Graphene nanoribbons as a drug delivery agent for lucanthone mediated therapy of glioblastoma multiforme.

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24h. However, their uptake was ~38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Structural disruption increases toxicity of graphene nanoribbons.

The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low-energy bath and high-energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 µg ml(-1) ) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post-production structural modifications for any application using nanomaterials.

Two-dimensional nanostructure-reinforced biodegradable polymeric nanocomposites for bone tissue engineering.

This study investigates the efficacy of two-dimensional (2D) carbon and inorganic nanostructures as reinforcing agents for cross-linked composites of the biodegradable and biocompatible polymer polypropylene fumarate (PPF) as a function of nanostructure concentration. PPF composites were reinforced using various 2D nanostructures: single- and multiwalled graphene oxide nanoribbons (SWGONRs, MWGONRs), graphene oxide nanoplatelets (GONPs), and molybdenum disulfide nanoplatelets (MSNPs) at 0.01-0.2 weight% concentrations. Cross-linked PPF was used as the baseline control, and PPF composites reinforced with single- or multiwalled carbon nanotubes (SWCNTs, MWCNTs) were used as positive controls. Compression and flexural testing show a significant enhancement (i.e., compressive modulus = 35-108%, compressive yield strength = 26-93%, flexural modulus = 15-53%, and flexural yield strength = 101-262% greater than the baseline control) in the mechanical properties of the 2D-reinforced PPF nanocomposites. MSNP nanocomposites consistently showed the highest values among the experimental or control groups in all the mechanical measurements. In general, the inorganic nanoparticle MSNP showed a better or equivalent mechanical reinforcement compared to carbon nanomaterials, and 2D nanostructures (GONPs, MSNPs) are better reinforcing agents compared to one-dimensional (1D) nanostructures (e.g., SWCNTs). The results also indicated that the extent of mechanical reinforcement is closely dependent on the nanostructure morphology and follows the trend nanoplatelets > nanoribbons > nanotubes. Transmission electron microscopy of the cross-linked nanocomposites indicated good dispersion of nanomaterials in the polymer matrix without the use of a surfactant. The sol-fraction analysis showed significant changes in the polymer cross-linking in the presence of MSNP (0.01-0.2 wt %) and higher loading concentrations of GONP and MWGONR (0.1-0.2 wt %). The analysis of surface area and aspect ratio of the nanostructures taken together with the above results indicated differences in nanostructure architecture (2D vs 1D nanostructures), and the chemical compositions (inorganic vs carbon nanostructures), number of functional groups, and structural defects for the 2D nanostructures may be key properties that affect the mechanical properties of 2D nanostructure-reinforced PPF nanocomposites and the reason for the enhanced mechanical properties compared to the controls.

Fabrication and Characterization of Three-Dimensional Macroscopic All-Carbon Scaffolds.

We report a simple method to fabricate macroscopic, 3-D, free standing, all-carbon scaffolds (porous structures) using multiwalled carbon nanotubes (MWCNTs) as the starting materials. The scaffolds prepared by radical initiated thermal crosslinking, and annealing of MWCNTs possess macroscale interconnected pores, robust structural integrity, stability, and conductivity. The porosity of the three-dimensional structure can be controlled by varying the amount of radical initiator, thereby allowing the design of porous scaffolds tailored towards specific potential applications. This method also allows the fabrication of 3-D scaffolds using other carbon nanomaterials such as single-walled carbon nanotubes, fullerenes, and graphene indicating that it could be used as a versatile method for 3-D assembly of carbon nanostructures with pi bond networks.

Synthesis and Characterization of Carbon Microbeads.

We report a microfluidic-based droplet generation platform for synthesizing micron-sized porous carbon microspheres. The setup employs carbon materials such as graphite, carbon nanotubes, graphene, fullerenes, and carbon black as starting materials. Custom composition, structure, and function are achieved through combinations of carbon materials, cross-linkers, and additives along with variations in process parameters. Carbon materials can be assembled into spheres with a mean diameter of units to hundreds of µm with relatively tight size distribution (<25% RSD). Pore structure and size (tens to hundreds of angstrom) can be modulated by incorporating porogen/coporogen dilutants during synthesis. The microbeads have excellent mechanical stability with an elastic modulus of hundreds of MPa. They can sustain high dynamic fluid flow pressures of up to 9000 psi. This work lays the foundation for synthesizing novel tailorable and customizable carbon microbeads. It opens avenues for applying these novel materials for composite and additive manufacturing, energy, life science, and biomedical applications.

