NANOMATERIAL STRUCTURES

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This disclosure was made with government support under grant number

1DP2OD007394 awarded by the National Institute of Health. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

[0002] Current processes for fabricating macroscopic 2D carbon nanomaterial structures, such as Chemical Vapor Deposition (CVD) and vacuum filtration, do not lend themselves easily to size scalability, utilizing large variety of substrates, and mechanical stability. CVD based film growth requires temperatures up to 1000°C and vacuum filtration requires flat substrates. CVD method requires very specific substrates for nanomaterial film growth or deposition. For example, direct growth of carbon nanotube forests or graphene coatings by CVD requires substrates that can withstand both high temperatures and pressures.

[0003] In vacuum filtration, as the filtrate passes through the membrane, it restricts flow as the film is deposited on the substrate, limiting the maximum film thickness. There are many drawbacks for these processes. Although micron-scale thick films of graphene, can be fabricated by vacuum filtration, this method requires flat substrates to maintain their structural features and cannot be easily applied to irregular or round shapes.

[0004] Another limitation of these methods is that in the absence of strong chemical bonds linking between the individual nanomaterials, the structural integrity of films and coatings relies mainly on physical entanglement of the nanoparticles or Van der Walls forces. Thus, these films and coatings could be prone to disassociation by compressive flexural or shear forces.

[0005] Yet another limitation of these methods is a high surface roughness of a formed material. For example, vacuum filtration of carbon nanotubes on mixed cellulose ester and transferred to a smooth silicon wafer results in about 8 nm r.m.s. surface roughness. This roughness is quite high and cannot be lowered in typical vacuum filtration processes.

[0006] Therefore, what is desired is the ability to form carbon nanomaterial films on a range of substrates at a range of temperatures. Embodiments of the present disclosure provide methods that address the above and other issues.

SUMMARY OF THE DISCLOSURE

[0007] The present disclosure is directed to methods of forming a carbon film and substrates, upon which carbon films can be formed. In certain embodiments, the method of the current disclosure includes forming a carbon film by mixing a carbon nanomaterial with an initiator to form a mixture of nanomaterial and initiator, placing the mixture on a surface of a substrate and maintaining the mixture and substrate at a temperature above room temperature for a period of time to form the film. The substrate includes a carbon film formed on a surface of the substrate and the substrate being thermally stable up to about 120°C.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The present disclosure will be better understood by reference to the following drawings of which:

[0009] FIG. 1 A is an illustration of an in situ carbon film forming process; [0010] FIG. IB is photograph of film standing vertically (top photograph) and tilted to illustrate substantial transparency (bottom photograph).

[0011] FIG.2A is a graphical illustration of Raman spectroscopy for Multi Walled Carbon Nanotube (MWCNT) crosslinked films with three different mass ratios of MWCNT:BP;

[0012] FIG. 2B is a graphical representation of the variation in Raman Band Ratio and

Resistivity based on three different mass ratios of MWCNT:BP;

[0013] FIGs. 3A-3F are SEM images of graphene nano-onions, nanoribbons and nanoplatelets;

[0014] FIG. 4 are Atomic Force Microscopy (AFM) images of ultrasonic spray coated carbon materials;

[0015] FIGs. 5A-5E are Scanning Electron Microscope (SEM) images of ultrasonic spray coated carbon materials;

[0016] FIGs. 6A-6F are representative SEM and TEM micrographs for 1 :4 MWCNT films. FIG. 6 A is an overview micrograph with a cross-sectional inset (scale bar ΙΟΟμπι), FIG. 6B indicates formed crosslinks between nanotubes, FIG. 6C and FIG. 6D are magnified views of what is shown in FIG. 6B, FIG. 6E is a TEM image of a single crosslink junction with FIG. 6F indicating the directions of intersecting MWCNT lattice shown by the arrows;

[0017] FIG. 7 is a representative AFM image of 1 :4 MWCNT films, on silicon wafer, indicating topography created by the mesh network of carbon nanotubes;

[0018] FIG. 8 is representative load-unloading curve from nanoindentiation of spray coated non-crosslinked pristine MWCNT and crosslinked MWCNT (1 :4); [0019] FIGs. 9A-9E are graphical representations of cell proliferation and toxicity for ADSCs (FIG. 9A and FIG. 9C) and MC3T3-E1 cells (FIG. 9B and FIG. 9D) on

MWCNT crosslinked substrates and control coverslips (CS), the graphical illustrations are presented as mean and standard deviation with significance shown (*between time points and ^# between groups) for p<0.05, FIGs. 9E and 9F are graphical representations of LDH release over time;

[0020] FIGs. 10A-10D are representative images of proliferation marker Ki-67 and actin immunofluorescence for ADSCs grown on glass coverslips (FIG.1 OA and FIG. 10B) and MWCNT crosslinked substrates (FIG. IOC and FIG. 10D);

[0021] FIGs. 1 1A— 1 IF are representative SEM images of adipose derived stem cells grown on MWCNT substrates, circles in FIG. 11 A and FIG. 11C, magnified in FIG. 1 IB and FIG. 1 ID respectively, with arrows showing cell adhesion by wrapping around nanotubes, FIG. 1 IB, or cell protrusions going underneath nanotube structures, FIG. 1 ID, FIGs. 1 IE and 1 IF illustrate the uniaxial cytoplasmic elongation on the MWCNT films;

[0022] FIGs. 12A-12C are representative confocal fluorescence microscopy of ADSCs stained with and Hoechst 33342 (two-photon λ- grown on glass coverslips (FIG. 12A) and MWCNT crosslinked substrates (FIG. 12B and FIG. 12C) for 5 days at 37°C;

[0023] FIG. 13 is a Raman spectra of SWCNT crosslinked with varying crosslinking agents; and

[0024] FIG. 14 is AFM images of SWCNT crosslinked with varying crosslinking agents. DETAILED DESCRIPTION OF THE DISCLOSURE

[0025] As used herein, the term "crosslinked" refers to a process, in which at least two molecules, or two portions of a molecule, are joined together by a chemical interaction. Such interactions may occur in many different ways including formation of a covalent bond, formation of hydrogen bonds, hydrophobic, hydrophilic, ionic or electrostatic interaction. Furthermore, molecular interaction may also be characterized by an at least temporary physical connection between at least one molecule of carbon nanomaterial with itself or between two or more molecules. Therefore, it is contemplated that a crosslinked nanomaterial may crosslink with itself.

