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Systemic Administration of Polymer-Coated Nano-Graphene to Deliver Drugs to Glioblastoma Thomas L. Moore, Rama Podilakrishna, Apparao Rao, and Frank Alexis* drug loading via π–π stacking and hydrophobic interactions while providing a large area for drug loading.[17,18] Furthermore, nGr has been shown to have high accumulation within tumors, and PEG-coated graphene sheets were injected intravenously into tumor-bearing mice.[19] Significant accumulation of nGr was shown in 4T1 breast cancer, KB human epidermoid carcinoma, and U-87 human glioblastoma subcutaneous tumor xenografts. We therefore pursued nGr sheets as a potential candidate for systemic drug delivery vehicle to intracranial glioblastoma tumor xenografts, and functionalized nGr with poly(lactide)-poly(ethylene glycol) (PLA-PEG) for the controlled delivery of paclitaxel (PTX). PLA and PEG are FDAapproved biomaterials, and PLA-PEG has been used clinically as a nanoparticle drug delivery system (NPDDS).[20–24] PLAPEG was coated onto nGr using a one-pot ring-opening polymerization directly from the hydroxyl-functionalized nGr surface. Coating with biocompatible, biodegradable PLA-PEG reduced the cytotoxicity of non-coated nGr in U-138 human glioblastoma in vitro. The polymer coating also enabled the encapsulation of PTX, and controlled release over a 19-d period. Finally, nGr-PLA-PEG was shown to passively accumulate in U-138 intracranial xenografts in athymic BALB/c mice. NPDDS are expected to improve the efficacy of conventional chemotherapy agents due to nonspecific accumulation of nanoparticles within tumors, active targeting of over-expressed cell-surface proteins on cancerous cells, improving circulation time of drugs, and protecting drugs from premature degradation. NPDDS may also reduce the systemic toxicity of drugs by reducing interaction with non-target tissues. Physicochemical properties (e.g., shape, size, and surface charge) of NPDDS have been shown to mediate their interaction with the body on cellular and systemic levels.[12,25–37] Specifically, shape has previously been shown to have a critical role in nanoparticle biodistribution, accumulation in tumors, interaction with the body, and an interesting attribute of nGr is its plate-like, 2D shape.[25,27–29,38–40] nGr has been shown to accumulate in tumors,[19] and has longer circulation time compared to singlewalled carbon nanotubes (CNTs) and fullerene.[41] Therefore, nGr offers interesting properties as a drug delivery vehicle. Drugs have previously been loaded onto nGr via noncovalent adsorption (π–π stacking and hydrophobic interactions), and

Graphene—2D carbon—has received significant attention thanks to its electronic, thermal, and mechanical properties. Recently, nano-graphene (nGr) has been investigated as a possible platform for biomedical applications. Here, a polymer-coated nGr to deliver drugs to glioblastoma after systemic administration is reported. A biodegradable, biocompatible poly(lactide) (PLA) coating enables encapsulation and controlled release of the hydrophobic anticancer drug paclitaxel (PTX), and a hydrophilic poly(ethylene glycol) (PEG) shell increases the solubility of the nGr drug delivery system. Importantly, the polymer coating mediates the interaction of nGr with U-138 glioblastoma cells and decreases cytotoxicity compared with pristine untreated nGr. PLAPEG-coated nGr is also able to encapsulate PTX at 4.15 wt% and sustains prolonged PTX release for at least 19 d. PTX-loaded nGr-PLA-PEGs are shown to kill up to 20% of U-138 glioblastoma cells in vitro. Furthermore, nGr-PLAPEG and CNT-PLA-PEG, two carbon nanomaterials with different shapes, are able to kill U-138 in vitro as well as free PTX at significantly lower doses of drug. Finally, in vivo biodistribution of nGr-PLA-PEG shows accumulation of nGr in intracranial U-138 glioblastoma xenografts and organs of the reticuloendothelial system.

