Nanoparticle-Facilitated Membrane Depolarization ... - ACS Publications

Article pubs.acs.org/journal/abseba

Nanoparticle-Facilitated Membrane Depolarization-Induced Receptor Activation: Implications on Cellular Uptake and Drug Delivery Sayan Mullick Chowdhury,† Shawn Xie, Justin Fang, Stephen K. Lee, and Balaji Sitharaman* Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York 11794, United States S Supporting Information *

ABSTRACT: Cell-specific uptake of drug delivery systems (DDSs) are crucial to achieve optimal efficacy of many drugs. Widely employed strategies to facilitate targeted intracellular drug delivery involves attachment of targeting ligands (peptides or antibodies) to DDSs. Target receptors mutations can limit the effectiveness of this approach. Herein, we demonstrate, through in vitro inhibitory and drug delivery studies, that graphene nanoribbons (GNRs), water dispersed with the amphiphilic polymer called PEG-DSPE ((1, 2-distearoyl-sn-glycero-3phosphoethanolamine-N [amino (polyethylene glycol)]) (induce membrane depolarization-mediated epidermal growth factor receptor (EGFR) activation. This phenomenon is ligand-independent and EGFR activation occurs via influx of Ca2+ ions from the extracellular space. We further provide evidence, through in vivo studies, that this mechanism could be exploited to facilitate efficacious drug delivery into tumors that overexpress EGFR. The results suggest that transient membrane depolarization-facilitated cell receptor activation can be employed as an alternate strategy for enhanced intracellular drug delivery. KEYWORDS: graphene nanoribbons, drug delivery, targeting, membrane depolarization, cancer

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potential between −60 and −100 mV. Cancer cells typically exhibit much different resting membrane potentials (−55 to −5 mV).15 It is postulated that a more depolarized cell membrane favors proliferation in cancer cells.15 Herein, we report that O-GNR-PEG-DSPEs (O-GNR, graphene oxide nanoribbons; PEG-DPSE, (1, 2-distearoyl-snglycero-3-phosphoethanolamine-N[amino(polyethylene glycol)]) activate EGFR by inducing transient membrane depolarization in cells that express these receptors. We further demonstrate in vitro and in vivo that this effect can be exploited as a novel mechanism to enhance anticancer drug efficacies in EGFR expressing tumors.

INTRODUCTION Nanoparticle drug delivery systems (NDDSs) typically accumulate at tumor sites via its leaky vasculature due to the enhanced permeability and retention (EPR) effect.1 Other novel strategies have also been explored to improve the tumor accumulation of nanoparticles.2,3 Once at the site of tumor, uptake of the NDDSs into tumor cells is crucial to achieve optimal efficacy of many anticancer drugs.4 Receptor-mediated endocytosis and pinocytosis are some common mechanisms by which nanoparticles that accumulate in the vicinity of cells get uptaken into cells.4 Attachment of targeting ligands (peptides or antibodies) that append to cell receptors could improve the accumulation of NDDSs into specific cancer cells and increases its probability of intracellular uptake.5,6 However, mutations in target receptors can limit the effectiveness of this approach.7,8 Thus, identification of novel nanoparticle cellular uptake pathways will not only improve drug delivery capabilities of nanoparticles but also help in designing NDDSs that can overcome cancer cell resistance to existing anticancer drugs. Cancer cells differ from normal cells in a variety of aspects including but not limited to metabolism, cellular structure and specific receptor overexpression.9−11 All these aspects have been utilized to identify cancer phenotype12,13 and more recently for targeted therapy.11,14 A less investigated yet critical characteristic of cancer cells is their membrane potential. Depending on the cell type, normal cells have a membrane © XXXX American Chemical Society

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MATERIALS AND METHODS

Cell Culture. HeLa and MCF-7 cell lines were used in the experiments. Both cell lines were obtained from ATCC (Manassas, VA, USA). We used DMEM as growth media for HeLa cells and RPMI1600 was used as growth media for MCF-7 cells. Both DMEM and RPMI 1600 were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The incubation temperature for the cell lines was set at 37 °C and the cells were kept in an incubator atmosphere of 5% CO2, and 95% air. HeLa and MCF7 cells used in all experiments were made resistant up to 3 μM doxorubicin by treating Received: June 19, 2016 Accepted: October 14, 2016

