetal tissue, or adult tissue, including adult (somatic) stem cells. Pluripotent stem cells are proliferated in vitro and differentiate into the three germ layers (ectoderm, mesoderm, and endoderm). Multipotent stem cells are unlikely to have the ability to differentiate into their precursor tissues, and their self-renewal capacity is limited. Sources of multipotent stem cells include any type of tissue, particularly bone marrow, blood, liver, skin, intestines, spleen, brain, skeletal muscle, and dental pulp.

[0049] Preferably, the stem cells used in the present invention are embryonic stem cells, adult stem cells, embryonic germ cells, and embryonic cancer cells, and more preferably, embryonic stem cells and adult stem cells.

[0050] ECMs may include discrete carbon nanotubes bonded to the matrix components so that dCNTs are not removed during washing or transfer of other forms of aqueous fluids. Nanotubes can bond to the ECM components at a single contact point, along the entire length of the nanotube, or for any amount in between. These bonding forces may include, but are not limited to, van der Waals forces, hydrogen bonds, depletion forces, and ionic bonds.

[0051] Unless the dCNTs are removed from the matrix during washing, the dCNTs may adhere to the components of the ECM in any orientation. These orientations include, but are not limited to, being wrapped around the ECM structure, adhering along one or more ECM structures, or being fixed to one end of the nanotube while the other is freely diffused.

[0052] A composition containing a mixture of ECM and discrete carbon nanotubes may contain discrete carbon nanotube lengths that are unimodal or multimodal (such as bimodal). A multimodal distribution can have a range of lengths that are evenly distributed (e.g., 50% of one length range and about 50% of another). The distribution may also be asymmetrical, meaning that a relatively small proportion of discrete nanotubes may have a particular length, while a larger proportion may contain other lengths.

[0053] A composition comprising a mixture of ECM and discrete carbon nanotubes, wherein the diameter distribution of the discrete carbon nanotubes may be unimodal or multimodal (e.g., bimodal). A multimodal distribution may have a uniformly distributed range of diameters (e.g., 50% of one diameter range and approximately 50% of another). The distribution may also be asymmetrical, meaning that a relatively small proportion of discrete nanotubes may have a particular diameter while a larger proportion may contain other diameters.

[0054] A composition comprising a mixture of ECM and discrete carbon nanotubes may include discrete carbon nanotubes, where the discrete carbon nanotubes may include combinations of functional groups. For example, a portion of discrete carbon nanotubes having 2% by weight of carboxylic acid groups may be mixed with a portion of discrete carbon nanotubes having 2% by weight of hydroxyl groups for the purpose of providing association or chemical bonding to different components of the ECM.

[0055] Carbon nanotubes can be contacted with surfactants or dispersants selected from the classes of anionic, cationic, nonionic, or zwitterionic molecules, and combinations thereof. An example of an anionic surfactant is sodium dodecyl sulfate. An example of a cationic surfactant is cetyltrimethyl bromide. An example of a nonionic surfactant is Pluronic F127, which is blocked polyethylene oxide-polypropylene oxide. An example of a zwitterionic surfactant is dioctanoylphosphatyl choline.

[0056] ECM containing dCNTs for stem cell transplantation exhibits superior properties in network formation between differentiated stem cells and surrounding cells. dCNT ECM shows improved cell adhesion for enhanced cell density and intercellular adhesion, and is non-cytotoxic. These characteristics of dCNTs contribute to the formation of a network between transplanted stem cells and surrounding tissue, enabling the transplanted stem cells to exert their functions and effects. Furthermore, these characteristics prevent the transplanted stem cells from being washed away.

[0057] In particular, stem cell transplantation mixtures containing dCNTs can be easily transplanted in a fluid form, for example, using a syringe. Stem cells may be surrounded by a dispersion of discrete carbon nanotubes to protect them during fluid transport.

[0058] dCNT-containing ECM can be well mixed with stem cells and injected into the target site, thereby reducing the adverse effects associated with surgery. The injected dCNTs can form structures suitable for the formation of intercellular networks over time. Due to the electrical conductivity of dCNTs, they can be delivered to the target site via electrical induction, thereby allowing them to be used as a stem cell composition in areas where tissue has been damaged.

[0059] The diseases or disorders treated by the compositions of the present invention include all diseases or disorders that can be treated by stem cell therapy. Preferably, the cell therapy compositions of the present invention are applied to the treatment of neuronal diseases, myocardial ischemic injury or cardiomyopathy, spinal injuries and degenerative rhinitis.