[0026] Crosslinking can be mediated by various reactive groups, and may occur by numerous mechanisms. If a covalent bond is formed between two reactive groups, it may be formed by a variety of chemical reaction mechanisms, including additions, eliminations or substitutions. Examples are nucleophilic or electrophilic addition, El- or E2-type eliminations, nucleophilic and aromatic substitutions. Crosslinking may be a spontaneous process or may require energy or a catalyst. Examples of such energy are thermal energy, radiation, mechanic, electric or electromagnetic energy. Examples of catalysts are acids, bases, and palladium-coated activated charcoal. Also, crosslinking may or may not involve extrinsic crosslinkers, and any extrinsic crosslinker may comprise single molecules, crosslinking molecules may also themselves be oligomeric or even polymeric.

[0027] As used herein, the term thermally stable refers to a substrate maintaining its shape and chemical structure after being heated to a given temperature for a period of time of a few hours to several days or weeks. [0028] As used herein, the term carbon nanotube refers to an elongated hollow structure having a cross section (e.g. angular fibers having edges) or a diameter (e.g. rounded) less than about 1 micron.

[0029] As used herein, the term multiwalled carbon nanotube (MWCNT) refers to carbon nanotubes which are substantially cylindrical, graphitic nanotubes of substantially constant diameter and comprise cylindrical graphitic sheets or layers whose c-axes are substantially perpendicular to their cylindrical axis.

[0030] As used herein, the term graphene refers to a two dimensional single sheet of carbon or multiple sheets of carbon.

[0031] As used herein, the term fullerene refers to a carbon compound in the form of a hollow sphere, ellipsoid, or tube.

[0032] As used herein, the term graphene oxide nanoribbon refers to, for example, single- or multiple layers of graphene (typically less than 10 carbon layers thick) that have an aspect ratio of greater than about 5, based on their length and their width.

[0033] As used herein, the term graphene oxide nanoplatelets refers to a planar-like nano-sized graphene oxide that is substantially solid.

[0034] As used herein the term grapheme oxide nano-onion shall mean hollow, porous, multi-wall carbon nanospheres or polyhedral structures with a narrow size distribution and an average particle size of approximately 80 nm and an average aspect ratio close to 7:5. Such structures are also referred to as carbon Q-dots or Q-graphene and can be any suitable sp ² hybridized carbon nanostructure.

[0035] As used herein, the term "substantially", or "substantial", is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a surface that is "substantially" flat would either completely flat, or so nearly flat that the effect would be the same as if it were completely flat.

[0036] The disclosure includes a method of forming a carbon film. The method for forming the carbon film includes several steps, the first being mixing a carbon nanomaterial with an initiator to form a mixture of nanomaterial and initiator. The carbon nanomaterial can be any suitable carbon nanomaterial, including but not limited to multi-walled carbon nanotubes (MWCNT), nanotubes, graphenes, fullerenes, graphene oxide nanoplatelets, graphene oxide nanoribbon, graphene oxide nano-ions and combinations thereof. The initiator can be any initiator that is suitable for forming crosslinked-carbon nanomaterial from the carbon nanomaterial, such as peroxides, or any radical initiator containing a peroxide functional group (ROOR'), hydroperoxides, peresters, and azo compounds, the like, and mixtures thereof. Examples of suitable free radical initiators include benzoyl peroxide, methyl ethyl ketone peroxide, Di-tert-butyl peroxide, acetone peroxide, dicumyl peroxide, di-t-butyl peroxide, t- butylperoxybenzoate, 2,5-dimethyl-2,5-di(t-butylperoxy)hexane, t-butyl

peroxyneodecanoate, 2,5-dimethyl-2,5-di(t-butylperoxy)hexyne, t-amyl peroxypivalate, l,3-bis(t-butylperoxyisopropyl)benzene, tert-amylperoxy 2 -ethyl hexanoate, t- butylperoxy 2-ethyl hexanoate, t-butyl peroxy isobutyrate, t-butylperoxy isopropyl carbonate, t-butylperoxy 3,5,5-trimethylhexanoate, 2,5-dimethyl-2,5- di(benzolyperoxy)hexane, n-butyl 4,4-di(t-butylperoxy)valcratic, t-butylcumyl peroxide, di(2 -t-butylperoxy isopropyl)benzene, t-butyl hydroperoxide, cumyl hydroperoxide and mixtures thereof. Examples of suitable azo compounds include 2,2'- azobisisopropionitrile, 2,2'-azobisisobutyronitrile (AIBN), dimethyl azoisobutyrate, 1,1'- azobis (cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropane), the like, and mixtures thereof.

