Graphene Nanoribbon-Based Platform for Highly Efficacious

Figure 2B shows the merged brightfield and fluorescence image that indicates colocalization of the O-GNR nanoparticles and FITC. Because FITC-labeled ...
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Graphene Nanoribbon-Based Platform for Highly Efficacious Nuclear Gene Delivery Sayan Mullick Chowdhury, Siraat Zafar, Victor Tellez, and Balaji Sitharaman* Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York 11794-5281, United States S Supporting Information *

ABSTRACT: Current efforts in the design and development of nonviral vectors for gene delivery and transfection have focused on the development of versatile agents that can load short or large sized genetic material, and are efficacious without eliciting toxicity in dividing and nondividing cells. Herein, we have investigated oxidized graphene nanoribbons (O-GNRs) as nonviral vectors for gene therapy and report in vitro studies that detail their cytotoxicity, intracellular and nuclear uptake, and gene delivery and transfection efficiencies. Our results indicate that, without additional functionalization with positively charged groups or other nonviral vectors, O-GNRs could load large amounts of small-sized single-stranded or large-sized double stranded genetic materials. O-GNRs at potential therapeutic doses (20−60 μg/ mL) elicited lower cytotoxicity compared to widely used commercial nonviral gene delivery vectors (Polyethylenimine and Fugene 6). The O-GNR-plasmid DNA complexes showed uptake into vesicular structures of dividing Henrietta Lacks (HeLa) and nondividing Human umbilical vein endothelial cells (HUVEC), release into the cell’s cytoplasm and entry into the nucleus. In these cells, O-GNRs loaded with enhanced green fluorescence protein (EGFP) plasmid or siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed a concentration- and time- dependent increase in gene delivery and gene transfection efficiencies up to 96−98%. The results suggest that O-GNRs are promising candidates as versatile and efficient nonviral vectors of small- or large-sized genetic material in primary and secondary cell types for gene therapy. KEYWORDS: graphene nanoribbons, cellular uptake, endosomal escape, nuclear entry, gene delivery

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transfection efficiency.13 However, viral agents are limited in terms to the size of genetic material they can carry (only small DNA/RNA (90%) © XXXX American Chemical Society

Received: December 29, 2015 Accepted: March 25, 2016

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ACS Biomaterials Science & Engineering on cell division (when the nuclear membrane disintegrates).26 Thus, in general, nonviral vectors release the genetic material in the cytoplasm and are more efficacious in adherent immortal or secondary cells which can undergo cell division and suboptimal for primary cells.28 To address some of the above issues, recent research has focused on chemical strategies to covalently and electrostatically append various synthetic (including nanoparticles) and biological moieties to existing nonviral vectors to mitigate toxicity without decreasing its transfection efficiency (by facilitating direct nuclear entry).29,30 Thus, there is a need for a versatile (that delivers large sized DNA and siRNA) gene delivery system that exhibits low cytotoxicity and high transfection efficiency (i.e., translocates into cells, escapes endosomes, and enters the nucleus to deliver loaded genetic material). Recently, graphene-based nanoparticles have shown promise in biomedical applications such as drug delivery and imaging.31−33 Various types of graphene nanoparticles such as Graphene oxide (GO) or graphene oxide nanoplatelets (GONPs), oxidized graphene nanoribbons (O-GNRs) and Graphene nano-onions (GNOs); each exhibiting different morphologies, and physiochemical properties, are available depending on the synthesis method and starting material.34 Yet, all previous in vitro or in vivo gene delivery studies have used graphite-derived graphene oxide (GO) or nano graphene oxide (nGO) which served as a scaffold35−39 (size: 50−800 nm diameter in the different studies) to load or covalently functionalize a known positively charged transfection agent (e.g., PEI) before being used for gene therapy applications. In case of functionalization with PEI, different molecular weights ranging from 1.8 kDa to 25 Kda and straight chained and well as branched PEI were used in the different studies. Feng et al. and Chen et al. employed PEI functionalized GO can be used to bind plasmid DNA and deliver it to cells.35,36 Feng et al. used 1.2KDa and 10KDa PEI for their experiments whereas Chen et al. utilized 25 kDa PEI. Zhang et al. used dual-functionalized GO (with poly(ethylene glycol)) (PEG) and PEI) for transfection of plasmid DNA into Drosophila S2 cells.37 PEG was used to increase the stability of graphene in physiological solutions (buffer, cell media). Zhou et al. used ultrasmall GO functionalized with PEI to transfect genes into zebrafish embyos.38 Yin et al. also showed that dual functionalized (PEG and PEI) graphene oxide can be used to transfect siRNA into mouse.39 However, these studies have mainly focused on proof-of-principle demonstration of these graphene complexes in secondary cell types. Their transfection efficiencies in primary cells is yet to be examined. Further, these studies until date have not shown nuclear entry of the graphene complexes; a critical requirement for establishing graphene as a stable gene transfection agent. A previous study has reported that GO does not enter A549 cells in vitro.40 Thus, the efficacy of GO by itself as an effective vector has still not been demonstrated. Recent studies show O-GNR-based nanoparticles elicit cellular response distinctly different from GO or any other carbon nanoparticles and exhibit promising drug delivery capabilities.41−43 Herein, we report that O-GNRs without the need of additional functionalization serves as a versatile platform to load large amounts of small- or large-sized genetic material (DNA or siRNA) and deliver them into the nucleus of dividing or nondividing cells to facilitate high transfection efficiencies.