1. Introduction Graphene, an atom-thin layer of sp2-bonded carbon, has been gaining much attention thanks to its unique mechanical, electronic, and optical properties. While graphene has been effectively used to realize novel electronic and optical devices, its applications in biomedical field are still in nascent stages.[1,2] Graphene is now slowly expanding its territory toward biomedical areas such as enhanced biosensing through surfaceplasmon-coupled emission, graphene-based stent coatings that could decrease protein adsorption, graphene-mediated photodynamic therapy, and enhance cell differentiation and growth.[3–16] Carbon nanomaterials, particularly nano-graphene (nGr), are attractive drug delivery vehicles because they allow hydrophobic

Dr. T. L. Moore, Prof. F. Alexis Department of Bioengineering Clemson University Clemson, SC 29634, USA E-mail: [email protected] Prof. R. Podilakrishna, Prof. A. Rao Department of Physics Clemson University Clemson, SC 29634, USA

DOI: 10.1002/ppsc.201300379

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Part. Part. Syst. Charact. 2014, 31, 886–894



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Normalized Intensity

Raman spectroscopic studies have shown the presence of an overtone of disorder band (referred to as the 2D band) for nGr samples that exhibited two peaks at ≈2690 cm−1 and 2740 cm−1. The 2D band in graphene is highly sensitive to the number of layers and exhibits 1, 4, and 6 distinguishable peaks for single-, bi-, and tri-layered graphene owing to the change in their electronic structure.[43–46] At a higher number of layers (>5), multiple vibrational modes of graphene are not easily resolved due to close spacing and appear as two broad prominent peaks, as shown in Figure 2. These nanoparticles were used for initiating PLA polymerization directly onto Figure 1. Schematic representation of nano-graphene (nGr) coated with poly(lactide)- hydroxyl-functionalized nGr (nGr-OH) surpoly(ethylene glycol) (PLA-PEG). Hydrophobic PLA is able to encapsulate the hydrophobic faces via a one-pot, room temperature ringanticancer drug paclitaxel (PTX), while hydrophilic PEG stabilizes the nanoparticle. opening polymerization. nGr-OH was mixed with D,L-lactide in anhydrous acetonitrile adsorption with electrostatic interaction.[4,6,7,11,12,42] However, (ACN), and PLA polymerization was catalyzed by the phospdrug adsorption on the surface of nGr is suitable for drugs hazene base P2-t-Bu. Hetero-bifunctional mPEG-isc was added that physically interact with graphene only. Here, we report a directly into this reaction without any washing. The rationale different approach to use nGr as a drug delivery vehicle based behind this step was to react the isocyanate group with on functionalizing nGr with a biocompatible, biodegradable hydroxyl-chain ends of PLA. This forms a urethane linkage and polymer. Coating with PLA-PEG enables the encapsulation of effectively terminates the polymerization reaction. GrLP was hydrophobic drugs such as PTX within the hydrophobic PLA characterized via thermogravimetric analysis (TGA). Samples matrix, while PEG improves the aqueous solubility of the were heated under a nitrogen gas stream, and mass loss was NPDDS. Figure 1 shows an illustrative representation of this measured (Figure 3A). Uncoated nGr showed only 5 wt% mass drug delivery vehicle. PLA was polymerized directly onto the loss. An aliquot of nGr-PLA was tested, showing a PLA coating hydroxyl-functionalized nGr surface via a room temperature of ≈20 wt%. After PEGylation, GrLP samples showed a mass ring-opening polymerization. This reaction was capped with loss of ≈80 wt%, indicating roughly 60 wt% PEG coating. Transa hetero-bifunctional methoxy-PEG-isocyanate (mPEG-isc) by mission electron microscopy also showed morphological differreacting the isocyanate group with terminal hydroxyl groups ence between uncoated and PLA-PEG-coated nGr (Figure 3B), from PLA to prepare the end product, nGr-PLA-PEG (GrLP).[17] and 1H nuclear magnetic resonance (NMR) spectroscopy verified the presence of a PLA coating. An aliquot of nGr-PLA showed characteristic peaks at 1.57 and 5.20 ppm. After addition of mPEG-isc, the final GrLP product was tested showing 2. Results and Discussion characteristic peaks for PLA, and a characteristic peak for PEG at 3.65 ppm. Fourier transform infrared spectroscopy (FTIR) 2.1. Nanomaterial Synthesis further verifies the presence of a polymeric coating. Figure 4 shows characteristic peaks for PLA-PEG, such as a carbonyl nGr was synthesized from bulk graphite, and hydroxylated stretch at ≈1780 cm−1. These methods of characterization prowith sulfuric acid. nGr was plate-shaped with 5 nm thickvide strong evidence of successful coating. ness, and was roughly 3 × 3 µm. As shown in Figure 2, our