[0037] In the method for forming the carbon film, the first step can also include mixing a solvent to form a mixture of nanomaterial, initiator and solvent. The solvent can be any suitable solvent, including organic solvents such as dimethylformamide (DMF), acetone, chloroform, ethyl acetate, tetrachloroethylene, carbon tetrachloride,

cyclohexene, methyl benzoate, anisole, ethylbenzene, chlorobenzene, nitrobenzene, benzene, toluene, allyl acetate, styrene, cumene, iodobenzene, carbon disulfide, ethyl iodide, methylene chloride, ethyl chloride, bromobenzene, t-butylbenzene, maleic anhydride, ethyl bromide, allyl bromide, acetic anhydride, cyclohexane, acetic acid, pyridine, dioxane, diethyl ether, ethyl alcohol, m-cresol, aniline and triethylamine.

[0038] The ratio of carbon nanomaterial to initiator in the mixture can vary based on characteristics of the final carbon film. Some of the characteristics that can be modified by altering the ratio of carbon nanomaterial to initiator include surface roughness of the film, sheet resistivity, cytotoxicity and cell proliferation. The ratio of carbon

nanomaterial to initiator in the mixture can be in any suitable ratio, such as about 1 :0.5, about 1 : 1, about 1.5: 1, about 2: 1, about 2.5: 1, about 3: 1, about 3.5: 1 and about 4: 1.

[0039] FIG. 2A and FIG. 2B illustrate how the ratio of carbon nanomaterial to initiator affects Raman shift, Raman band ratio and Resistivity. Therefore, based on the prospective use of the mixture, the ratio can be modified based on the desired qualities of the carbon film.

[0040] Carbon nanotubes are known to be excellent conductors of electricity, and disruptions (due to functionalization of structural defects) to the sp ² carbon network are known to decrease electrical conductivity of carbon nanotubes. As shown in FIG. 2B, although 1 : 1 (MWCNT:BP) samples showed greater sheet resistivity than pristine MWCNT coatings, a decrease in sheet resistivity was observed with increase in the defect sites in the MWCNT films to the point of recovery by 1 :4 (MWCNT:BP) (FIG. 2B).

[00 1] In other embodiments, one or more crosslinkers can be added to the mixture of carbon nanomaterial and initiator prior to the mixture being placed on a surface of a substrate. These crosslinkers can include any suitable crosslinker, including but not limited to ethylene diacrylate, methylene bisacrylamide, divinyl benzene and mixtures thereof. In this embodiment, after mixture of the crosslinker with the carbon

nanomaterial and initiator, the combined mixture can be placed on a surface of a substrate and formed into a film as discussed below when referring to the mixture of carbon nanomaterial and initiator.

[0042] After the mixture of carbon nanomaterial and initiator are formed, the mixture is placed on a surface of a substrate. The mixture can be placed on the surface of the substrate in any suitable way, such as by a pressure driven or ultrasonic driven spraying device. In some embodiments, the substrate is thermally stable up to about 60°C, in other embodiments, the substrate is thermally stable up to about 120°C.

[0043] The substrate can be any suitable substrate for forming a carbon film and is at least one of a material with one or more substantially flat surfaces, a flexible material, a material with one or more curved surfaces or a material with one or more erratically shaped surfaces. The suitable substrates include a whole or a part of a substrate selected from the group consisting of an implantable defibrillator, an artificial joint, a pacemaker, an implantable fixation element, a stent, a catheter, an artificial bone, an implantable drug delivery device, a cochlear implant, a suture, a valve, a tube, a guidewire, such as an ear tube, an implant such as a breast implant or chin implant, a balloon, a magnet, a power supply, a port, a sensor, a seed, a shunt and combinations thereof.

[0044] Included in the suitable artificial joints for forming a carbon film thereon are an artificial hip ball, an artificial hip socket, an artificial knee, an artificial finger joint, an artificial toe joint, an artificial shoulder, an artificial elbow, an artificial ankle.

[0045] Included in the suitable implantable fixation devices for forming a carbon film thereon are one or more of screws, wires, rods, pins, plates, discs, meshes, dowels, cuffs, pegs, washers, bolts, nuts, anchors, clips, clamps and staples.

[0046] Included in the suitable implantable drug delivery device for forming a carbon film thereon are an intra-uterine device, a catheter, an implantable insulin pump, an implantable drug pump and any suitable transdermal drug delivery system.

[0047] After the mixture of the carbon nanomaterial and initiator is placed on a surface of a substrate, the mixture of nanomaterial and initiator is maintained at a temperature above room temperature for a period of time. After the period of time, the film is formed. The mixture of nanomaterial and initiator can be maintained at the temperature above room temperature for any suitable amount of time, for instance up to about 24 hours. Included in this suitable amount of time is any time from about 1 minute to about 24 hours, including about 1 minute, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours, about 23 hours, about 23.5 hours and about 24 hours.

[0048] The mixture of carbon nanomaterial and initiator can be maintained at any suitable temperature above room temperature that effects at least some crosslinking of the nanomaterial, including about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C or about 100°C.

[0049] The method of forming the carbon film includes an additional, optional step of increasing the temperature above room temperature to an increased temperature above room temperature for a period of time after the maintaining step. This optional step can be used to remove any remaining initiator present in the carbon film. The increased temperature above room temperature can be any temperature useful in removing a suitable initiator, including about 100°C, about 105°C, about 110°C, about 115°C, about 120°C, about 125°C, about 130°C, about 135°C, aboutl40°C, about 145°C, about 150°C, about 155°C, about 160°C, about 165°C, about 170°C, about 175°C, about 180°C, about 185°C, about 190°C, about 195°C or about 200°C.

[0050] The method of forming the carbon film includes another additional, optional step of contacting the carbon film with at least one reducing agent, such as hydrazine hydrate and ascorbic acid after the maintaining step. This additional step may be useful in correcting or reducing oxidation defects that arise by reestablishing the sp ² hybridization of the carbon film.

[0051] The present disclosure is also directed to a substrate that can have a carbon film formed on a surface of the substrate. In this disclosure the substrate can be thermally stable up to about 120°C. In another embodiment, the substrate can be thermally stable up to about 60°C.