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synthesize the oxidized graphene nanoribbons (O-GNRs) from multiwalled carbon nanotubes (MWCNTs; Sigma-Aldrich, New York).34 Briefly, 300 mg of MWCNTs were dissolved in concentrated sulfuric acid (60 mL) for 2 h and 1.5 g of potassium permanganate (KMnO4) was added to the dispersion. This mixture was stirred for 60 min at room temperature following which it was heated, on an oil bath, to 55 °C for 45 min and an additional 15 min at 70 °C. The mixture was cooled to 25 °C, and washed with water and dilute aqueous hydrochloric acid. The O-GNR nanoparticles were flocculated via addition ethanol and ether, and separated from the dispersion by centrifugation at 3000 rpm for 30 min. The O-GNR pellet was dried overnight in an oven (at 60 °C) before being used for experiments. Cell Culture. Henrietta Lacks (HeLa) cells, MRC5 fibroblasts derived from normal lung tissue and Human umbilical vein endothelial cells (HUVEC) were obtained from ATCC (Manassas, VA, USA). HeLa and MRC5 cells were grown in Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum.1% penicillinstreptomycin was used as antibiotic. HUVEC cells were grown in F12K medium supplemented with 10% FBS, 100 μg/mL heparin and 30 μg/mL endothelial cell growth supplement. All cell lines were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cell Synchronization. Unsynchronized HeLa and HUVEC cells were blocked at the G2/M stage of growth by treatment with nocodazole followed by release to obtain cellular synchronization. Briefly, 2 × 105 cells were plated in 75 cm2 flasks and allowed to grow for 24 h in normal media. The media was then removed and the cells were washed with phosphate buffered saline. Growth medium containing 200 ng/mL nocodazole was added to the flask and the cells were allowed to grow in it for 30 h. After 30 h the media was removed, cells were collected by trypsinization and centrifuged at 500g three times accompanied by resuspension of the cell pellet with fresh media after every centrifugation. The cell suspensions were then plated in 75 cm2 plates and allowed to move into G1 phase together. This process was repeated several times to obtain ∼90% synchronized cells for each cell line. Post synchronization, HUVEC cells had a doubling time of ∼27 h and HeLa cells ∼20 h. All experiments were done with synchronized cells. Transmission Electron Miscroscopy. Transmission electron microscopy (TEM) was performed on O-GNR samples prepared by dispersing 1 mg of the particles in a water−ethanol mixture (1:1) mixture by probe sonication for 2 min (Cole Parmer Ultrasonicator LPX, 750 W, 2 s on and 1 s off cycle). The O-GNR suspension was centrifuged at 3000 rpm for 3 min. The supernatant after centrifugation was dropped onto holey lacey carbon grids on a copper support (Ted Pella, Inc., Redding, CA). TEM of these grids were performed using a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR), at 80 kV. Digital images were acquired using an XR-60 CCD digital camera system. (AMT, Woburn, MA) Gene Loading. A 1.2 mg/mL dispersion of O-GNR was obtained by bath sonicating (Ultrasonicator FS30H, Fischer Scientific,Pittsburgh, PA) 4.8 mg of O-GNR in 4 mLphosphate buffered saline (PBS) for 30 min. Post sonication, 1 μg of EGFP plasmid (Addgene) or 50 μM siRNA against GADPH was added to the dispersion and stirred for 60 min on an ice bath. After the stirring, O-GNR loaded with EGFP or siRNA was centrifuged at 3000 rpm for 30 min in a cold centrifuge (4 °C) and the plasmid or siRNA left in the supernatant was quantified using a Nanodrop ND-100 (Nanodrop Technologies, Wilmington, DE). The plasmid loaded onto the O-GNRs was calculated by subtracting DNA or siRNA left in the supernatant from the total genetic material added. The EGFP or siRNA-loaded O-GNR pellet was resuspended in DMEM at 100, 200, 400, and 600 μg/mL. O-GNR Tagging with FITC. Five milligrams of solid O-GNRs were suspended in 5 mL of FITC-PEG-DSPE (Fluorescein isothiocyanate1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))]) solution in PBS and bath sonicated (Ultrasonicator FS30H, Fischer Scientific, 250 W,Pittsburgh, PA) for 15 min. Following sonication, the suspension was stirred at room temperature for 4 h. Next, the suspension was centrifuged at 3000 rpm for 30 min. The supernatant was discarded and the pellet containing O-GNR-FITC particles were resuspended in DMEM to achieve a concentration of 400 μg/mL