Delivery of long chain C16 and C24 ceramide in HeLa cells using oxidized graphene nanoribbons.

The bioactive sphingolipid ceramide has many important roles in cell signaling processes, particularly in signaling programmed cell death in cancer. However, ceramide levels are often impaired in multi-drug resistant and radiation resistant cancers due to the dysregulation of ceramide metabolism. Restoration of ceramide levels through external delivery therefore represents a potential therapeutic target for the treatment of resistant cancers. However, as a lipid, ceramide is extremely hydrophobic and requires a delivery system to enter cells. Here we report the development of a method to load significant amounts of the long chain C16 and C24 ceramides onto oxidized graphene nanoribbons (O-GNRs) derived from carbon nanotubes. Using O-GNRs as a delivery system for these ceramides, we were able to induce significant biological effects in HeLa cells in conjunction with C6 ceramide and ultraviolet radiation treatment. However, we found that O-GNRs themselves exert significant biological effects and can interfere with the actions of these ceramides and ultraviolet treatment. Loading of ceramides onto O-GNRs did not have a significant effect on the entry of the nanoparticles into cells. Despite the need for further improvement, these data represent an important first step in the development of O-GNRs as a delivery system for long chain ceramides.

Nanofilm generated non-pharmacological anabolic bone stimulus.

Stimulus-responsive nanomaterials have mainly been employed to ablate or destroy tissues or to facilitate controlled release of drugs or biologics. Herein, we demonstrate the potential of stimulus-responsive nanomaterials to promote tissue regeneration via a non-pharmacological and noninvasive strategy. Thin nanofilms of an optically-absorbing organic dye or nanoparticle (single-walled graphene nanoribbons [SWOGNR]) were placed over (without touching the skin) a rodent femoral fracture site. A nanosecond pulsed near-infrared laser diode was employed to generate photoacoustic (PA) signals from the nanofilms. X-ray micro-computed tomography (microCT), histology, and mechanical testing results showed that daily PA stimulations of upto 45 min for 6 weeks (complete fracture healing) do not adversely affect bone regeneration and quality. Further, microCT and histological analysis showed 10 min daily stimulation for 2 weeks significantly increases bone quantity at the fracture sites of rats exposed to the nanoparticle-generated PA signals. In these rats, up to threefold increase in bone volume to callus volume ratio and twofold increase in bone mineral density within the callus were noted, compared to rats that were not exposed to the photoacoustic signals. The results taken together indicate that nanofilm-generated photoacoustic signals serve as an anabolic stimulus for bone regeneration. The results, in conjugation with the ability of these nanofilms to serve as PA contrast agents, present opportunities toward the development of integrated noninvasive imaging and noninvasive or invasive treatment strategies for bone loss due to disease or trauma.

Single-walled carbon nanotubes as a multimodal-thermoacoustic and photoacoustic-contrast agent.

We have developed a novel carbon nanotube-based contrast agent for both thermoacoustic and photoacoustic tomography. In comparison to deionized water, single-walled carbon nanotubes exhibited more than twofold signal enhancement for thermoacoustic tomography at 3 GHz. In comparison to blood, they exhibited more than sixfold signal enhancement for photoacoustic tomography at 1064 nm wavelength. The large contrast enhancement of single-walled carbon nanotubes was further corroborated by tissue phantom imaging studies.

In vivo carbon nanotube-enhanced non-invasive photoacoustic mapping of the sentinel lymph node.

Sentinel lymph node biopsy (SLNB), a less invasive alternative to axillary lymph node dissection (ALND), has become the standard of care for patients with clinically node-negative breast cancer. In SLNB, lymphatic mapping with radio-labeled sulfur colloid and/or blue dye helps identify the sentinel lymph node (SLN), which is most likely to contain metastatic breast cancer. Even though SLNB, using both methylene blue and radioactive tracers, has a high identification rate, it still relies on an invasive surgical procedure, with associated morbidity. In this study, we have demonstrated a non-invasive single-walled carbon nanotube (SWNT)-enhanced photoacoustic (PA) identification of SLN in a rat model. We have successfully imaged the SLN in vivo by PA imaging (793 nm laser source, 5 MHz ultrasonic detector) with high contrast-to-noise ratio (=89) and good resolution ( approximately 500 microm). The SWNTs also show a wideband optical absorption, generating PA signals over an excitation wavelength range of 740-820 nm. Thus, by varying the incident light wavelength to the near infrared region, where biological tissues (hemoglobin, tissue pigments, lipids and water) show low light absorption, the imaging depth is maximized. In the future, functionalization of the SWNTs with targeting groups should allow the molecular imaging of breast cancer.