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DOI: 10.1021/acsbiomaterials.6b00338 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Representative fluorescence, bright-field, and merged microscopy images. All cells were treated with O-GNR-PEG-DSPE for 15 min. (A− C) Untreated HeLa cells and (D−F) HeLa cells exposed to 25 μg/mL O-GNR-PEG-DSPEs that indicate activated EGFRs (green fluorescence). (G−I) HeLa cells exposed to 50 μg/mL O-GNR-PEG-DSPEs that indicate activated EGFRs (green fluorescence). width, and L is the length. For analysis of tumor angiogenesis, 2 mice containing 2 week old HeLa cell xenografts were injected with 25 μL of 200 μg/mL doxorubicin-loaded O-GNR-PEG-DSPE three times every 3 days. The tumors were harvested 20 days after the first injection and their blood vessel density was observed. Histology. Tumors removed from mice were fixed for by immersing into 4% paraformaldehyde. Post fixation, the tumors were cut into 3 mm segments. All the tumors were dissected symmetrically for consistency. Tumor segments were then dehydrated using graded ethanol washes and then paraffin-embedded for further processing. Five μm sections from each paraffin embedded tumor sample were sliced using a microtome and stained with Hematoxylin and Eosin (H&E) for histological evaluation. Digital photo microscopy was performed using a bright field microscope at 400× and 100× magnification. Drug Delivery Studies. Cell death was used as an indirect measure to assess the efficacy of drug delivery by the nanoparticles. Assessment of cell death was conducted using a TOX-7 assay kit that estimates lactate dehydgenase (LDH) released from the cells (SigmaAldrich, St Louis, MO). Briefly, Dox-resistant HeLa cells were plated, at a density of 1 × 104 cells per well, in 96-well cell culture plates. The cells were incubated for 24 h. After 24 h, the media was changed to either normal DMEM or DMEM without Ca2+ and the cells were treated with 1) different concentrations of Dox-loaded O-GNR-PEGDSPE (10, 25, 50, and 75 μg/mL), for 24 h at 37 °C. (2) Dox-loaded O-GNR-PEG-DSPE at 50 μg/mL, O-GNR-PEG-DSPE at 50 μg/mL, PEG-DSPE and Dox in PEG-DSPE (at the same concentration as loaded and double the concentration of loaded), (3) Dox-loaded OGNR-PEG-DSPE at 50 μg/mL after pretreatment of HeLa cells with 65, 130, and 200 nM anti EGFR antibody specific to the ligand binding pocket of EGFR(Santa Cruz Biotechnology,Santa Cruz, CA). After 24 h, the 96-well plates were centrifuged at 1200 rpm for 5 min. Postcentrifugation, 50 μL of the media from each well was collected. This media was incubated with LDH assay reagent for 45 min in a fresh 96 well plate. All absorbance measurements were done at 490 nm. Cells incubated with 10 μL of lysis solution 45 min before centrifugation

them with increasing concentration of doxorubicin and isolating the resistant populations. Animal Care and Induction of Xenograft Tumors. The guidelines of Institutional Animal Care and Use Committee at Stony Brook University, NY for all animal experiments. Doxorubicin resistant HeLa cells and MCF7 cells were plated at a 3 × 106 cell density in 10 cm plates and grown in Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) media, respectively. The cells were grown to confluency after which the media was removed and replaced with fresh media. The cells were allowed to grow for 6 h in fresh media. They were then trypsinized and resuspended in equal volumes of DPBS and Corning matrigel matrix to a concentration of 5 × 107 cells/mL. Immunocompromised male mice were injected with 100 μL of each cell suspension on each flank and allowed to grow for 2 weeks. Drug Loading onto Graphene Nanoribbons. We employed a previously reported protocol to load the drug doxorubicin (Dox) onto the graphene nanoribbons.16 Briefly, 2 mL of 500 μg/mL O-GNRPEG-DSPE was added to 1 mg of Dox and stirred for 2 min. This solution was then bath sonicated for 30 min. Following sonication, a magnetic stirrer was used to stir the solution for 2 days. The mixture was next centrifuged at 14000 rpm for 45 min to pellet drug loaded OGNR-PEG-DSPE. The supernatant containing unloaded Dox was carefully pipetted out. A Dox standard curve was used to calculate its amount in the supernatant. The amount of drug loaded was calculated by subtracting the total drug at the beginning of loading from the unloaded drug. The drug-loaded loaded O-GNR nanoparticles were resuspended in PEG-DSPE. O-GNR-DSPE stock solutions (500 μg/ mL) for all experiments. Tumor Injection Studies. Post 2 weeks of tumor growth the size of each tumor was measured using calipers and 50 μL of 500 μg/mL Dox-loaded O-GNR-PEG-DSPE, 50 μL of PEG-DSPE (1.2 mg/mL), 50 μL of 500 μg/mL O-GNR-PEG-DSPE and 50 μL of free Dox in PEG-DSPE were injected directly into 3 tumors for each injection type every 3 days (n = 3). Tumor volume was calculated according to the formulas V = [W(2)L]/2 where V is the volume of the tumor, W is the B