[0060] The amount of dCNTs in the cell therapy composition per 1 ml of aqueous ECM composition is in the range of 0.002 to 60 mg / ml, preferably 0.01 to 10 mg / ml, more preferably 0.01 to 1 mg / ml, and most preferably 0.01 to 0.3 mg / ml.

[0061] In the cell therapy compositions of the present invention, pharmaceutically acceptable carriers may be conventional for formulation and may include, but are not limited to, those conventional for formulation, including carbohydrates (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose), acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, salt solutions, alcohols, gum arabic, syrup, vegetable oils (e.g., corn oil, cottonseed oil, peanut oil, olive oil, coconut oil), polyethylene glycol, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical compositions of the present invention may further include wetting agents, lubricants, stabilizers, or mixtures thereof. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0062] The precise dosage of the pharmaceutical composition of the present invention varies depending on the specific formulation, mode of application, the patient's age, weight and sex, diet, administration time, route of administration, patient's condition, excretion rate, response sensitivity, and other factors.

[0063] According to the prior art known to those skilled in the art, the cell therapy compositions of the present invention may be formulated with pharmaceutically acceptable carriers and / or vehicles, ultimately providing several forms including unit dosage forms or multi-unit dosage forms. The dosage forms may include solutions, suspensions or emulsions in oily or aqueous media, as well as further dispersions or stabilizers.

[0064] The cell therapy composition of the present invention promotes the formation of a network between transplanted stem cells and surrounding tissue, enabling the transplanted stem cells to fully exert their functions and effects.

[0065] Another aspect of the present invention, involving discrete carbon nanotubes in ECM applications, comprises a controlled drug delivery system containing dCNTs and at least one active drug component, preferably the active drug component substantially associated with DCNTs targeting a specific site in the body (e.g., a protein). The controlled drug delivery method is not limited to but can be activated by methods such as heat, static or vibrational electromagnetic fields.

[0066] Another embodiment of ECM applications is a bone adhesive composition comprising dCNTs and a biocompatible adhesive, such as polyvinyl alcohol (PVA), calcium phosphate, or polyurethane, preferably activated by electromagnetic radiation (e.g., X-rays, infrared rays, or ultraviolet rays).

[0067] Structural bone systems and compositions containing dCNTs, nerve repairs containing dCNTs, and biocompatible coverings containing dCNTs are also within the scope of the present invention. These systems and compositions can be activated by radiation such as electromagnetic radiation, X-rays, infrared radiation, or ultraviolet radiation.

[0068] In addition to the scaffold, the present invention also includes a stem cell proliferation and / or delivery substrate comprising dCNTs.

[0069] ECMs containing dCNTs may also include films, cellular structures, or fibrous structures suitable for cell proliferation. However, fibrous polymers such as electrospun PVA, silk, or fibroin may be included in compositions containing discrete carbon nanotubes, although these are not limited to these.

[0070] Films, fibers, or foam ECM structures containing discrete carbon nanotubes may also contain layers with different compositions. For example, in a tape, the layers may consist of different amounts of discrete carbon nanotubes, different types of discrete carbon nanotubes, or different additives or concentrations of additives.

[0071] A useful method for differentiating stem cells may first include placing the stem cells in or on a selected extracellular medium (ECM) containing discrete carbon nanotubes; culturing the stem cells to induce the desired type of differentiated cell growth; and subsequently transplanting the differentiated cells into a living organism. [Examples]

[0072] The following examples describe a method for preparing the compositions of the present invention. The synthetic ECM material used is a commercially available product known as a vitrogel, which is a matrix material mainly composed of cross-linked polysaccharides. Discrete carbon nanotubes can be prepared by means of high-pressure mixing or sonication, but are not limited to these. In this example, discrete oxide multiwalled carbon nanotubes (dMWCNTs) with a diameter of approximately 13 nm were prepared using concentrated nitric acid at 95°C for 2.5 hours, followed by washing with deionized water to pH 3.5. 2.3% (by weight) of the oxidation species was determined by thermogravimetric analysis in nitrogen at a temperature range of 200-600°C. The oxide carbon nanotubes were then made into a fluid slurry in water and subjected to high-pressure mixing to obtain discrete oxide carbon nanotubes. Discrete oxide single-walled carbon nanotubes can be prepared using a similar procedure, as in the case of discrete oxide multiwalled carbon nanotubes. Other functionalization chemistry, such as nitrene insertion reactions, can be used, but are not limited to these. Discrete oxidized multilayer carbon nanotubes were dispersed in water using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)DSPE-PEG(NH2) with a molecular weight of 2 kDa, in a dMWCNT:DSPE-PEG(NH2) ratio of 1:0.55. The cells incorporated into the ECM were mesenchymal stem cells.