Oxidized graphene nanoparticles as a delivery system for the pro-apoptotic sphingolipid C6 ceramide.

Sphingolipids such as ceramide have attracted much attention as possible anticancer agents due to their potent pro-apoptotic effects. However, due to their extreme hydrophobicity, there is currently no clinically approved delivery method for in vivo use as a therapeutic agent. To this end, we have developed a novel method for loading the short-chain C6 ceramide onto oxidized graphene nanoribbons (O-GNRs) and graphene nanoplatelets (GNPs). Mass spectrometry revealed loading efficiencies of 57% and 51.5% for C6 ceramide onto O-GNRs and GNPs, respectively. The PrestoBlue viability assay revealed that 100 µg/mL of C6 ceramide-loaded O-GNRs and C6 ceramide-loaded GNPs reduced HeLa cell viability by approximately 93% and approximately 76%, respectively, compared to untreated HeLa cells, while equal concentrations of these nanoparticles without C6 ceramide did not significantly reduce HeLa cell viability. We confirmed that this cytotoxicity was apoptotic in nature via capase-3 activity and Hoechst staining. Using live-cell confocal imaging with the fluorescent NBD-ceramide loaded on O-GNRs, we observed robust uptake into HeLa cells within 30 min while NBD-ceramide on its own was uptaken much more rapidly. Transmission electron microscopy confirmed that C6 ceramide-loaded O-GNRs were actually entering cells. Taken together, these data show that O-GNRs are a promising delivery agent for ceramide. To our knowledge, this study is the first to use such a loading method. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 25-37, 2019.

Two-dimensional graphene oxide-reinforced porous biodegradable polymeric nanocomposites for bone tissue engineering.

This study investigates the mechanical properties and in vitro cytotoxicity of two-dimensional (2D) graphene oxide nanoribbons and nanoplatelets (GONRs and GONPs) reinforced porous polymeric nanocomposites. Highly porous poly(propylene fumarate) (PPF) nanocomposites were prepared by dispersing 0.2 wt % single- and multiwalled SONRs (SWGONRs and MWGONRs) and GONPs. The mechanical properties of scaffolds were characterized using compression testing and in vitro cytocompatibility was assessed using QuantiFlour assay for cellularity and PrestoBlue assay for cell viability. Immunofluorescence was used to assess collagen-I expression and deposition in the extracellular matrix. Porous PPF scaffolds were used as a baseline control and porous single and multiwalled carbon nanotubes (SWCNTs and MWCNTs) reinforced nanocomposites were used as positive controls. Results show that incorporation of 2D graphene nanomaterials leads to an increase in the mechanical properties of porous PPF nanocomposites with following the trend: MWGONRs > GONPs > SWGONRs > MWCNTs > SWCNTs > PPF control. MWGONRs showed the best enhancement of compressive mechanical properties with increases of up to 26% in compressive modulus (i.e., Young's modulus), ~60% in yield strength, and ~24% in the ultimate compressive strength. Addition of 2D nanomaterials did not alter the cytocompatibility of porous PPF nanocomposites. Furthermore, PPF nanocomposites reinforced with SWGONRs, MWGONRs, and GONPs show an improvement in the adsorption of collagen-I compared to PPF baseline control. The results of this study show that 2D graphene nanomaterial reinforced porous PPF nanocomposites possess superior mechanical properties, cytocompatibility, and increased protein adsorption. The favorable cytocompatibility results opens avenues for in vivo safety and efficacy studies for bone tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1143-1153, 2019.

In vivo biocompatibility of ultra-short single-walled carbon nanotube/biodegradable polymer nanocomposites for bone tissue engineering.