METHODS

Synthesis of Graphene Nanoribbons. An oxidative method first developed by Kosynkin et al.34 and adapted by us41−44 was employed to B

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Figure 1. (A) Low-resolution TEM image of O-GNR’s produced from unzipping of MWCNT’s. (B) Schematic showing mechanism of loading of dsDNA onto O-GNR suspension in phosphate buffered saline. (C) Loading efficiency of EGFP plasmid onto O-GNR (1.2 mg/mL) in phosphate buffered saline. (D) Loading efficiency of siRNA against GAPDH onto O-GNR (1.2 mg/mL) in phosphate buffered saline. (E) Comparison of toxicity of O-GNR, Fugene 6, and Polythyleneimine in MRC5 fibroblast cells at maximum concentration used for transfection. Toxicity Analysis Using LDH Assay. 5 × 103 MRC5 cells were plated per well in a 96-well plate and grown for 18 h. Following this incubation, media was removed from the wells, and replaced with 180 μL of fresh media. Cyto-toxicity of O-GNR (at 100 μg/mL), Fugene 6 and PEI were then compared using the lactate dehydrogenase assay. To the fresh media, 20 μL of O-GNR at 1 mg/mL was added to give a final concentration of 100 μg/mL in solution. Fugene 6, a commercially available gene transfection agent was added to the cells according to manufacturer’s instructions (24 μL of the agent was added to 200 μL of media and 20 μL of this solution was added to each well). Twenty microliters of 5 μg/mL PEI (branched, 25 kDa, Sigma-Aldrich), was added to each well to give a final concentration of 0.5 μg/mL in solution. The cells along with transfection agents were incubated for 72 h. After 72 h, the 96-well plates were centrifuged at 1200 rpm for 5 min. Fifty microliters of media from each well of the centrifuged 96-well plate was removed and added to a fresh 96-well plate. 100 μL of LDH reagent (Tox7 reagent, Sigma-Aldrich) was added to each well and incubated for 45 min. Absorbance readings of the plate were taken in a BIOTEK ELx 800 absorbance micro plate reader at 490 nm. Untreated cells were

treated as control. Lysed control was prepared by adding 20 μL of lysis solution to the untreated cells, 45 min before centrifugation of the plate. The LDH release (% of lysed control) is expressed as the percentage of (ODtest − ODblank)/(ODlysed − ODblank), where ODtest is the optical density of the control cells, or cells exposed to gene delivery agents, ODlysis is the optical density of the positive control cells, and ODblank is the optical density of the wells without cells. Confocal Microscopy. 25 × 103 HeLa and HUVEC cells were plated per well in glass bottom confocal plates and grown for 18 h. Following this incubation, media was removed and replaced with 360 μL of fresh media. Forty microliters of either O-GNR loaded with EGFP at the three different concentrations (200, 400, and 600 μg/mL) or OGNR-FITC at 400 μg/mL was added to each plate. The plates were incubated for 30 min or 12 h, following which EGFP-loaded O-GNR particles or O-GNR-FITC were washed away using PBS washes and the cells were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatford, PA) for 30 min. Hoescht 3342 stain (Invitrogen) was used to stain the nuclei of cells. Images were obtained using a Zeiss LSM 510 META NLO two-photon laser scanning confocal microscope system. C

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chemical methods.34 Although, reduction of graphene oxide usually results in increased efficacy of drug loading due to more available surface area,48 it also results in decreased stability in physiological solvents.49 As such, for applications involving intravenous injection of a drug delivery agent, oxygen containing graphene oxide is more suited. However, graphene nanoribbons without oxidized functional groups maybe more suited for other high impact applications such as in graphene based biosensor technologies where surface area becomes an important factor for assembly of polymers and DNA on the nanoribbon surface.50,51 Figure 1B is a depiction of the genetic material loaded onto the O-GNR nanoparticles. The schematic shows the possible π−π interactions between nitrogenous bases (purines and pyrimidines) associated with DNA with the graphene’s π bond network that could facilitate the loading of these biomacromolecules as reported in literature.31,52 The figure also shows positively charged metal ions in buffer solutions neutralizing the negative charges on the DNA backbone (phosphate groups) to aid the loading of the genetic material onto the negatively charged graphene nanostructures as previously reported.52,53 Figure 1C, D shows the amount of EGFP plasmid and siRNA that could be loaded onto the nanoparticles when incubated with 1.2 mg/mL O-GNRs for 60 min. EGFP could be loaded onto O-GNRs at ∼74.5% of the starting weight (1000 ng) (Figure 1C). siRNA could be loaded onto O-GNRs at ∼63% of the initial concentration (50 μM) (Figure 1 D) . Resuspension did not significantly decrease loaded genetic material (EGFP plasmid) on the O-GNRs (Figure S1). Additionally, functionalizing the OGNR with PEG-DSPE significantly decreased gene (EGFP) loading probably due to lesser space available for π stacking interactions (Figure S2). The loading results (Figure 1C, D) taken together suggest variety of genetic material could be loaded efficiently onto OGNRs. The direct loading of genetic material onto O-GNRs is interesting because it has not been reported for other graphene nanoparticles explored for gene delivery. For instance, the widely studied GO nanoparticles has been reported to be highly unstable in physiological buffer solutions and typically fall out of solution.37,39 Thus, GO is functionalized with amphiphilic macromolecules such as poly ethylene-glyol-1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))] (PEG-DSPE) to increase its stability in buffers and cell culture media.37,39 Because nucleic acid loading is dependent on the available graphene surface area, functionalization with macromolecular groups could also result in a decrease of available GO surface area, and hence lower its loading efficiency. On the other hand, compared to GO, we and others have shown that the oxidative functional groups on the surface and edges of O-GNRs, generated during its synthesis, improves the stability O-GNR aqueous dispersions even in the presence of biological buffers containing charged ions.34,41,53 This aqueous stability perhaps improves the gene loading capability of O-GNRs (without the need for additional functionalization) compared to GO. Figure 1E shows the cytotoxicity assessment of O-GNRs at a potential treatment dose compared to standard treatment protocols of commonly used transfection agents. LDH assay was employed to evaluate the cytotoxicity of 100 μg/mL OGNRs after 72 h of incubation with normal lung fibroblasts (MRC5). MRC5 cells were chosen for the cytotoxicity analysis since they are widely used for cytotoxic screening of nanoparticles. Further, MRC5 cells are lung fibroblast cells, which are more representative of normal cells compared to the two other