[0073] The introgel solution (500 micrograms in 400,000 micrograms of water) was brought to room temperature. A cell suspension was prepared at 1 × 10⁶ cells / ml. 150 μl of a mixture of the cell suspension and introgel solution was plated into each well of a 48-well cell culture plate, ensuring uniform coverage of the mixture at the bottom of the wells. A gel formed after approximately 15 minutes. Without disturbing the gel, an additional 150 μl of cell medium containing 0.002 mg / ml of discrete carbon nanotubes was added to the top of the gel. The final concentration of dMWCNTs incorporated into the ECM structure was 0.001 mg per ml of gel (0.06% by dry weight of the introgel). The introgel constituted 0.125% by weight of the total solution. The masses of discrete carbon nanotubes, introgel, and water are shown in Table 1.

[0074] [Table 1]

[0075] The control was prepared in the same manner as in Example 1, but without discrete multilayer carbon nanotubes. Figure 1 shows that when Example 1 was incubated for 10 days in a 3D ECM-like matrix containing discrete carbon nanotubes, it provided approximately 60% improvement in mesenchymal stem cell proliferation compared to the control without discrete carbon nanotubes after 10 days of incubation. Cell counts were measured using a colorimetric assay. Figure 2 shows bright-field images of mesenchymal stem cells incubated with and without discrete carbon nanotubes after 9 days.

[0076] One example involves the preparation of an extracellular matrix containing discrete carbon nanotubes (dMWCNTs) in which no living cells are present and the nanotubes remain discrete only at specific concentrations. Vitrogel matrices were prepared in a manner similar to that outlined in Table 1, and mixed with discrete multiwall carbon nanotubes dispersed in DSPE-PEG(NH2) (molecular weight 2 kDa) and branched polyethyleneimine (PEI, molecular weight 270 kDa) at various concentrations in a dMWCNT:DSPE-PEG(NH2):PEI ratio of 10:7:3. The dMWCNT concentration was varied in the ranges of 0.01, 0.1, 0.5, 1, and 2 mg / ml, while the concentration of the vitrogel components was kept constant. The mixtures were incubated at 37°C for 24 hours. Figure 3 shows bright-field microscopy images of dMWCNTs at each concentration in the vitrogel. The scale bar is 100 μm. Significant aggregation was observed at all concentrations except 0.01 mg / ml, indicating that the MWCNTs used under these conditions were not adequately dispersed. However, no visible aggregation occurs at a concentration of 0.01 mg / ml, thus providing an example of well-dispersed dMWCNTs incorporated into the ECM described by this patent.

[0077] Further examples are shown in Figure 4, in which single-walled carbon nanotubes (SWCNT:DSPE-PEG) were non-covalently dispersed in DSPE-PEG(NH2). These (SWCNT:DSPE-PEG mass ratio 1:0.55) were incorporated into a synthesized ECM via a vitrogel at three concentrations (0.001, 0.01, and 0.05 mg / ml) as described above. MSCs were seeded onto the vitrogel, incubated at 37°C for 3 days, and imaged under a bright-field microscope. At concentrations of 0.001–0.01 mg / ml, the number of visible cells was higher than in the untreated group. Furthermore, cell elongation (a measure of MSC viability) in the 0.001–0.01 mg / ml range was significantly higher than in the untreated group, suggesting a positive effect of dSWCNTs on MSC proliferation.

[0078] Embodiment One embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, wherein the extracellular matrix comprises components selected from the class of proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof, and the discrete carbon nanotubes are less than about 1% by weight of the dry weight of the whole composition.

[0079] Another embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, wherein at least a portion of the extracellular matrix, or at least a portion of the discrete carbon nanotubes, or both, are at least partially oriented.

[0080] A further embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, wherein at least a portion of the discrete carbon nanotubes are physically attached directly or indirectly to at least one component of the composition, or optionally at least a portion of the discrete carbon nanotubes are chemically bonded to at least one component of the composition.