Scaffolds play a pivotal role in the tissue engineering paradigm by providing temporary structural support, guiding cells to grow, assisting the transport of essential nutrients and waste products, and facilitating the formation of functional tissues and organs. Single-walled carbon nanotubes (SWNTs), especially ultra-short SWNTs (US-tubes), have proven useful for reinforcing synthetic polymeric scaffold materials. In this article, we report on the in vivo biocompatibility of US-tube reinforced porous biodegradable scaffolds in a rabbit model. US-tube nanocomposite scaffolds and control polymer scaffolds were implanted in rabbit femoral condyles and in subcutaneous pockets. The hard and soft tissue response was analyzed with micro-computed tomography (micro CT), histology, and histomorphometry at 4 and 12 weeks after implantation. The porous US-tube nanocomposite scaffolds exhibited favorable hard and soft tissue responses at both time points. At 12 weeks, a three-fold greater bone tissue ingrowth was seen in defects containing US-tube nanocomposite scaffolds compared to control polymer scaffolds. Additionally, the 12 week samples showed reduced inflammatory cell density and increased connective tissue organization. No significant quantitative difference in polymer degradation was observed among the various groups; qualitative differences between the two time points were consistent with expected degradation due to the progression of time. Although no conclusions can be drawn from the present study concerning the osteoinductivity of US-tube nanocomposite scaffolds, the results suggest that the presence of US-tubes may render nanocomposite scaffolds bioactive assisting osteogenesis.

Synthesis and conformational evaluation of a novel gene delivery vector for human mesenchymal stem cells.

We have synthesized a novel gene delivery vector by covalently combining branched polyethylenimine (bPEI) and hyaluronic acid (HA) with the aim of improving transfection of bPEI into human mesenchymal stem cells (hMSCs) while maintaining cell viability. Because of the opposite charges on bPEI and HA, the bPEI-HA vector forms a zwitterionic polymer capable of inter- and intramolecular interactions. We have characterized the hydrodynamic radius of bPEI-HA and bPEI-HA/DNA complexes at ambient and physiological temperatures, as well as at a range of salt concentrations using light scattering, and investigated the effect of the size of transfecting complexes on gene delivery. We found that by increasing the salt concentration from 150 to 1000 mM of NaCl, the mean hydrodynamic radius (R(h)) of bPEI-HA increases from 2.0 +/- 1.1 to 366.0 +/- 149.0 nm. However, increasing the salt concentration decreases the mean R(h) of bPEI-HA/DNA complexes from 595.0 +/- 44.6 to 106.0 +/- 19.2 nm at 25 degrees C and from 767.0 +/- 137.2 to 74.0 +/- 23.0 nm at 37 degrees C. hMSCs transfected with smaller complexes showed a significant increase in transfection from 3.8 +/- 1.5% to 19.1 +/- 4.4%. Similarly, bPEI-HA performed significantly better than bPEI in terms of cell viability (86.0 +/- 6.7% with bPEI-HA versus 7.0 +/- 2.8% with bPEI, 24 h post exposure at the highest concentration of 500 mg/mL) and maximum transfection efficiencies (12.0 +/- 4.2% with bPEI/DNA complexes and 33.6 +/- 13.9% with bPEI-HA/DNA complexes). Thus, modifying bPEI by covalent conjugation with HA improves its performance as a gene delivery vector in hMSCs. This presents a promising approach to altering hMSCs for tissue engineering and other applications.

In Vitro Bioactivity of One- and Two-Dimensional Nanoparticle-Incorporated Bone Tissue Engineering Scaffolds.

This study investigates the effect of incorporation of one- or two-dimensional nanoparticles with distinct composition and morphology on the bioactivity of biodegradable, biocompatible polymer matrices. 0.2 wt% multiwalled carbon nanotubes, multiwalled graphene nanoribbons, graphene oxide nanoplatelets (GONPs), molybdenum disulfide nanoplatelets (MSNPs), or tungsten disulfide nanotubes (WSNTs) were uniformly dispersed in poly(lactic-co-glycolic acid) (PLGA) polymer. PLGA or nanoparticle-incorporated PLGA were then incubated with simulated body fluid (SBF) under physiological conditions for 1, 3, 7, or 14 days. Apatite collection on control and incorporated scaffolds was assessed. All groups showed apatite precipitate on the surface after 1 day of SBF incubation. After 14 days of SBF incubation, scaffolds incorporated with GONPs, MSNPs, or WSNTs showed significantly higher phosphate accumulation compared to PLGA scaffolds. Scaffolds incorporated with GONPs, MSNPs, or WSNTs should be studied in vivo to further investigate potential bioactivity, leading to enhanced integration and tissue repair at the bone-implant interface.