[0052] The carbon film formed on the substrate can comprise any suitable carbon nanomaterial, including but not limited to multi-walled carbon nanotubes (MWCNT), nanotubes, graphenes, fullerenes, graphene oxide nanoplatelets, graphene oxide nanoribbon, graphene oxide nano-ions and combinations thereof.

[0053] The substrate can be any suitable substrate for forming a carbon film and is at least one of a material with one or more substantially flat surfaces, a flexible material, a material with one or more curved surfaces or a material with one or more erratically shaped surfaces.

[0054] The suitable substrates include a whole or a part of a substrate selected from the group consisting of all implantable or external, medical or therapeutic devices, such as an implantable defibrillator, an artificial joint, a pacemaker, an implantable fixation element, a stent, an ear tube, an artificial bone, an implantable drug delivery device, a cochlear implant and combinations thereof. Included in the suitable artificial joints for forming a carbon film thereon are an artificial hip ball, an artificial hip socket, an artificial knee, an artificial finger joint, an artificial toe joint, an artificial shoulder, an artificial elbow, an artificial ankle. Included in the suitable implantable fixation devices for forming a carbon film thereon are screws, wires, rods, pins, plates and discs.

Included in the suitable implantable drug delivery device for forming a carbon film thereon are an intra-uterine device, a catheter, an implantable insulin pump, an implantable drug pump and any suitable transdermal drug delivery system.

[0055] The implantable drug delivery device can be coated fully with the carbon film, or a portion of the implantable drug delivery device can be coated with the carbon film. The drug to be delivered can be adsorbed or absorbed to the carbon film, the drug can be encapsulated in or on the carbon film, including encapsulation within polymeric spheres, and the drug can be included in or on the carbon film in fluorinated microbubbles.

Delivery of the drug from the carbon film can be facilitated in any suitable way, such as by diffusion, thermal agitation by variety of sources (e.g., optical, ultrasound, microwave and radiofrequency sources) and by photoacoustic disruption of the carriers (e.g.

fluorinated microbubbles). Any suitable drug could be delivered by this delivery device, including hydrophobic drugs and topical drugs.

[0056] Although the examples discussed below refer to carbon films using carious carbon nanotubes as the carbon nanomaterial, different sp ² hybridized allotropes of carbon including, but not limited to, various types of graphene, including graphene nano- onions, nanoribbons and nanoplatelets can also be formed into carbon films. As shown in FIGs.3A-3F, these other films are shown. FIGs. 3A, 3C and 3D are low- magnification SEM images of crosslinked graphene oxide nano onions, graphene oxide nanoplatelets and graphene oxide nanoribbons. FIGs. 3B, 3D and 3F are high magnification SEM images of crosslinked graphene oxide nano onions, graphene oxide nanoplatelets and graphene oxide nanoribbons.

[0057] The methods and compositions of the present disclosure will be better understood by reference to the following Examples, which are provided as exemplary of the disclosure and not by way of limitation.

Example 1

[0058] The carbon films of the disclosure are versatile and can be adapted for different sp ² hybridized allotropes of carbon including, but not limited to, various types of graphene (e.g., graphene nano-onions, nanoribbons and nanoplatelets, as shown in FIG. 4. FIG. 4 is AFM images of ultrasonic spray coated graphene nanoplatelets (GONP), graphene oxide nanoribbons (GONR), single walled carbon nanotubes (SWCNT ) and MWCNT. In FIG. 4, carbon nanomaterials and benzoyl peroxide, dispersed in ethyl acetate, were mixed by bath sonication. The resulting mixture was sent through a glass syringe and injected at a constant rate through an ultrasonic spray nozzle. An xyz gantry was used to raster the spray coating in x-y directions as well as maintain constant height from the coating substrate.

[0059] FIGs. 5A-5E includes scanning electron microscope (SEM) images of ultrasonic spray coated: SWCNT (FIG. 5 A); MWCNT of low diameter (FIG. 5B); MWCNT of high diameter (FIG. 5C); graphene nanoplatelets (FIG. 5D); and graphene oxide nanoribbons (FIG. 5E).

[0060] It appears that covalent bonding between the MWCNT when combined with spray coating allows for a layer-by-layer assembly, which results in thick coatings (> lOOum in thickness). This method yields coatings with nano- and micro-pores and high surface roughness. These features are especially advantageous for applications such as biosensing which require high charge storage capacity and self-cleaning hydrophobic substrates.

[0061] Spray coating allows coating of irregular shapes, and ability to create a continuous network (FIG. 2A) with a relatively high surface roughness (~730nm). The droplet size inhomogeneity (as depicted in FIG. 1 A), and nanomaterial aggregation may be responsible for this roughness.

[0062] The use of the xyz gantry enable more control over the following three parameters: (1) the number of layers sprayed is controlled by the number of passes the xyz gantry, which holds the spray head, makes over the substrate; (2) the volume of nanomaterial suspension sprayed is controlled at a substantially constant rate by a syringe pump; and (3) there is a low operating pressure compared to air pressure driven spray techniques, resulting in less wasted material (for example about 2 p.s.i. for ultrasonic spray coating vs 30 p.s.i. for airbrush coating).

[0063] For applications requiring crosslinked MWCNT films with a smoother surface, more homogenous spray coating techniques such as ultrasonic spray coating could be used. Ultrasonic spray coating can create functionalized-MWCNT films of 3 nm average surface roughness. The chemical crosslinking of MWCNTs, substantially enhances the mechanical properties of the films and thus, their structural stability compared to pristine non-crosslinked MWCNT films. This enhancement should prevent the films from disintegration under compressive flexural or shear forces under physiological conditions.