[0081] Another embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, further comprising living cells. The living cells may be stem cells, preferably primary stem cells.

[0082] One embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes for use in injury, burn, and wound care, sensors, and treatment of bone, skin, muscle, nerve, blood, and organs, which enables accelerated repair of injury compared to similar compositions of extracellular matrix that do not contain discrete carbon nanotubes.

[0083] A further embodiment of the present invention is that the composition can be molded, injected, implanted, or fitted. The composition may take the form of closed-cell foam, open-cell foam, film, strand, particles, or mixtures thereof.

[0084] Another embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, which is subjected to a static current, an oscillating current, a static voltage, an oscillating voltage, or any combination thereof. The supplied static or oscillating voltage is preferably less than about 100 millivolts, more preferably less than about 50 millivolts, and most preferably less than about 10 millivolts.

[0085] Another embodiment of the present invention is a composition comprising stem cells, wherein the stem cells grow at least about 10% faster, preferably at least about 20% faster, and more preferably at least about 50% faster, than equivalent compositions without discrete carbon nanotubes, under substantially the same conditions and in substantially the same culture medium.

[0086] One embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, wherein the discrete carbon nanotubes are substantially individualized from other discrete carbon nanotubes in the extracellular matrix.

[0087] A further embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, wherein the composition further comprises a dispersant that substantially prevents aggregation of the discrete carbon nanotubes. The dispersant is selected from the classes of anionic, cationic, nonionic, and zwitterionic molecules.

[0088] A further embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, wherein at least a portion of the discrete carbon nanotubes further comprises molecules that differ in composition and structure from the extracellular matrix, the molecules being at least partially bound to the nanotube surface and selected from a class of molecules that function as antimicrobial agents, growth factors, growth stimulants, nutrients, or a combination thereof.

[0089] Another embodiment of the present invention is a composition comprising a mixture of an extracellular matrix and discrete carbon nanotubes, further comprising graphene, graphene oxide, carbon nanofibers, cellulose nanofibers, hydroxyapatite, silk, and mixtures thereof.

[0090] Yet another embodiment of the present invention is a composition comprising a mixture of extracellular matrix and discrete carbon nanotubes, further comprising water.

[0091] Further embodiments relate to a method for growing living cells comprising an extracellular matrix and a mixture comprising discrete carbon nanotubes and living cells, wherein the extracellular matrix comprises components selected from proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof, the discrete carbon nanotubes constitute less than 1% of the dry weight of the total composition, the amount of living cells is sufficient to enable growth and proliferation, provides sufficient conditions to promote the growth of living cells, and allows for the growth of further living cells. At least a portion of the discrete carbon nanotubes may coat the extracellular matrix or exist as a more concentrated layer.

[0092] A further embodiment of the present invention is a method for collecting at least a portion of grown living cells or one or more biological products produced by living cells. Optionally, living cells are stem cells, preferably primary stem cells. Stem cells may be differentiated or undifferentiated.

[0093] Another embodiment of the present invention is a method comprising injecting at least a portion of grown living cells into an animal.

[0094] A further embodiment of the present invention, comprising a mixture of extracellular matrix and discrete carbon nanotubes, is a method for adding molecules structurally different from the components of the extracellular matrix, the molecules being selected from a class of molecules that function as antimicrobial agents, growth factors, growth stimulants, nutrients, or a combination thereof. Furthermore, a dispersant may optionally be added to the discrete carbon nanotubes before, simultaneously with, or after the addition of molecules structurally different from the components of the extracellular matrix.

[0095] Specific Embodiments 1. A composition,

[0096] A composition comprising a mixture of extracellular matrix and discrete carbon nanotubes, wherein the extracellular matrix comprises components selected from the class of proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof, and the discrete carbon nanotubes constitute less than about 1% by weight of the dry weight of the total composition.

[0097] 2. The composition according to Embodiment 1, wherein at least a portion of the extracellular matrix, or at least a portion of the discrete carbon nanotubes, or both, are at least partially oriented.

[0098] 3. The composition according to Embodiment 1, wherein at least a portion of the discrete carbon nanotubes are physically attached directly or indirectly to at least one component of the composition.

[0099] 4. The composition according to Embodiment 1, wherein at least a portion of the discrete carbon nanotubes are chemically attached to the components of the composition.