Osteogenic differentiation of human adipose derived stem cells on chemically crosslinked carbon nanomaterial coatings.

Carbon nanomaterial coatings have been widely investigated for many biomedical applications including bone tissue engineering. Current methods to fabricate carbon nanomaterial coatings are limited by specific substrate requirements and the lack of strong bonds between the nanomaterials. Furthermore, few studies compare the effect of carbon nanoparticle architecture on stem cell differentiation and mineralization for osteogenic differentiation. Herein, we report a study comparing chemically crosslinked carbon nanotubes (of various diameters), graphene nanoplatelets, and graphene nanoribbons coatings for adipose derived stem cell differentiation toward an osteogenic lineage. We observed greatest autodeposition of calcium on graphene nanoribbon surfaces, while multiwalled carbon nanotubes of high diameter had the greatest influence on stem cell fate (by alkaline phosphatase activity, calcium deposition, and osteocalcin measurements). Studies indicate the cause for multiwalled carbon nanotube related stem cell differentiation, may be related to early timepoint toxicity as indicated by lactose dehydrogenase release. These results indicate suggestions for orthopedic tissue engineering applications for carbon nanomaterial coatings. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1189-1199, 2018.

Fabrication of porous ultra-short single-walled carbon nanotube nanocomposite scaffolds for bone tissue engineering.

We investigated the fabrication of highly porous scaffolds made of three different materials [poly(propylene fumarate) (PPF) polymer, an ultra-short single-walled carbon nanotube (US-tube) nanocomposite, and a dodecylated US-tube (F-US-tube) nanocomposite] in order to evaluate the effects of material composition and porosity on scaffold pore structure, mechanical properties, and marrow stromal cell culture. All scaffolds were produced by a thermal-crosslinking particulate-leaching technique at specific porogen contents of 75, 80, 85, and 90 vol%. Scanning electron microcopy, microcomputed tomography, and mercury intrusion porosimetry were used to analyze the pore structures of scaffolds. The porogen content was found to dictate the porosity of scaffolds. There was no significant difference in porosity, pore size, and interconnectivity among the different materials for the same porogen fraction. Nearly 100% of the pore volume was interconnected through 20microm or larger connections for all scaffolds. While interconnectivity through larger connections improved with higher porosity, compressive mechanical properties of scaffolds declined at the same time. However, the compressive modulus, offset yield strength, and compressive strength of F-US-tube nanocomposites were higher than or similar to the corresponding properties for the PPF polymer and US-tube nanocomposites for all the porosities examined. As for in vitro osteoconductivity, marrow stromal cells demonstrated equally good cell attachment and proliferation on all scaffolds made of different materials at each porosity. These results indicate that functionalized ultra-short single-walled carbon nanotube nanocomposite scaffolds with tunable porosity and mechanical properties hold great promise for bone tissue engineering applications.

In Vivo Hard and Soft Tissue Response of Two-Dimensional Nanoparticle Incorporated Biodegradable Polymeric Scaffolds.

Current efforts in the design of bone tissue engineering scaffolds have focused on harnessing the physiochemical properties of two-dimensional organic and inorganic nanoparticles to improve bulk and surface properties of biodegradable polymers. Herein, we investigate the hard and soft tissue in vivo biocompatibility of two such constructs: 90% porous poly(lactic-co-glycolic acid) (PLGA) nanocomposite scaffolds incorporated with 0.2 wt % graphene oxide nanoplatelets (GONPs) or molybdenum disulfide nanoplatelets (MSNPs). Scaffolds were implanted in a noncritical sized monocortical defect in the tibia or subcutaneously on the dorsum of a rat model for 2 or 6 weeks. Hard and soft tissue in vivo biocompatibility of the nanoparticle reinforced scaffolds was comparable to that of the PLGA control. In addition, 2 weeks after implantation, significantly less bone growth (∼35%) was observed for the PLGA group compared to that of the empty defect group; it was not observed for the experimental groups which showed 20% and 15% greater bone growth compared to that of the PLGA group. This may indicate that the nanoparticles do play a role in assisting bone regeneration. Taken together, the results suggest that scaffolds incorporated with GONPs or MSNPs show promise for bone tissue engineering applications.