[0064] The mean surface roughness measurements for ultrasonic spray coated for GONP was 159.07 nm, for GONR was 169.5 nm, for SWCNT was 167.05 nm and for MWCNT was 120.51 nm.

[0065] The method discussed herein allows fabrication of MWCNT substrates with micro- and macro porous architecture, which should allow cell infiltration. Additionally, carbon nanotubes assembled in 2D films and substrates, may exhibit multifunctional capabilities for regenerative medicine applications. Further, the crosslinked MWCNT films remained intact during the entire duration (5 days) of static culture experiments.

[0066] Radical initiated thermal crosslinking of carbon nanomaterials combined with air- pressure driven spray coating technique, allows in situ assembly of MWCNTs into chemically-crosslinked and mechanically robust MWCNT films. This protocol can be adapted for other carbon nanostructures such as graphene (e.g., graphene nano onions, graphene nanoribbons and graphene nanoplatelets). The crosslinked MWCNT films were found to be cytocompatible for human ADSCs. The results introduce a method to fabricate robust carbon nanotubes nanofiber mats for use in various medical applications.

Example 2

[0067] In the present example, the following materials were used. Multi-walled carbon nanotubes (MWCNT) were purchased from Sigma Aldrich with the outer wall diameters of about 110-170 nm and lengths of about 5-9μπι. Graphene oxide nanoplatelets (GONP) were synthesized and characterized. Graphene oxide nanoribbons (GONR) were synthesized by longitudinal unzipping of multiwalled carbon nanotubes (Sigma Aldrich). Graphene oxide nano-onions (GONO), also known as Q-graphene, were purchased from Graphene Supermarket (Calverton, NY).

Using the above materials, forming of a film was conducted. Nanoparticle suspensions at 1 mg/mL in anhydrous ethyl acetate were dispersed by sonication for 15 minutes. MWCNT to benzoyl peroxide (BP) mass ratios of 1 : 1, 1 :2, and 1 :4 were used for initial characterization. All cell studies, discussed below, were performed on the 1 :4 ratio samples. Suspensions were sprayed with an air pressure driven spray device (Iwata HP- CS) onto 12mm diameter round glass coverslips (Electron Microscopy Sciences). A graphical representation of this is shown in FIG. 1A. As shown in this graphical representation, the thickness can be modulated from thin to thicker through the application process. The MWCNTs completely coated the coverslips (FIG. IB, top) and were semi-transparent (FIG. IB, bottom).

[0068] Prior to spraying, the coverslips were cleaned with acetone and autoclaved.

During the spraying process the coverslips were heated on a hotplate to about 60 C. This temperature is a cause of in situ crosslinking and prevents the liquid suspension from accumulating on the surface of the coverslips. In this example, samples were further thermally crosslinked in an oven at 60 C for 12 hours. In this example, excess BP was removed by heating the coated coverslips at 150 C for an additional 30 minutes.

[0069] This spraying method leads to the generation of substantially heterogeneously- sized droplets of MWCNT and benzoyl peroxide which deposit onto the heated coverslip. The solvent (ethyl acetate) evaporates and initiates the free radical crosslinking process, which in turn leads to the in situ crosslinking of MWCNTs, and fabrication of the films.

[0070] Raman spectroscopy (Enwave Optronics) was performed in three regions of each sample under a 40x objective using a 532nm laser source. Point spectra scanning from 100 to 3,100 cm ^"1 at room temperature were acquired.

[0071] Scanning electron microscopy (SEM) was performed using a JEOL 7600F analytical high resolution SEM. Samples were sputter coated with a 3ng layer of Au to prevent surface charge accumulation. Transmission electron microscopy (TEM) samples were prepared by fragmenting crosslinked films by scratching the surface with sharp tweezers and placing them on a conductive carbon TEM porous grid (PELCO, Ted Pella). TEM was performed using a JEOL JEM2100F high resolution analytical TEM. Both electron microscopy techniques were performed at Brookhaven National

Laboratory in Upton, NY.

[0072] Sheet resistivity was assessed by a four probe resistance measurement technique (Signatone S302-4, SP-4 probe) at Center for Functional Nanomaterials (CFN), Brookhaven National Laboratory, New York. Four spring-loaded probes, spaced equally by 1.25mm distances, were lowered onto glass coverslips coated with MWCNT (1 :4) to measure sheet resistance and resistivity.

[0073] Crosslinked MWCNT (1 :4) on freshly cleaved silicon wafers (Ted Pella, USA) were prepared for Atomic Force Microscopy (AFM). AFM images were obtained with a Nano Surf Easy Scan 2 Flex AFM (NanoScience Instruments Inc., Phoenix), in air by tapping a V-shaped cantilever (APP Nano ACL - 10, frequency fc = 145-230 kHz, L = 225 μηι, W= 40 um, tip radius < 10 nm, spring constant k = 20-95 N/m). Nanosurf Easy Scan 2 Software was utilized to calculate the root mean square (r.m.s.) surface roughness of the coatings. Raman spectroscopy (Enwave Optronics, Irvine, CA) was performed in three regions of each sample (after thermal treatment to remove residual BP) under a 40x objective using a 532nm laser source. Point spectra scanning from 100 to 3,100 cm-1 at room temperature were acquired.