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2.2. In Vitro Cytotoxicity, Drug Release, and Therapeutic Efficacy

nGr 514.5 nm

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Wavenumber(cm ) Figure 2. The shape of 2D band in the nGr sample Raman spectrum confirms the presence of few-layer graphene in our samples.


Both PLA and PEG are FDA-approved polymers, and PLA-PEG nanoparticles have entered either clinical trials in the United States.[20–22,47] Thus, the rationale for this polymeric coating was to mitigate potential carbon nanomaterial-related toxicity. Carbon-based nanomaterials have been extensively studied for biomedical applications and toxicity.[17–19,36,48–52] Graphene nanosheet toxicity has been shown to be related to size, especially with respect to the lateral dimension.[53,54] Akhavan et al.[53] have previously shown that graphene oxide nanoplatelets with a lateral dimension of 11.4 nm caused cytotoxicity at 1 µg mL−1, and were able to penetrate the nucleus and



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Figure 3. Materials characterization of coated nano-graphene (GrLP). A) Thermogravimetric analysis shows a 20 wt% PLA coating, and a 60 wt% PEG coating. B) Raman spectrum of nGr before and after coating with PLA-PEG. The strong graphitic band (≈1585 cm−1) indicated that the nGr samples exhibited excellent crystallinity with relatively low-defect density, as suggested by a weak and broad defect band at 1350 cm−1. C,D) Transmission electron microscopy images of uncoated nGr (C), and PLA-PEG-coated nGr (D) show clear morphological differences. Scale bars represent 500 nm. E,F) NMR spectroscopy of nGr coated with only PLA (E) and with PLA-PEG (F) shows characteristic peaks for the two polymers.

pathways.[55,56] Moreover, systemic toxicity after intravenous injection at 20 mg kg−1 was measured by changes in body mass, and several blood markers such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase (ALP), and albumin/globulin ratios. No systemic toxicity was observed up to 90 d following injection. In another study, male Wistar rats were exposed to various carbon nanomaterials via inhalation.[49] Bronchoalveolar lavage analysis at 7 d following inhalation at 10 mg m−3 showed significant increase in expression of lymphocytes, and polymorphonuclear neutrophils compared with concurrent control groups. Lung tissue showed increased expression of activity of γ-glutamyltranspeptidase, lactate dehydrogeFigure 4. FTIR analysis of PLA-PEG-coated nGr confirms the presence of a polymer coating. nase, and ALP. While nanomaterial toxicity is related to a number of factors, including * Indicates a carbonate impurity in the KBr pellet that serves as an internal reference.

show signs of genotoxicity. Previously, PEGylated nGr has also been injected into mice, and biodistribution and toxicity were evaluated.[51] As expected, nGr accumulated primarily in the liver and spleen. This study suggested that nGr was cleared via renal excretion, or potentially degraded via oxidative metabolic

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FULL PAPER Figure 5. Cytotoxicity of uncoated nGr, and GrLP with U-138 glioblastoma. A) Live/Dead microscopy showed live cells in green, and dead cells in red. Microscopy higher concentrations of nGr resulted in lower cell density, and an increased number in dead cells. Qualitatively, higher concentrations of GrLP showed less decrease in cell density, and less dead cells. Scale bars represent 500 µm. B) PrestoBlue cell viability assays showed quantitatively that only nGr at a concentration of 250 µg mL−1 was significantly toxic compared with cells treated with only media. Significance was determined with a Tukey’s honestly significant difference test.