[0100] 5. The composition according to Embodiment 1, further comprising living cells.

[0101] 6. The composition according to Embodiment 5, further comprising stem cells.

[0102] 7. The composition of Embodiment 1 for use in injury, burn, or wound care.

[0103] 8. A composition of Embodiment 1 for use in a sensor.

[0104] 9. The composition of Embodiment 1 for use in the treatment of bone, skin, muscle, nerve, blood, and organs to promote the repair of damage, compared to similar compositions of extracellular matrix that do not contain discrete carbon nanotubes.

[0105] 10. The composition according to Embodiment 1, wherein the composition can be molded, injected, implanted, or fitted.

[0106] 11. The composition according to Embodiment 1, wherein the composition is used for static current, oscillating current, static voltage, oscillating voltage, or any combination thereof.

[0107] 12. The composition according to Embodiment 6, wherein the stem cells are primary stem cells.

[0108] 13. The composition according to Embodiment 6, wherein stem cells grow at least 10% faster in substantially the same medium and under substantially the same conditions than in equivalent compositions that do not contain discrete carbon nanotubes.

[0109] 14. The composition according to Embodiment 6, wherein stem cells grow at least 50% faster in substantially the same medium and under substantially the same conditions than in an equivalent composition that does not contain discrete carbon nanotubes.

[0110] 15. The composition according to Embodiment 1, wherein the majority of the discrete carbon nanotubes, or at least 60%, or at least 70%, or at least 80%, or more, are substantially individualized from other discrete carbon nanotubes in the extracellular matrix and / or remain substantially individualized from other discrete carbon nanotubes in the extracellular matrix.

[0111] 16. The composition according to Embodiment 1, further comprising one or more dispersants, the dispersants substantially preventing aggregation of discrete carbon nanotubes.

[0112] 17. The composition according to Embodiment 16, wherein one or more dispersants are selected from the classes of anionic, cationic, nonionic, amphiphilic, and zwitterionic molecules.

[0113] 18. The composition according to Embodiment 1, wherein at least a portion of the discrete carbon nanotubes further comprises molecules having different components and structures of the extracellular matrix, where the molecules are at least partially bound to the nanotube surface and are selected from a class of molecules that function as antimicrobial agents, growth factors, growth stimulants, nutrients, or a combination thereof.

[0114] 19. The composition according to Embodiment 1, further comprising graphene, graphene oxide, carbon nanofibers, cellulose nanofibers, hydroxyapatite, silk, and mixtures thereof.

[0115] 20. The composition according to Embodiment 1, which is in the form of closed-cell foam, open-cell foam, film, strand, particles, or mixtures thereof.

[0116] 21. The composition according to Embodiment 1, further comprising water.

[0117] twenty two.

[0118] A method for growing living cells comprising a mixture of an extracellular matrix, discrete carbon nanotubes, and living cells, wherein the extracellular matrix comprises components selected from the class of proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof, the discrete carbon nanotubes constitute less than 1% of the dry weight of the total composition, the amount of living cells is sufficient to enable growth and proliferation, and the method further comprises the step of providing sufficient conditions to promote the growth of living cells.

[0119] 23. The method according to Embodiment 22, wherein at least a portion of the discrete carbon nanotubes coat or form a layer on the extracellular matrix.

[0120] 24. The method according to Embodiment 23, further comprising collecting at least a portion of the grown living cells.

[0121] 25. The method according to Embodiment 24, further comprising collecting one or more biological products produced by living cells.

[0122] 26. The method according to Embodiment 25, wherein the living cells are stem cells.

[0123] 27. The method according to Embodiment 25, wherein the living cells are primary stem cells.

[0124] 28. The method according to Embodiment 26, wherein the stem cells have been differentiated.

[0125] 29. The method according to Embodiment 26, wherein the stem cells have not been differentiated.

[0126] 30. The method according to Embodiment 26, further comprising injecting at least a portion of the grown stem cells into an animal.

[0127] 31. The method according to Embodiment 22, further comprising a dispersant and molecules having different components and structures of the extracellular matrix, wherein the molecules are selected from a class of molecules that function as antimicrobial agents, growth factors, growth stimulants, nutrients, or a combination thereof.

[0128] The method according to 31, wherein the dispersant is optionally added to discrete carbon nanotubes before, at the same time as, or after the addition of molecules that have different components and structures from the extracellular matrix.