[0074] Samples were also tested to be in contact with various cell cultures. Primary human adipose derived stem cells (ADSCs) were cultured to passage 3 in ADSC basal media supplemented with heat inactivated FBS and ADSC Growth Media Bulletkit™ (Lonza). Cells were grown in tissue culture treated polystyrene at 95% humidity, 5% C0 ₂ , at 37°C with media changes every three days. Nanoparticle coated coverslips and plain coverslips (control) were washed with a sterile phosphate buffered saline solution (Gibco, New York) and sterilized under ultraviolet radiation for two hours. Cells were plated on the coverslips (n=6), kept in an un-treated non-adherent 24 well plate, at a density of 4 10 ⁴ cells per well. Cells were incubated for 24 hours to allow their attachment, after which the coverslips were transferred to a new 24 well plate

(considered as the Day 1 time point). Cells were kept plated for three time points; Day 1, 3 or 5. At each time point, the cells were washed twice in phosphate buffered saline solution and used for viability and cytotoxicity assays.

[0075] Cytotoxicity of MWCNT 1 :4 films was assessed with ADSCs by measuring lactate dehydrogenase (LDH) release from cells as a function of membrane integrity (Sigma Aldrich). Media was collected from MWCNT films and control coverslips from each cell line at Day 1, 3 and 5. For each sample (n=6), 200 μΐ _^ of the extracted media was incubated for 45 minutes with LDH reagent and absorbance at read at 450 nm. Positive control of 100% dead cells was performed by adding 10 μΐ. of kit-supplied lysis buffer to the control cells. Cell death was calculated from measured optical density of experimental groups, coverslip control:

„ _{„ n} OD _3ampls - OD _{CDi;e sitp}

Ceil Death =— -

[0076] Cell proliferation of ADSCs on MWCNT 1 :4 films was assessed using CellTiter 96 Cell Proliferation MTS Assay (Promega). Cell proliferation was assessed according to the formula below:

Ceil Proliferation =—— — -

ODE½ _5;g - 0D _ec09grsSip

[0077] Live cells were washed three times with PBS, treated with calcien-AM

(0.5mg/mL) for 30 minutes and Hoechst 33342 (2 for 30 minutes. For immunofluorescence microscopy, glutaraldehyde fixed cells were washed with PBS, incubated with 2% glycine for 5 minutes, and permeabilized using 0.5% Triton-X-100 permeabilizing buffer (10.3 g sucrose, 0.4 g HEPES buffer, 0.29 g NaCl, 0.06 g MgCl ₂ , and 0.5 mL Triton-X-100 in 100 ml DI water) for 25 minutes. Samples were washed using immunofluorescence buffer (IFB, 0.1% BSA and 0.1% Triton-X-100 in PBS) and incubated with commercially available monoclonal anti-proliferating i-67 antibody raised in mouse (2 μΙ/mL in IFB, Cat. No. P8825, Sigma Aldrich, New York, USA) for 1 hour. Samples were washed with IFB (3X) and incubated with anti-mouse rhodamine conjugated secondary antibody (2 yLlmL in IFB, Cat. No. SAB3701218, Sigma Aldrich, New York, USA) for 1 hour. Samples were washed with IFB (3X) and stained with FITC conjugated phalloidin (2 ^LIvaL in PBS) for 1 hour to visualize cytoskeleton (actin filaments). Samples were then imaged using a confocal laser scanning microscope (Zeiss LSM 510 Two-Photon LSCM).

[0078] Specimens for scanning electron microscopy (SEM) were prepared as follows. MWCNT 1:4 samples with ADSCs were dehydrated by serial ethanol wetting steps from 50% to anhydrous ethanol. The samples were then air dried for one day and vacuum dried overnight at room temperature. A 3nm layer of gold sputter was applied prior to SEM. SEM was performed on a high resolution analytical JOEL 7600F SEM at the Center for Functional Nanomaterials (Brookhaven National Laboratories).

[0079] All plots for cell studies present a mean and standard deviation. Statistical analysis for cell studies was performed with one-way ANOVA followed by Tukey- Kramer post hoc analysis (Graphpad Prism). Statistical analysis for nanoindentation was performed using the Mann- Whitney test (Graphpad Prism). A 95% confidence interval (p<0.05) was used for all statistical analysis.

[0080] The results of the above described steps in Example 2 are discussed below.

[0081] Normalized Raman spectra of crosslinked MWCNT films are presented in FIG. 2A. Each mass ratio showed the characteristic Raman peaks of MWCNT with D, G, and G' bands at ~ 1345 cm ^"1 , 1560 cm ^"1 , and 2670 cm ^"1 , respectively. Pristine graphitic networks are characterized by the G band (intensity represented by I _G ) generated by in- plane vibrations of C=C carbon atoms and the D band (intensity represented by ID) is generated by structural defects or disorder features in graphitic network generate. The I _D /I _G ratio increased with increase in MWCNT: BP ratio (FIG. 2B). Further, weakly defined peaks at 802 cm ^"1 to 915 cm ^"1 were observed and assigned to C-O-C bond vibrations and asymmetrical stretching, respectively. These peaks imply presence of covalent carbonyl functional groups most probably formed during the crosslinking reaction.

[0082] Electrical resistivity measurements allow evaluation of the changes in the electrical properties of the MWCNT after the crosslinking reaction. The changes provide surrogate information about the interconnectivity between the MWCNTs. FIG. 2B shows the bulk electrical resistivity of the MWCNT films as a function of MWCNT: BP ratio. Pristine MWCNT coatings had a resistivity of 29.45 Ω-cm. Adding BP

(MWCNT:BP) to samples lead to an initial increased in sheet resistivity to 35.3 Ω-cm for 1 : 1 (MWCNT:BP) mass ratios and reduction thereafter in sheet resistivity from to 29.2 Ω-cm for 1 :4 (MWCNT:BP) mass ratios.