route of administration, previous work with multiwalled carbon nanotubes has shown that PLA-PEG coatings can reduce toxicity.[17] Toxicity from nGr may be due to its hydrophobicity and tendency to aggregate in aqueous solutions. A variety of covalent and noncovalent approaches for nGr surface functionalization have been explored to improve their aqueous dispersion. Surface modifications include Pluronic,[57] PEG,[4,5,51] chitosan,[8] folic acid,[6] poly(N-isopropylacrylamide),[7] poly(2(diethylamino) ethyl methacrylate),[10] and so on. However, to our knowledge, this is the first report of nGr covalently functionalized with the biodegradable, biocompatible copolymer PLA-PEG. Here, cytotoxicity was measured in vitro against U-138 glioblastoma cells using Live/Dead and PrestoBlue cell viability assays (Figure 5). Cells were treated with cell culture media (live cell control), 70% ethanol (dead cell control), increasing doses of uncoated nGr, or increasing doses of GrLP. Except for the dead cell control, cells were exposed to nanomaterials for 24 h. For the dead cell control, cells were treated with 70% ethanol for 5 min immediately before beginning the assay. In this assay, live cells were green, and dead cells were red. Uncoated nGr showed toxicity at doses higher than 50 µg mL−1. This was noted by both the increase in dead cells, and also the decrease in overall cell population density. However, the coated GrLP did not show signs of dose-dependent toxicity up to 250 µg mL−1.


It appears that coating nGr with the multifunctional PLA-PEG decreases cytotoxicity in U-138 cells, and from this Live/Dead data the GrLP did not show toxicity at a fivefold increase in concentration compared with nGr. Thus, it is apparent that coating with PLA-PEG improves the safety of nGr as a drug delivery vehicle. This is possibly due to the biocompatible polymer coating mediating the interaction between the carbon-based nGr, and also the PEG increasing the solubility of nGr. The PrestoBlue cell viability assay showed similar results, where only the nGr sample at 250 µg mL−1 was significantly more toxic than the control. Means were compared using a Tukey’s honestly significant difference test. PTX release was measured over a 19-d period (Figure 6). GrLP was able to load ≈4.15 wt% PTX, while CLP loaded ≈0.4 wt%. Differences in loading were potentially due to different specific surface areas of the nanomaterials. GrLP also released ≈6% of total loaded PTX within 19 d. Thus, it appears that GrLP is a capable vehicle for sustained drug delivery. PTX was encapsulated within the PLA coating via a solvent evaporation approach. Briefly, GrLP and PTX were dissolved together in ACN. This solution was dropped into water and stirred, allowing ACN to evaporate and for hydrophobic–hydrophobic interactions to encapsulate PTX within the PLA matrix. PTX-loaded GrLP at 25 µg mL−1 was used to treat U-138 glioblastoma in vitro. Efficacy data for PTX-loaded GrLP were compared with PTX-loaded



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CLP, respectively. While these estimations are based on release kinetics of PTX in H2O, these data suggest that GrLP and CLP were able to use significantly less drug to achieve the same efficacy as free PTX at 140 × 10−9 M. Differences in efficacy of GrLP may also be attributable to low cellular uptake of the 2D nGr. It has been previously shown that size plays a significant role in the uptake kinetics of nGr.[58] Large and small nGr sheets, with mean equivalent diameters of 860 and 420 nm, respectively, were dispersed by adsorbing plasma proteins to the surface. Kinetic uptake studies showed that significantly higher amounts of small nGr were uptaken after 14 h, compared with large nGr. Thus, further investigation into the influence of nGr dimensions into cellular uptake is required to optimize the therapeutic efficacy of these GrLP NP. 2.3. In Vivo Biodistribution Figure 6. Controlled release of paclitaxel (PTX) from GrLP. PTX could be loaded at 4.15 wt%, and release was sustained over a 19-d period.