Assay for GAPDH Activity. GAPDH activity was assayed using KDalert GAPDH Assay Kit (Life Technologies, Foster City, CA). Briefly, 5 × 103 HeLa and HUVEC cells per well were plated in a 96-well plates and grown for hours. This was followed by removal of media from the wells and addition of 180 μL of fresh media. Twenty microliters of O-GNRs loaded with siRNA at various concentrations (100, 200, 400, and 600 μg/mL was then added to the wells to achieve final treatment concentrations of 10, 20, 40, and 60 μg/mL, respectively, and incubated for 48 h. Following this incubation, the media containing the loaded OGNRs was removed and the cells were then treated with 200 μL cell lysis buffer provided with the kit for 20 min. Post cell lysis 10 μL of the lysate from each well was moved to a fresh 96-well plate. Ninety microliters of the diluted master mix from the kit was added to the wells and the fluorescence in the wells was measured using a an Infinite M200 multiwell plate reader (Tecan Group, Morrisville, NC) at 545 nm excitation and 575 nm emission after 5 min. Untreated cells and cells treated with O-GNRs (60 μg/mL) loaded with negative control siRNA were used as controls. Transmission Electron Microscopy of Cells. 25 × 104 HeLa and HUVEC cells were plated per well in 6 well plates covered with ACLAR film (Electron Microscopy Sciences, Hatford, PA) and grown for 22 or 30 h, respectively, to allow one cycle of cell division. Following this incubation the media was removed and replaced with media containing O-GNR at 20 μg/mL The cells were incubated in the media for 30 min or 12 h after which the excess O-GNR was washed away using PBS. The films containing cells were either trypsinized for cell counting or fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatford, PA). The fixed films were then placed in a solution of 2% Osmium Tetroxide (in PBS) given ethanol washes and embedded in Durcupan resin (Sigma-aldrich, St. Louis, USA). Cells on the film were then blocked, cut into thin sections (∼80 nm) using an Ultracut E microtome (ReichertJung, Cambridge, UK), and placed on copper grids coated with formavar. This obtained cell sections were viewed with a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR), at 80 kV. Digital images were acquired using an XR-60 CCD digital camera system. (AMT, Woburn, MA) Flow Cytometry. 5 × 104 HeLa cells were grown in 10 cm dishes for 20 h. Following this incubation, one dish was trypsinized and the cell number was determined. Media was removed and replaced with 9 mL of fresh media in the other dishes. O-GNRs were loaded with propidium iodode (PI) by sonicating the particles (5 mL at a concentration of 1 mg/mL) with 10 mg PI and stirring the mixture for 4 h at room temperature. The stirred mixture was centrifuged at 3000 rpm for 30 min. The supernatant was discarded and the pellet was resuspended in DMEM to give O-GNR-PI at a concentration of 400 μg/mL. The HeLa cells were treated with 1 mL of O-GNR-PI to give a final concentration of 40 μg/mL in solution. Untreated cells were used as control. After 2, 12, and 24 h, cells were trypsinized, resuspended in FACS buffer (Phosphate buffered saline containing 20% fetal bovine serum), and placed on ice. Flow cytometry to detect PI fluorescence was performed on all samples using a FACS Calibur Cell Sorter (BD Biosciences, San Jose, CA). Cell number was determined at all time points tested to determine if cell divison had taken place.