Boron nitride nanotubes and nanoplatelets as reinforcing agents of polymeric matrices for bone tissue engineering.

This study investigates the mechanical properties and in vitro cytotoxicity of one- and two-dimensional boron nitride nanomaterials-reinforced biodegradable polymeric nanocomposites. Poly(propylene fumarate) (PPF) nanocomposites were fabricated using crosslinking agent N-vinyl pyrrolidone and inorganic nanomaterials: boron nitride nanotubes (BNNTs) and boron nitride nanoplatelets (BNNPs) dispersed at 0.2 wt % in the polymeric matrix. The incorporation of BNNPs and BNNTs resulted in a ∼38 and ∼15% increase in compressive (Young's) modulus, and ∼31 and ∼6% increase in compressive yield strength compared to PPF control, respectively. The nanocomposites showed a time-dependent increased protein adsorption for collagen I protein. The cytotoxicity evaluation of aqueous BNNT and BNNP dispersions (at 1-100 µg/mL concentrations) using murine MC3T3 preosteoblast cells showed ∼73-99% viability. The cytotoxicity evaluation of media extracts of nanocomposites before crosslinking, after crosslinking, and upon degradation (using 1×-100× dilutions) showed dose-dependent cytotoxicity responses. Crosslinked nanocomposites showed excellent (∼79-100%) cell viability, cellular attachment (∼57-67%), and spreading similar to cells grown on the surface of tissue culture polystyrene control. The media extracts of degradation products showed a dose-dependent cytotoxicity. The favorable cytocompatibility results in combination with improved mechanical properties of BNNT and BNNP nanocomposites opens new avenues for further in vitro and in vivo safety and efficacy studies towards bone tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 406-419, 2017.

Gene delivery to mammalian cells using a graphene nanoribbon platform.

We have developed a novel oxidized graphene nanoribbon-based platform (O-GNR) for gene delivery of double-stranded DNA into mammalian cells. O-GNRs, synthesized via longitudinal unzipping of multi-walled carbon nanotubes (MWCNTs), exhibited efficient DNA loading of small dsDNA fragments. Fourier Transform Infrared Spectroscopy identified stretching peaks in the O-P-O and DNA sugar phosphate backbone that were consistent with DNA loading onto O-GNRs. The presence of salts in the loading buffer promoted DNA loading and effective dispersion of O-GNRs. DNA:O-GNR complexes were stable upon treatment with surfactants Tween 20 and Triton-X100. O-GNRs did not impact the viability of mammalian cells. Last, the detection of GFP expression upon transfection of the DNA:O-GNR complex indicated that the cargo DNA is expressed in the nucleus. Taken together, O-GNRs function as a platform for gene delivery to mammalian cells.

Three-dimensional macroporous graphene scaffolds for tissue engineering.

The assembly of carbon nanomaterials into three-dimensional (3D) porous scaffolds is critical to harness their unique physiochemical properties for tissue engineering and regenerative medicine applications. In this study, we report the fabrication, characterization, and in vitro cytocompatibility of true 3D (>1 mm in all three dimensions), macroscopic (3-8 mm in height and 4-6 mm in diameter), chemically cross-linked graphene scaffolds prepared via radical initiated thermal cross-linking of single- and multiwalled graphene oxide nanoribbons (SWGONRs and MWGONRs). SWGONR and MWGONR scaffolds possess tunable porosity (∼65-80%) and interconnected macro-, micro-, and nanoscale pores. Human adipose derived stem cells (ADSCs) and murine MC3T3 preosteoblast cells show good cell viability on SWGONR and MWGONR scaffolds after 1, 3, and 5 days comparable to 3D poly(lactic-co-glycolic) acid (PLGA) scaffolds. Confocal live-cell imaging showed that cells were metabolically active and could spread on SWGONR and MWGONR scaffolds. Immunofluorescence imaging showed the presence of focal adhesion protein vinculin and expression of cell proliferation marker Ki-67 suggesting that cells could attach and proliferate on SWGONR and MWGONR scaffolds. These results indicate that cross-linked SWGONR and MWGONR scaffolds are cytocompatible and opens-avenues toward the development of 3D multifunctional graphene scaffolds for tissue engineering applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 73-83, 2017.