PLA-PEG-coated carbon nanotubes (CLP), and free PTX at 140 × 10−9 M. We previously explored PLA-PEG-coated CNT as a drug delivery vehicle.[17] GrLP was loaded with PTX as described, and incubated with U-138 cells for 6 or 24 h. After the initial incubation, cells were washed gently with phosphatebuffered saline (PBS) and allowed to grow for a total of 72 h following initial exposure. At 72 h, cell viability was measured using a PrestoBlue assay (Figure 7). Cells were treated with free PTX similarly, and cells treated with only media were used as a control. Neither NP formulation was shown to be significantly improved compared with free PTX at 140 × 10−9 M. Using release kinetics data, we estimated the amount of PTX released at 72 h to be ≈24.6 and 12.1 × 10−9 M for GrLP and

Figure 7. Efficacy of paclitaxel (PTX)-loaded PLA-PEG-coated nanographene (GrLP-PTX) at PTX concentration approximately equivalent to 24.6 × 10−9 M, and PTX-loaded PLA-PEG-coated carbon nanotubes (CLP-PTX), PTX concentration equivalent to 12.1 × 10−9M was compared with U-138 glioblastoma treated with free PTX at 140 × 10−9M. Cells were treated with free drug or PTX-loaded nanoparticles for 6 or 24 h, and cell viability was measured at 72 h following initial exposure to treatment. Nanoparticle formulations showed equivalent efficacy even when administered at doses six- to tenfold lower.

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In order to evaluate the potential benefit of GrLP as a drug delivery vehicle, the in vivo biodistribution of GrLP was investigated in athymic BALB/c mice bearing intracranial U-138 xenografts, as nGr has previously been shown to accumulate significantly in subcutaneous tumor models in mice.[19] Here, mice were implanted with tumors following the procedure of Ozawa et al.[59] We employed an intracranial U-138 glioblastoma model to more closely estimate the behavior of GrLP in vivo using a relevant model. Alexa fluor 647 fluorescent dye was loaded into GrLP using a solvent evaporation procedure similar to the PTX loading. Dye-loaded GrLP was systemically administered to tumor-bearing Balb/c mice via tail vein injection, and fluorescent images of mice were taken at 0.5, 1, 3, 6, 12, and 24 h following injection (Figure 8A). Live animal fluorescent images showed accumulation of GrLP in the brain, and also possibly in organs of the RES system such as the spleen or liver. CLP was injected to compare biodistribution between CNT and nGr, two carbon-based nanomaterials with different shape. Live animal fluorescent images qualitatively show higher localization of CLP in the brain tumors compared with GrLP. Figure 8B (inset) shows ex vivo fluorescent images of brains, and regionof-interest (ROI) measurements of fluorescent intensity were used to semiquantitatively analyze relative fluorescent signal. CLP showed an ≈40% increase of fluorescent signal compared with the mouse that was injected with only saline. GrLP showed only an ≈1% increase compared with saline mouse. We hypothesize that CLP accumulated more in intracranial xenograft tumors compared with GrLP due to NP size and shape. GrLP was larger and plate-shaped, therefore may not have extravasated into the tumor as easily as CLP. Yang et al.[19] examined tumor accumulation of PEGylated nGr with nGr sheets that were ≈10–50 nm, and showed high relative accumulation of nGr in tumors compared with other organs. However, these nGr sheets were two orders of magnitude smaller than the nGr sheets we used. Thus, the differences in nGr size may account for differences in intratumoral accumulation. Furthermore, NP uptake may be different for a subcutaneous tumor model versus an intracranial tumor model. Therefore, further investigation into the effects of nGr size and differences due to tumor model are needed. Explanted fluorescent images show accumulation of NP in the brain, kidney, liver, and some in the





FULL PAPER Figure 8. In vivo biodistribution of PLA-PEG-coated carbon nanotubes (CLP), or nano-graphene (GrLP) in U-138 glioblastoma intracranial xenograft Balb/c mice was imaged using Alexa Fluor 647 fluorescent dye-loaded nanoparticles (NP). A) Fluorescent images were taken at 0.5, 1, 3, 6, 12, and 24 h following intravenous tail vein injection. Intracranial accumulation of NP was apparent due to passive accumulation in tumors. B) Relative amount of NP accumulated in tumors was calculated by measuring fluorescent intensity in the brain. Fluorescence measurements from CLP and GrLP were normalized by the mouse that received only saline as an injection.