[0083] All films created a continuous coating on 12mm diameter glass coverslips with a porous network as shown in FIG. 6A. The films had a relatively high surface roughness with a mean height of 75 um (FIG. 6 A inset). Ultra-high magnification under SEM showed MWCNT networks with connectivity, micro- and nano-porosity with many junctions, as shown in FIG. 6B, and individual MWCNT and bundles crosslinking with each other, as shown in FIGs. 6C and 6D. TEM analysis illustrated junctions between two MWCNTs, as shown in FIG. 6E, leading to a checkerboard pattern, as shown in FIG. 6F, at the location of the crosslink. Atomic force microscopy, as shown in FIG. 7, revealed a high r.m.s. area surface roughness of 730.667nm (s.d. 124.720 nm, n=3). [0084] Mechanical properties of spray coated pristine MWCNT and crosslinked

MWCNT (MWCNT to benzoyl peroxide (BP) mass ratio of 1 :4) were determined using nanoindentation (Triboindenter; Hysitron, Minneapolis, MN) with a Berkovich indenter tip. AFM specimen discs (Ted Pella) of 15mm diameter, which can stick firmly to the magnetic triboindenter base, were coated with either MWCNT or crosslinked MWCNT and mounted into the indenter. After careful analysis of the disks under the imaging system of the triboindenter, points of indentation were selected at a distance no less than 100 μιη away from each other.

[0085] The imaging system of the triboindenter consisted of an objective of

magnification 10X and an end zooming lens of magnification 2X. A further zoom of 5X magnification was used to decide the final selection of indentation points through the special electronically controlled magnification of the triboindenter. Samples were indented 7 times to determine elastic modulus (Er) and material hardness (H). Each indentation further comprised of 9 sub-indents in a 3 x 3 pattern and thus, the total number of indents each sample were 63. Due to the porous nature of the coatmgs, mdents resulting in outlier points were removed individually from each 3*3 indent.

[0086] Due to the porous nature of the coatings, some of the indents were not made on the thin carbon films but on the pores. Such indentations localized in holes or resulting in poor curves were not included in the analysis. The tip area function was calibrated from indentation analysis on fused quartz, and drift rates in the system were measured prior to each indentation. First, a preload of 3μΝ was applied to the system followed by a constant loading rate (lOuN/second). Next, a hold segment at a fixed system load was applied, followed by a constant unloading rate to retract the tip (-ΙΟμΝ/second), and finally another hold segment was applied (3 μΝ). Each sample was indented with peak loads ranging from » 15 μΝ to 100 μΝ. The elastic response was calculated from the 20- 90% portion of the unloading curve.

[0087] The nanoindentation protocol yielded the values of elastic modulus (Er) and hardness (H) of the films, and are summarized in Table 1. Representative force- displacement curves for the chemically-crosslinked MWCNT and pristine MWCNT films are shown in FIG. 8. Data is reported in mean (μ), median (mdn.), standard deviation (S.D.) and interquartile range (i.q.r.). The median Er value of the chemically- crosslinked MWCNT films (mdn=376 MPa, μ=424 MPa, S.D.=287 MPa) were -232% greater than that of pristine MWCNT (mdn=162 MPa, μ=232 MPa, S.D.=299MPa) (p< 0.0001). The crosslinked MWCNT films also exhibited statistically significant

(p<0.0001) -242% increase in hardness (mdn=5.15 MPa, μ=5.83 MPa, S.D.=3.84MPa) compared to the pristine MWCNT films (mdn=2.13 MPa, μ=2.37 MPa, S.D.=1.61MPa).

Table 1

Mechanical properties of spray coated pristine MWCNT and crosslinked MWCNT (MWCNT: BP = 1 :4) determined by nanoindentation.

Pristine MWCNT Crosslinked MWCNT

Er (MPa) H (MPa) Er (MPa) H (MPa)

Median 162 2.13* 376 ^# 5.15* I.Q.R. 173 1.53 307 5.16 min. 20.7 0.452 59.8 0.881 max 1762 8.87 1604 22.3

(^represents p<0.0001 between groups and represents p<0.0001 between groups ) [0088] Crosslinked MWCNT films (1:4 of MWCNT:BP) were also assessed for human adipose derived stem cell (ADSC), murine preosteoblast (MC3T3) proliferation and cytotoxicity. Proliferation and cellularity were assessed by measuring mitochondrial activity on cells adhered to the MWCNT films and control glass coverslips. Day 1 cell attachment of ADSCs on MWCNT scaffolds was approximately the same as the control group, as shown in FIG. 9A, however, less initial attachment (p<0.01) was observed for MC3T3 cells, as shown in FIG. 9B. Furthermore, from Day 1 to Day 3, both ADSCs and MC3T3 cells proliferate slower (p<0.05) than the control samples, as shown in both FIGs. 6A and 6B. Cytotoxicity, measured by the release of LDH by compromised cell membranes, showed little to no cell death for ADSCs and MC3T3 cells at Day 3 and Day 5 timepoints. Furthermore, these assays also suggest lower proliferation as the LDH release is decreasing and negative in values over at Day 3 and Day 5 for MC3T3 cells, as shown in FIG. 9D, and at Day 5 for ADSCs, as shown in FIG. 9C.

[0089] FIG. 9E illustrates the cell death on crosslinked MWCNT substrates. The results are normalized to a positive control of 100% dead cells by using an LDH assay lysis buffer. The ADSCs grown on coverslips released approximately 35% and 45% of LDH at days 1 and 3 as compared to the positive control while the cells on the crosslinked MWCNT substrates released approximately 50% and 43% LDH at days 1 and 3 with no statistical differences. A statistical increase by 10% (p<0.01) in LDH release was observed at day 5 timepoint LDH assay (FIG. 9B). This also corresponds to the timepoint where cell proliferation increased as observed by the MTS assay.