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RESULTS AND DISCUSSION

Characterization, Gene Loading Efficiencies, and Cytotoxicity. Figure 1A shows representative low resolution TEM image of multiple O-GNRs. The figure shows O-GNRs approximately 300−500 nm in length and 80−125 nm in breadth (red arrows) having high aspect ratio(Ratio of length to breadth is >5). The images indicate that O-GNRs have a large flat surface area to load the genetic material. Previously, characterization of O-GNRs used in this study has revealed the presence of abundant oxygen containing functional groups which provide high aqueous stability to the nanoparticles.45 In the past, many studies have focused on reducing graphene oxide nanoparticles before using them for drug delivery applications.46,47 Reduction of O-GNRs has been achieved already through hydrazine based D

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Figure 2. (A−C) Representative bright-field, merge, and fluorescence images of HeLa cells treated with FITC-PEG-DSPE-loaded O-GNR particles. Red arrows indicate cells and white arrows indicate nanoparticles in the figures. (D−F) Representative brightfield, hoechst fluorescence, and merge of Hoechst and FITC fluorescence images of HeLa cells treated with FITC-PEG-DSPE-loaded O-GNR’s for 12 h. (G−K) Flow cytometry-based analysis showing PI fluorescence in 1 × 104 HeLa cells either left (G) untreated or incubated with PI-loaded O-GNRs for (H) 2, (I) 12, and (J) 24 h. (K) Percentage of cells showing PI fluorescence at the three time points tested calculated from the flow cytometry data.

cells. Compared to untreated control, treatment of cells with OGNRs at 100 μg/mL did not show a significant increase in cell death. Cells incubated with Fugene 6 showed ∼25% increase in LDH release compared to untreated control. Treatment with PEI shows ∼60% increase in LDH release compared to untreated control suggesting that O-GNRs are potentially safe for use up to the tested concentration (100 μg/mL). These results are in agreement with our previous toxicity studies with O-GNRs in HeLa cells where we have shown that unfunctionalized O-GNRs remain nontoxic to HeLa cells as long as it is not processed by high intensity probe sonication.44

cell lines (HeLa and HUVEC) used in this study. Additionally, non-specific nanoparticle accumulation and toxicity after intravenous injection is very commonly observed in lung tissue and thus evaluating the cytotoxicity of O-GNRs in fibroblast cells derived from lung tissue is an important assessment. Two commonly used transfection agents, Fugene 6 and PEI, incubated at maximum suggested dose (0.5 μg/μL for PEI and 2.4 μL per well total for Fugene 6) for the same duration, were used as controls. GO nanoparticles could not be used as controls due to its instability in buffer solutions, and propensity to aggregate in cell growth media. Figure 1B shows the cytotoxicity of O-GNRs and transfection agents Fugene 6 and PEI on MRC5 E

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Figure 3. Representative TEM of HeLa and HUVEC cells showing (A, D) invagination of cell membrane and formation of vesicles at the surface of the membrane in contact with O-GNR. (B, E) Vesicle containing large O-GNR aggregates. (C) Representative TEM of a HeLa cell showing membrane extensions taking up O-GNR aggregates. (F) Representative TEM of a HUVEC cell showing uptake of O-GNR into large number of vesicular structures throughout the cytoplasm. In A−F, blue arrows indicate cell membrane, black arrows indicate nanoparticles, red arrows indicate formation of vesicles, and green arrows indicate the nuclear membrane. (G) Representative TEM images of a HeLa cell showing O-GNR aggregates escaping a vesicle. (H) Representative TEM image of a HeLa cell showing an escaped O-GNR aggregate outside the nuclear membrane. (I) Representative TEM image of a HeLa cell showing two O-GNR aggregates released from lysed vesicles. (J) Representative TEM image of a HeLa cell showing a large O-GNR aggregate released from a lysed vesicle. (K, L) Representative TEM image of HUVEC cells showing escaped O-GNR aggregates outside the nuclear membrane. In G−L, blue arrows indicate O-GNR particles inside vesicles, yellow arrows indicate nuclear membrane, red arrows indicate escaped or escaping nanoparticles, and green arrows indicate the vesicles. Yellow arrows indicate darkened area of the nucleus near the nanoparticle aggregates. (M−O) Representative TEM images of HeLa cells treated with 20 μg/mL O-GNR for 12 h. (M) O-GNR aggregates inducing formation of nuclear invagination. Image also shows presence of O-GNR’s in the nucleus. (N) O-GNR aggregates entering the cell and nucleus. (O) O-GNR aggregates inside the nucleus. (P−R) Representative TEM images of HUVEC cells treated with 20 μg/mL O-GNR for 12 h. (P) O-GNR aggregates inducing formation of nuclear invagination. (Q) Creation of a gap in the nuclear membrane. O-GNR’s can also be seen near the gap (yellow arrows). (R) Presence of O-GNR inside F