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Figure 9. Fluorescent images of explanted organs for CLP (left), and GrLP (right) show accumulation of NP in organs of the reticulo-endothelial system, primarily the liver and spleen. GrLP qualitatively showed less accumulation in the spleen, compared with CLP. Explanted organs were: i) fat, ii) spleen, iii) liver, iv) kidneys, v) heart, vi) lungs, and vii) brain.

lungs and spleen (Figure 9). Compared with CLP, GrLP also showed less accumulation in the spleen. Nanoparticle clearance via the reticulo-endothelial system (RES), for example, the liver and spleen, remains a challenge in drug delivery, and reduction of NP accumulation in the organs of the RES may result in higher accumulation of drug-loaded NP at the target site/ organ.[60–62] Thus, identifying factors that may result in higher NP accumulation in tumors is a pressing goal for NP-mediated drug delivery system. The difference in biodistribution between the two carbon nanomaterials, and lower accumulation of GrLP compared with CLP in the spleen may be attributable to differences in shape between these two NP.

3. Conclusion Here, we have demonstrated a method to coat nGr with PLAPEG using a one-pot ring-opening polymerization of PLA directly on the surface of hydroxyl-functionalized nGr. PEG was attached by reacting isocyanate-terminated PEG with the hydroxyl-end groups of PLA. TGA of GrLP showed a 20 wt% coating of PLA, and a 60 wt% coating of PEG. Polymer coatings were further verified via NMR spectroscopy. Live/Dead and PrestoBlue cell viability assays of U-138 glioblastoma treated with nGr and GrLP showed that GrLP was not significantly more toxic than control cells treated with media only up to 250 mg mL−1. GrLP was able to load 4.15 wt% PTX and sustain release over a 19-d period. GrLP was shown to be as effective as free PTX at an estimated sixfold lower concentration when treating U-138 glioblastoma in vitro. In vivo biodistribution studies of GrLP in U-138 xenograft Balb/c mice showed that CLP qualitatively accumulated more in tumors compared with GrLP. Graphene is a potentially beneficial nanomaterial for the controlled delivery of drugs due to high specific surface area, and unique shape. Further work is required to optimize the internalization of 2D nGr for controlled drug delivery, investigate cyto- and even geno-toxicity, in vivo blood circulation time, biodistribution, and tumor to normal organ uptake ratios. However, this is the first proof of concept to demonstrate PLA-PEG multifunctional nGr for controlled drug delivery.