[0090] LDH cytotoxicity assay (FIG. 9F) indicated that cells remained comparably viable on the MWCNT substrates and the coverslip controls at days 1 and 3. The increase in LDH release at day 5 could be attributed to one of two factors: 1) an increase in basal LDH release for the increasing ADSC proliferation on crosslinked MWCNT substrates or 2) increasing cell death as the cells were in critical density without media changes for 5 days. The latter may be less likely because an increase in LDH release for ADSCs on glass coverslips was not observed. Cell staining and immunofluorescence was performed on ADSCs grown on coverslips and MWCNT films grown for 5 days. Cellular proliferation marker, Ki-67, was used to evaluate if the ADSC were still dividing or they entered a Go resting phase. Ki-67 expression was observed through the cell cytoplasm and nucleus for glass coverslips (controls, FIG. 10A and FIG. 10B) and MWCNT (FIG. IOC and FIG. 10D) coated on glass coverslip substrates. While glass coverslips had a more even biaxial growth pattern, as shown in FIGs. 10A and 10B, longer uniaxial cytoplasmic elongation was observed on the MWCNT films than glass coverslips, as shown in FIGs. IOC and 10D. Immunohistochemistry analysis in FIGs. 10A-10D also showed cell spreading and proliferation on the MWCNT mats and provided further evidence that these mats did not affect the various phases of cell growth cycle.

[0091] The cytoplasmic prolongations were also corroborated by SEM, FIGs. 1 1 A-l IF. Interactions with the crosslinked nanotube bundles and cell prolongations from the circled areas of FIG. 11A, and FIG. 11C show 'wrapping' form of cell adhesion (magnified in FIGs. 1 IB and 1 ID respectively).

[0092] Cell viability was further assessed using calcein AM live cell stain and Hoechst 33342 nuclear stain (FIGs. 12A-12C). Cellular uptake of calcein AM by living cells leads to intracellular esterase cleavage, and enhanced green fluorescence due to calcein. Hoechst 33342 is a nucleic acid stain which emits blue fluorescence upon binding to double-stranded DNA (dsDNA), and provides evidence on the presence of dsDNA within intact nuclear membrane of non-apoptotic cells. Calcein AM verified the presence of live cells on glass coverslips (FIG. 12A) and on crosslinked MWCNT substrates (FIG. 112B). The Hoechst 33342 stains indicated that dsDNA was confined within the cell in the nucleus. Together the stains provided surrogate confirmation of live cells well spread on the glass coverslips (FIG. 12A) and MWCNT substrates (FIGs. 12B and 12C).

[0093] The present carbon nanomaterial films showed relatively high orders of interconnectivity and crosslinking in SEM and TEM images as well as Raman spectroscopy. Increased electrical conductivity was observed when increasing the concentration of crosslinking reagent. Further, cytocompatibility analysis towards MC3T3-E1 osteoblasts and ADSCs were alive and proliferating on crosslinked MWCNT substrates.

[0094] The disclosed method creates chemical bonds to adjoin sp ² -hybridized nanomaterials while substantially maintaining their architectural features and electrical conductivity. This method and resultant carbon film can be useful in many fields, including but not limited to fields where carbon nanotubes and graphene thin films are used such as electronics and biomedical applications.

[0095] The assays, immunochemistry and SEM analysis together indicate that, even though the initial cell proliferation rates on the MWCNT substrates were slower compared to coverslip controls, the MWCNTs were not cytotoxic and allowed cell attachment and proliferation over the 5 day period. These results indicate methods for the fabrication of robust carbon nanotube mats with chemically cross-linked junctions between sp ² carbon atoms, which can be adapted for other carbon nanostructures such as graphene and a variety of substrates with different shapes. Furthermore, with the advent of commercialized 3D printing, the mechano-structural benefits provided by the chemical crosslinks should allow for 3D-printed all-carbon nanomaterial structures.

Example 3

[0096] In this example, single walled carbon nanotubes (SWCNT) were dispersed in ethyl acetate with benzoyl peroxide and one of three crosslinkers (ethylene diacrylate, methylene bisacrylamide, and divinyl benzene). The concentration of the nanomaterials to solvent was lmg/mL. The mass ratio for the nanoparticle (SWNCT), free radical initiator (benzoyl peroxide), and crosslinker in the solution was 1 : 1 :2 respectively.

[0097] After dispersion, the mixture was spray coated, as discussed in Examples 1 and 2 above, onto titanium discs (12mm diameter) and heated to 120°C by an automated ultrasonic spray coating system. After heating, the nanomaterial films were characterized by Raman spectroscopy and atomic force microscopy (AFM).

[0098] As shown in FIG. 13, Raman spectra of SWCNT crosslinked with various crosslinking agents, including ethylene diacrylate, methylene bisacrylamide, and divinyl benzene, is illustrated. All values are normalized to the G band of the carbon nanotubes.

[0099] FIG. 14 includes AFM images of SWCNT crosslinked with varying crosslinking agents, including from the left of FIG. 14 to the right, divinyl benzene, ethylene diacrylate and methylene bisacrylamide. As can be seen from these figures, crosslinking occurs in films created from mixtures of carbon nanomaterials (SWCNT in this example), an initiator (BP in this example) and crosslinkers.

[0100] The described embodiments and examples of the present disclosure are intended to be illustrative rather than restrictive, and are not intended to represent every embodiment or example of the present disclosure. While the fundamental novel features of the disclosure as applied to various specific embodiments thereof have been shown, described and pointed out, it will also be understood that various omissions, substitutions and changes in the form and details of the devices illustrated and in their operation, may be made by those skilled in the art without departing from the spirit of the disclosure. For example, it is expressly intended that all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, various modifications and variations can be made without departing from the spirit or scope of the disclosure as set forth in the following claims both literally and in equivalents recognized in law.