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the nucleus. In M−R, red arrows indicate O-GNR particles, green arrows indicate nuclear membrane, yellow arrows indicate invaginations of the nuclear membrane, and blue arrows indicate darkening of the nucleus.

results suggest that ∼50% of HeLa cells showed endosomal escape of O-GNRs within 12 h of treatment. TEM analysis was further performed to qualitatively corroborate cellular uptake, endosomal escape and nuclear transport of O-GNRs. Figure 3A−F are representative TEM images of HeLa and HUVEC cells treated with 20 μg/mL OGNRs for 30 min. HUVEC cells were included in this analysis to qualitatively examine whether these cells of primary endothelial origin can also uptake O-GNRs. Figure 3 A-C show HeLa cells treated with O-GNRs. Figure 3D−F are representative TEM images of HUVEC cells treated with O-GNRs. Figure 3A, D shows invagination of the cellular membrane (red arrows) near O-GNRs (black arrow) aggregates. Figure 3B, E shows the formation of large vesicular structures (red arrow) within HeLa and HUVEC cells containing O-GNR aggregates (black arrow). Figure 3C shows the disruption of large portions of a HeLa cell membrane (red arrows) that are in contact or proximity with OGNR aggregates (black arrows). Figure 3F shows multiple OGNR- (black arrow) filled vesicular structures (red arrows) in the cytoplasm of HUVEC cells. Analysis of multiple TEM images for each cell type indicated higher amounts on O-GNRs in HUVEC cells (average ∼23 aggregates/cell) compared to HeLa cells (average ∼7 aggregates/cell). The images suggested that within 30 min of incubation, the O-GNR nanoparticles are either in the process of being uptaken into the cells or inside the cells. Vesicular escape of delivery agents is necessary for their successful delivery of genetic material. Endosomal vesicles are often recycled back to membrane surface, and the delivery agents could never be released in the cytoplasm.26 Figure 3G−J are representative TEM images of HeLa cells incubated with 20 μg/ mL O-GNRs for 12 h. Figure 3G shows an O-GNR aggregate (red arrows) outside a vesicle (green arrows). Figure 3H shows an O-GNR aggregate (red arrow) which may have possibly escaped from the large vesicle containing O-GNR aggregates (green arrow) at the nuclear periphery. Invagination of the nucleus is also observed in the figure (yellow arrow). Figure 3I shows two O-GNR aggregates (red arrows) near lysed vesicles (green arrows). Figure 3J also shows a large O-GNR aggregate (red arrow) near a lysed vesicle (green arrow). Figure 3K, L shows representative TEM images of HUVEC cells incubated with 20 μg/mL O-GNRs for 12 h. Both images show presence of O-GNR aggregates (red arrows) in vesicular structures. A dark region in the nucleus (yellow arrows) near the released nanoparticles is also noted. This dark region was not noted in the HeLa cells. Once released into the cytoplasm, the gene delivery agent has to reach the nucleus. To determine if O-GNR could actually enter the nucleus, we performed additional histological analysis by TEM of cells treated with 20 μg/mL O-GNRs for 12 h. Synchronized cell populations were used to ensure no cell division took place within the 12 h incubation period; confirmed by cell counts before and after the 12 h incubation (Figure S3). Figure 3M−O shows representative TEM images of HeLa cells treated with O-GNRs. Figure 3M shows an O-GNR aggregate (red arrow outside nucleus, nuclear periphery demarcated by green arrow) outside another O-GNR-containing vesicle. The image further shows nuclear invaginations (yellow arrows) near the vesicle, and presence of O-GNR aggregates both inside and