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4. Experimental Section Materials Characterization: 1H NMR spectra were recorded on a Bruker Avance 300 MHz spectrometer. Samples were dissolved at 1 mg mL−1 in deuterated chloroform, and chemical shifts (δ) were expressed in ppm. 1H NMR (300 MHz, CDCl3, δ): 7.26 (s, CDCl3), 5.17 (q, C( O) CH(CH3) ), 3.65 (s, CH2CH2 O ), 1.59 (d, CH(CH3) ). Thermogravimetric analysis was recorded on a TA Instruments Hi-Res TGA 2950 thermogravimetric analyzer under nitrogen from 25 to 600 °C at 20 °C min−1. Transmission electron microscope images were taken on a Hitachi H7600T at 115kV. Raman spectra of nGr were recorded using a TE-cooled Dilor XY spectrometer with a 514.5-nm excitation. Fourier transform infrared spectroscopy was recorded on a Bruker IFS v66 vacuum FTIR at 1 mbar pressure with a globar source in the transmission mode using sample encased with a KBR pellet. Nano-Graphene Synthesis and Hydroxylation: For synthesizing nGr, bulk graphite (≈1 g) was dispersed in 100 mL of N-methyl-2-pyrrolidinone (NMP) and sonicated using 1/8” tip sonicator (Branson 250) at 100 W for 2 h. The resulting dispersion was filtered through a 0.45-µm nylon filter and resuspended in 100 mL of fresh NMP. Subsequently, the solution was bath sonicated for 6 h and centrifuged at 500 rpm for 45 min. The supernatant was vacuum filtered using a 0.45-µm nylon filter. Finally, the filtered powder was washed several times using deionized water to remove residual NMP. For hydroxylation, exfoliated graphene (2 g) was first re-dispersed in 70% H2SO4 (46 mL) and refluxed for 12 h. This reaction was terminated by the addition of a large amount of deionized water (300 mL) and 30% H2O2 solution (5 mL). Subsequently graphene was filtered and washed with deionized water. Graphene was then suspended in aqueous KOH (40 mg mL−1) and sonicated for 12 h. Finally, the suspension was filtered and washed with deionized water several times to remove any residual chemicals. The paste collected from the filter paper was dried at 60 °C, until it became agglomerated. The agglomeration was washed several times with deionized water and air-dried to obtain hydroxylated samples. PLA-PEG Coating of Nano-Graphene: Nano-graphene (nGr) was covalently coated with poly(lactide) (PLA) using a room temperature, ringopening polymerization. All reagents were dried under vacuum at 32 in Hg prior to use. 40 mg of nGr and 576 mg (4 mmol) of D,L-lactide (Purac Biomaterials, Lincolnshire, IL, USA) were dissolved in 4 mL of anhydrous ACN (Sigma–Aldrich) for 30 min under N2. Phosphazene base P2-t-Bu (2 M in tetrahydrofuran) (Sigma–Aldrich) was added as a catalyst. This reaction was continued with stirring under N2 for 24 h. Methoxy-PEGisocyanate (Nanocs Inc., New York, NY, USA) was dissolved in 2 mL of anhydrous ACN, and directly added to the reaction mixture. This reaction proceeded under N2 for 6 h. Next, NP were washed via centrifugation, the product was lyophilized, and stored under N2 at −20 °C.