[0129] 33. The composition according to Embodiment 1, wherein the discrete carbon nanotubes are selected from the classes of single-walled, double-walled, multi-walled carbon nanotubes or any combination thereof.

[0130] 34. The composition according to Embodiment 1, wherein discrete carbon nanotubes are functionalized in the range of 1 μmol / g to 1 mmol / g dry nanotubes with a functional group selected from the class of carboxyl, hydroxyl, ester, ether, imine, amine, amide, phosphate, sulfate, nitrate, or combination thereof.

[0131] 35. The composition according to Embodiment 5, wherein at least some living cells maintain viability for at least two more weeks, or at least three more weeks, or at least four more weeks or longer, after integration into the extracellular matrix, compared to the extracellular matrix of a similar composition that does not contain discrete carbon nanotubes.

Claims

1. Extracellular matrix and A composition containing discrete carbon nanotubes, The extracellular matrix comprises one or more components selected from the class of proteins, proteoglycans, polysaccharides, lipids, peptides, messenger molecules, signaling molecules, or any mixture thereof. The discrete carbon nanotubes constitute less than approximately 1% by weight of the dry weight of the entire composition. composition.

2. The composition according to claim 1, wherein at least a portion of the extracellular matrix, or at least a portion of the discrete carbon nanotubes, or both, are at least partially oriented.

3. The composition according to claim 1, wherein at least a portion of the discrete carbon nanotubes are physically attached directly or indirectly to at least one component of the composition.

4. The composition according to claim 1, wherein at least a portion of the discrete carbon nanotubes are chemically attached to the components of the composition.

5. The composition according to claim 1, further comprising living cells.

6. The composition according to claim 5, wherein at least a portion of the living cells comprises stem cells.

7. The composition according to claim 1, wherein the composition is in a molded form, an injectable form, an implantable form, or a fitted form.

8. The composition according to claim 6, wherein the stem cells are primary stem cells.

9. The composition according to claim 6, wherein the stem cells grow at least 10% faster in substantially the same culture medium and under substantially the same conditions than equivalent compositions that do not contain discrete carbon nanotubes.

10. The composition according to claim 5, wherein there is a sufficient amount of the discrete carbon nanotubes substantially individualized from other discrete carbon nanotubes in the extracellular matrix, so that the living cells grow at least 10% faster than equivalent compositions that do not contain the discrete carbon nanotubes in substantially the same medium under substantially the same conditions.

11. The composition according to claim 1, further comprising one or more dispersants, wherein the one or more dispersants substantially prevent aggregation of the discrete carbon nanotubes.

12. The composition according to claim 11, wherein one or more dispersants are selected from the classes of anionic, cationic, nonionic, amphiphilic, and zwitterionic molecules.

13. The composition according to claim 1, wherein at least a portion of the discrete carbon nanotubes further comprises molecules whose structure differs from the components of the extracellular matrix, wherein the molecules are at least partially bound to the surface of the discrete carbon nanotubes and are selected from a class of molecules that function as antimicrobial agents, growth factors, growth stimulants, nutrients, or a combination thereof.

14. The composition according to claim 1, further comprising graphene, graphene oxide, carbon nanofibers, cellulose nanofibers, hydroxyapatite, silk, and mixtures thereof.

15. The composition according to claim 1, wherein the discrete carbon nanotubes are single-walled carbon nanotubes, double-walled carbon nanotubes, thin-walled carbon nanotubes, multi-walled carbon nanotubes, or any combination thereof.

16. The composition according to claim 1, wherein at least a portion of the discrete carbon nanotubes are functionalized with one or more functional groups.

17. The composition according to claim 16, wherein one or more of the functional groups are selected from the classes of carboxyl, hydroxyl, ester, ether, imine, amine, amide, phosphate, sulfate, nitrate, or any combination thereof.

18. The composition according to claim 16, wherein a portion of the functionalized discrete carbon nanotubes is functionalized in a range of 1 μmol per gram of dry nanotubes to 1 mmol per gram of dry nanotubes.

19. The composition according to claim 17, wherein a portion of the functionalized discrete carbon nanotubes is functionalized in a range of 1 μmol per gram of dry nanotubes to 1 mmol per gram of dry nanotubes.

20. The composition according to claim 5, wherein at least a portion of the living cells maintain their viability for at least two more weeks after integration into the extracellular matrix compared to an extracellular matrix of a similar composition that does not contain discrete carbon nanotubes.

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