Uptake into Cells and Nucleus. Uptake of O-GNR’s into cells and nucleus was initially confirmed by confocal microscopy and flow cytometry (Figure 2). Figure 2A−C are representative confocal microscopy images of HeLa cells exposed to 40 μg/mL FITC-labeled O-GNRs (green fluorescence) for 30 min. HeLa cells are very commonly used for transfection studies and established protocols to assess nanoparticle entry has already been reported extensively for these cell lines.54−58 Figure 2A and 2C show the brightfield and fluorescence images, respectively, of the cells (red arrows) and O-GNRs (white arrows). Figure 2B shows the merged brightfield and fluorescence image that indicates colocalization of the O-GNR nanoparticles and FITC. Because FITC-labeled O-GNRs show the presence of O-GNRs inside the cells after 30 min of exposure, the result suggests a quick uptake of the nanoparticles. Figure 2D−F shows brightfield, nuclei fluorescence, and merged nuclei and FITC fluorescence images of a HeLa cells treated with 40 μg/mL FITC labeled GNRs for 12 h. Figure 2D shows presence of OGNR aggregates (red arrows) in and around the nucleus (yellow arrow). Figure 2E shows the same cell with nuclei stain (Hoechst stain, yellow arrow). Figure 2F shows the same cell with green fluorescence from the FITC-loaded O-GNRs. The images indicate that the nanoparticles are mostly localized inside the nucleus (demarcated by blue fluorescence), suggesting that the nanoparticles could enter the nucleus of the cells within 12 h of incubation. It should be clarified that the dark structures shown in the images are O-GNR aggregates which are visible under low power bright-field microscope. It is usually more difficult to visualize single layered O-GNRs using low power microscopes (electron miscroscopy is needed), because their size is lower than the diffraction limit. Thus, only the FITC can be visualized in some portions of these images without any visible O-GNR overlap. Propidium Iodide (PI) is a dye that is normally excluded from membrane of living cells and can only enter dead cells.59 Once PI is uptaken into the cells, it can enter the nucleus and bind to DNA to give its characteristic fluorescence.59 Thus, live cells exposed to PI loaded onto O-GNRs at a nontoxic concentration of O-GNRs (40 μg/mL) would only show fluorescence before the cells divide if the O-GNR can get into the cells, escape the vesicles and enter the nucleus. Flow cytometry was used to initially confirm and provide a preliminary estimate of the time taken for vesicular escape and uptake of the O-GNRs into the nucleus. Figure 2G− K shows the number of HeLa cells (out of 10 000 cells) that exhibited PI fluorescence in its nucleus. Figure 2G-J show the distribution of fluorescent and nonfluorescent cells after 2−24 h of treatment. Figure 2K shows the percentage of cells that showed presence of PI fluorescence at the different time intervals. After 2 h of incubation, ∼21% cells (Figure 2K) showed PI fluorescence (right shift in the fluorescent cell population (Figures 2H). After 12 h, ∼51% cells (Figure 2K) showed PI fluorescence as evident from a further increase in the number of fluorescent cells (Figure 2I). After 24 h, ∼ 60% cells (Figure 2K) showed PI fluorescence (Figure 2J). Because flow cytometry analysis showed that ∼51% of cells were fluorescent after 12 h (before the cells have divided, cell counts shown in Figure S1), and ∼60% of the cells were fluorescent after 24 h, the G

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ACS Biomaterials Science & Engineering

Figure 4. (A−C, F−G) Representative confocal microscopy images of Hela and HUVEC cells treated with various concentrations of EGFP plasmid loaded O-GNRs. Images represent nuclear staining with Hoechst stain and EGFP expression respectively in cells treated with (A) 20, (B) 40, and (C) 60 μg/mL GNR. (D, I) Quantification of EGFP fluorescence in HeLa and HUVEC cells treated with various concentrations (20−60 μg/mL) of EGFPloaded O-GNR’s. (E, J) Quantification of GAPDH activity in HeLa and HUVEC cells treated with various concentrations (10−60 μg/mL) of siRNAloaded (against GAPDH) O-GNR’s.

outside the nucleus (red arrows). Figure 3N shows representative TEM image of O-GNRs getting uptaken into HeLa cells (red arrow outside nucleus, nuclear periphery demarcated by green arrow). The image further shows O-GNR aggregates inside the nucleus (red arrows) and formation of nuclear invaginations. Figure 3O is a representative TEM image of HeLa cell nuclei (green arrow) showing presence of O-GNR aggregates (red arrow). No vesicular structures associated with the particles were noted inside the nuclei. Figure 3P-R shows representative TEM images of HUVEC cells treated with 20 μg/mL O-GNR for 12 h. Figure 3P shows a large O-GNR aggregate containing vesicle (red arrow). Additionally, nuclear invagination (blue arrow) similar to those observed in HeLa cells (Figure 3M) are noted. Furthermore, darkening of the nucleus near the O-GNR aggregate is noted; similar to those seen in Figure 3K, L. O-GNR aggregate is also observed inside the nucleus (red arrow). Figure 3Q shows an opening in the nucleus (gap in the nucleus indicated by yellow arrows, nucleus indicated with green arrows) in the presence of O-GNR aggregates in the vicinity of a vesicle (red arrows). The opening of the nucleus is noted near a dark spot (blue arrow) in the nucleus close to the O-GNR aggregates. Figure 3R shows the absence of dark spots in the nuclear membrane (green arrow) when no O-GNR aggregates are present in vicinity of the nucleus. An O-GNR aggregate is seen inside the nucleus (red arrows) in this image. EGFP-loaded O-GNRs showed similar uptake properties into HeLa and HUVEC cells, indicating that EGFP loading does not significantly affect uptake of the nanoparticles (Figure S4). The above results (Figure 3) taken together suggest that OGNRs could be uptaken and transported to the nucleus of these cells and thus, could serve as gene delivery agents. The exact mechanism of uptake needs further investigation. It is possible