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Paclitaxel-Controlled Release: PTX release kinetics were measured using high-performance liquid chromatography (HPLC). PTX (LC Laboratories, Woburn, MA, USA) was encapsulated by dissolving GrLP and PTX in ACN, and rotating on a rotisserie protected from light. The GrLP/PTX solution was then dropped in HyPure deionized water, and PTX was encapsulated via solvent evaporation. Samples were washed three times in HyPure water via centrifugation. CLP/PTX was re-dispersed in water and added to the top of a 3.5-kD Slide-a-Lyzer MINI dialysis unit. Each dialysis unit was placed in a 2-mL microcentrifuge tube with HyPure water. Five repeats were used in this study, and samples were placed in an incubator at 37 °C for the duration of the study. At each time point, dialysate was removed and frozen, and fresh HyPure water was replaced. Lyophilized aliquots were then analyzed via HPLC on a Waters 1525 Binary HPLC pump with a 2998 photodiode array detector. An Alltima C18 column (Grace Davison, Deerfield, IL, USA) was used that was 4.6 mm × 25 mm with 5 µm pores. Samples and standards were dissolved in ACN. The mobile phase used was 60% ACN and 40% water. Mobile phase was 1 mL min−1, and PTX was detected at a wavelength of 227 nm with an average elution time of 11.5 min. In Vitro Cytotoxicity: Human U-138 glioblastoma (ATCC, Manassas, VA, USA) were grown in Eagle’s modified essential medium (EMEM) supplemented with 10% fetal bovine serum (Atlanta Biologics, Lawrenceville, GA, USA), and penicillin/streptomycin (CellGro, Manassas, VA, USA) at 100 I.U. mL−1 and 100 µg mL−1, respectively. For Live/Dead cytotoxicity assays, cells were seeded at 12 500 cells per chamber in an 8-chambered glass slide. Next, nGr and GrLP were prepared by dissolving at 5 mg mL−1 in ACN, dropping into sterile HyPure water, and stirring for 2 h. Particles were washed twice with sterile HyPure water, and once with sterile phosphate-buffered saline (PBS). nGr and GrLP were then dispersed at varying concentrations in EMEM, and added to cells for 24 h. After incubating, cells were gently washed with sterile PBS and treated with 250 µL of 2 × 10−6 M calcein AM, and 4 × 10−6 M ethidium homodimer in sterile Dulbecco’s PBS (D-PBS) for 45 min. Cells were then gently washed twice with sterile D-PBS, and fixed with a 4 v/v% solution of formaldehyde in D-PBS for 30 min. Slide chambers were removed, and cells mounted and cover slipped in VectaShield (Vector Laboratories, Burlingame, CA, USA) fluorescent mounting media. Widefield fluorescent Live/Dead images were taken on a Nikon Eclipse Ti confocal microscope. To measure efficacy of PTX-loaded GrLP, U-138 were seeded at 10 000 cells per well in a black, clear-bottom 96-well plate. GrLP was loaded with PTX using the previously described solvent evaporation method. GrLP and CLP were added to cells at a concentration of 25 µg mL−1. Cells treated with only EMEM were used as a control. Free PTX at 140 × 10−9 M (the IC50 of free PTX) was added to compare PTXloaded NP versus free drug. Treatments were incubated with cells for 6 or 24 h to allow for uptake of the nanomaterial into cells. After the initial NP incubation, cells were gently washed twice with sterile PBS, and fresh EMEM was added. Cells were then allowed to grow for 72 h following initial exposure to NP. After the 72 h period, cells were treated with a 1:9 solution of PrestoBlue in EMEM for 45 min. Fluorescent signal was measured using a plate reader with excitation/emission wavelengths of 560/590 nm. Cell viability was determined by normalizing fluorescence values to the average value of all control cells. Five repeats were used for every treatment group. In Vivo Biodistribution: Animals were housed at Clemson University’s Godley-Snell Research Center. All animal work was done in accordance with Clemson University Institutional Animal Care and Use Committee (IACUC)-approved protocols. Athymic nude Foxn1nu mice (Harlan) were implanted with intracranial tumors of U-138 glioblastoma following the protocol of Ozawa and James.[59] U-138 cells were washed with sterile PBS. Cells were collected and concentrated in serum-free EMEM at 40 million cells mL−1. 100 µL of cell suspension was added to 100 µL of Matrigel (Becton Dickinson). Mice were anesthetized with ketamine–xylazine, skin was disinfected with chlorohexidine, and eyes were lubricated with PuraLube eye ointment. A 1-cm-sagittal incision was made across the top of the skull, and the skull was sterilized with hydrogen peroxide. A hole was made 2 mm anterior, and 1 mm lateral of

the bregma using a 25-gauge needle. 3 µL of cell solution was injected 3 mm deep into the brain over a 1 min period. The skull was closed using dental cement and the incision site was stapled shut. Tumors were allowed to grow for 10 d. After this growth period, biodistribution studies were performed. NP were prepared by loading with Alexa Fluor 647 in a solvent evaporation procedure similar to the PTX-loading approach. Dye-loaded NP were re-dispresed at 1 mg mL−1 in sterile PBS, and 200 µL was administered to mice via tail vein injection. A mouse injected with only saline was used as a control. Fluorescent images were taken with an IVIS Lumina XR live animal imaging system at 0.5, 1, 3, 6, 12, 24 h. After 24 h, mice were euthanized following IACUC-approved protocols, organs were explanted, and fluorescent images were taken.

Acknowledgements The authors thank Kim Ivey for use of the TGA, Dr. John Parrish and Travis Pruitt at the Godley-Snell Research Center for their help with the in vivo work, Dr. Terri Bruce and the Clemson Light Imaging Facility (CLIF) for use of the confocal microscope, and the Advanced Materials Research Lab (AMRL) for assistance with the electron microscope. Received: December 15, 2013 Revised: January 28, 2014 Published online: March 11, 2014

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