that these particles activate specific receptors on the cell surface, or induce local changes in membrane cytoskeleton due to their hydrophobicity to induce the uptake mechanism.60 The results further provide evidence that O-GNRs can potentially escape endosomes. Lipoplexes containing the pH sensitive fusogenic lipid DOPE have been used for endosomal release of the DNA into the cytoplasm by fusion of the liposomes with the endosomal membranes.26 Some reports have shown polyplexes can release DNA into the cytoplasm by a process called “proton− sponge” effect where polyplexes get protonated inducing Cl− and water influx, and ultimately lysis of the endosome.26,61 However, others reports show that no such mechanism exists.62 Delivery agents lacking either fusogenic or endosomal lytic capabilities are considered poor gene delivery agents.26 Our results suggest that O-GNRs lyse the endosomes to escape into cytoplasm perhaps in a manner similar to those other graphene structures that can spontaneously penetrate membranes.63 Finally, the above results corroborate the confocal microscopy and flow cytometry results (Figure 2) and confirm that O-GNRs can eventually enter nucleus of both cell lines. Nuclear entry of DNA (even when condensed by polyplexes) is difficult because of the small size of nuclear pores ∼10 nm).26,27 Most nonviral gene delivery agents depend on cell division (when the nuclear membrane disintegrates) for DNA to enter the nucleus.26 Other studies show that, without a nuclear localization signal attached, nonviral vectors could excluded from the nucleus even after mitosis.28 These issues could limit the transfection efficiency of nonviral gene delivery agents in dividing cells and restrict their use for gene transfection in differentiated nondividing cells. On the basis of our results, we hypothesize, that entry of the O-GNRs into the nucleus is initiated by release of the nanoparticles from vesicles and induction of invaginations on the nuclear membrane. Nuclear invaginations are usually actin rich structures observed H

DOI: 10.1021/acsbiomaterials.5b00562 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering in many cells.64,65 However, the functions of these structures are still unclear.64 Studies have showed that formation of such invaginations during cell division generates tensional forces along the nuclear membrane, and ultimately creates of gaps in the nuclear membrane to balances these forces.66 Such gaps were noted in HUVEC cells near released O-GNR inside the cells (yellow arrows, Figure 3Q). It is possible that O-GNRs pass through the gaps created in the nuclear membrane due to formation of such invaginations. In few images, we noted, post formation of nuclear invaginations and entry of O-GNRs into them, the nuclei membrane reforms around the invaginations (Figure 3N). This phenomenon is surprising because such a mechanism of nuclear entry has not been reported before and warrants further investigation. Future studies would test our hypothesis and provide better understanding of the mechanism of nuclear entry. Gene Delivery and Transfection. Figure 4 shows the gene delivery efficiency results from HeLa and HUVEC cells treated with EGFP plasmid and siRNA (against GAPDH) loaded OGNRs for 12 h. Gene delivery efficiency is typically calculated by the percentage of cells that show presence of the gene product out of the total number of cells exposed to the delivery agent. HeLa and HUVEC cells were used for the these experiments since previous studies with graphene-based and other commercially available gene delivery agents have frequently used these two cell lines, thus, allowing us to follow established protocols for calculating transfection efficiency.35,36,46,57,62,67 Figure 4A−C and Figure 4F−H shows nuclear staining and EGFP fluorescence of the treated HeLa and HUVEC cells, respectively. These cells were treated with EGFP loaded O-GNR at 20, 40, and 60 μg/mL. All cells in the representative images show green fluorescence due to expression of EGFP. Figure 4D shows the fluorescence quantification of O-GNR treated HeLa cells. The results indicated a concentration-dependent increase in fluorescence intensity with 60 μg/mL treated cells exhibiting maximum fluorescence. However, similar statistically significant dose-dependent increases were not observed for the fluoresecence quantification in HUVEC (Figure 4I) cells. Figure 4 E shows the GAPDH enzyme activity of HeLa cells exposed to siRNA (against GAPDH) loaded O-GNRs with increase in treatment concentrations (0−60 μg/mL) for 12 h. The results indicated a dose-dependent decrease in GADPH activity; decreases to ∼78, ∼69, ∼57, and ∼51% of untreated cells at treatment concentrations of 10, 20, 40, and 60 μg/mL loaded O-GNRs. However, cells treated with O-GNRs (at 60 μg/ mL) without loaded siRNA did not show similar dose-dependent decreases in GAPDH activity. HUVEC cells showed similar dose-dependent response to siRNA loaded O-GNRs (Figure 4J); GADPH activity decreased to ∼84, ∼77, ∼69, and ∼60% of untreated cells at treatment concentrations of 10, 20, 40, and 60 μg/mL loaded O-GNRs. The gene delivery results clearly implied that that exposure to more O-GNRs leads to more EGFP plasmid and siRNA reaching the nucleus of the cells. Further, for both cell types, gene transfection efficiency (i.e., percentage of cells with visible GFP fluorescence) at all treatment concentrations (obtained by observing fluorescence in 300 cells exposed to each concentration) were high (96−98%). In the past, GO-PEI has been utilized for several gene transfection studies (GO size: 50−800 nm, PEI: 1.8−25 kDa, either straight chained or branched) in HeLa cells. Feng et al. had previously showed that GO-PEI exhibit relatively low transfection efficiency (assessed qualitatively) at lower nanoparticle incubation concentrations (nitrogen/phosphate ratio