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Figure 2. Representative fluorescence, bright-field, and merge images of HeLa cells treated with 130 nM anti-EGFR antibody and exposed to (A−C) EGF and (D−F) O-GNR-PEG-DSPE. Red arrows point to EGFR activation and yellow arrows point to cells. was used as positive control. The formula (Abtest − Abblank)/(Abpositive − Abblank)100 was used to calculate LDH secretion from treated and control cells as percentage of LDH released from positive control cells. In this formulas, Abtest is the absorbance reading obtained for either untreated control cells or cells exposed to O-GNR-PEG-DSPE. Abpositive is the absorbance of the lysis buffer treated cells. Abblank is the absorbance from blank wells. Baseline correction was performed by measuring the absorbance from wells containing only PEG-DSPE in cell growth medium. LDH secretion (as percentage of cells treated with Dox-loaded O-GNR-PEG-DSPE) was calculated using the formula (Abtest − Abblank)/(Abtreatment − Abblank)100, where Abtest is the absorbance reading of the anti-EGFR antibody treated wells exposed to Dox-loaded O-GNR-PEG-DSPE, Abtreatment is the absorbance reading from wells treated with only Dox-loaded OGNR-PEG-DSPE, and Abblank is the optical density of blank wells. Confocal Microscopy. Confocal microscopy was performed on cell incubated in 35 mm plates. Briefly, 7 × 105 cells was plated for 48 h with 1 media change after 24 h. Next: (1) Cells were either treated with anti EGFR antibody (65−200 nM) or left untreated and then exposed to either 25 and 50 μg/ mL O-GNR-PEG-DSPE or EGF (10 nM) (Life Technologies, Carlsbad, CA) for 15 min. The cells were then washed with saline and 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield,PA) was used to fix the cells for 30 min. The fixed cells were then incubated with 0.5% Triton X-100, washed multiple times with saline, and incubated with Alexa fluor 488 tagged antiphospho EGFR antibody (Millipore, Billerica, MA) (Excitation 490/Emission 525). For Figure 1, 100 cells were analyzed from 3 different replicates and total of 15 fields were analyzed. For Figure 2, 50 cells were analyzed from 3 different replicates and total of 10 fields were analyzed. A Zeiss LSM 510 META NLO two photon laser microscope system was used for imaging. Quantification of fluorescence in confocal images was done using ImageJ with 15 cells per treatment group. Quantification provided in Figure S1 and S2. (2) Media was replaced with 200 μL of a 20 μM working solution of 2′, 7′-dichlorofluorescin diacetate (Sigma-Aldrich, Grand Island, New York) in each well and incubated for 1 h. Following this incubation, the solution was removed and the wells were washed thrice with DPBS. The cells were then treated with 10, 25, 50, and 75 μg/mL O-GNR-PEG-DSPE for 15 min. The nanoparticles were then removed using 3 washes of DPBS and the cells were fixed with 2.5% glutaraldehyde before imaging (Excitation 490/Emission 525). Image of untreated cell provided in Figure S3.

(3) Cells were treated with 10, 25, 50, and 75 μg/mL O-GNRPEG-DSPE followed by immediate addition of 50 nM voltage sensitive dye DiBAC4(3) (Life Technologies, Carlsbad, CA) in KRH buffer for 5 min. This step was followed by removal of the nanoparticles and DiBAC4 and three washes with phosphate buffered saline. The cells were then fixed for 30 min with 2.5% glutaraldehyde before imaging (Excitation 490/Emission 525). (4) Cells were loaded with Fura 2-AM (Sigma-Aldrich, Grand island, New York) by treating them with a 0.02 mM Fura 2-AM solution in DPBS for 60 min followed by its removal and three washes with DPBS. The cells were then treated with 50 μg/mL and 100 μg/mL O-GNR-PEG-DSPE for 5 min followed by their removal with multiple washes of DPBS. The cells were then fixed for 30 min with 2.5% glutaraldehyde before imaging. Although unconventional for Fura-2AM protocol, we used fixed cells for our study so that the calcium entry immediately after O-GNR-PEG-DSPE exposure can be qualitatively estimated following a previously reported protocol.17 Furthermore, we used a single excitation wavelength (Excitation 340 nm/ Emission 510 nm) because at this wavelength, emission from Fura-2 increases with increasing Ca2+ concentration.18 A Zeiss LSM 510 META NLO two photon laser microscope system was employed. Quantification of fluorescence in confocal images was done using ImageJ with 15 cells per treatment group per field (n = 15). A total of 6 fields were analyzed from 4 different treatments. Oxidative Stress. Oxidative stress in terms of ROS generation after 15 min incubation with O-GNR-PEG-DSPE was investigated in HeLa cells. For this study, 5 × 104 cells were plated per well in 96 well plates and incubated for 24 h. After 24 h, the old media was removed and 200 μL of a 20 μM working solution of 2′, 7′-dichlorofluorescin diacetate (Sigma-Aldrich, Grand Island, New York) was added in each well, and incubated for 1 h. This solution was replaced by 200 μL of DPBS (containing 10% FBS) followed by addition of 50 μL of of OGNR-PEG-DSPE to each well for final treatment concentrations of 10, 15, 25, and 50 μg/mL. The O-GNR solutions were incubated for 15 min, and aspirated out. The wells were washed with DPBS, and 200 μL of DPBS (with 10% FBS) was added to each well. Fluorescence readings were taken at an excitation at 485 nm and emission at 530 nm using a Cytofluor fluorescence multiwell plate reader (Series H4000 PerSeptive Biosystems, Framingham, MA). Cells without treatment were used as a control. Statistics. Experimental data are represented as mean + standard deviation. Student’s t test was used to evaluate statistical differences among experimental groups. All statistical analyses were performed using a p < 0.05. n for each experiment are provided in the figure legends. C


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RESULTS AND DISCUSSION Nanoparticle-Facilitated Membrane Depolarization Induces Receptor activation. O-GNRs were coated (noncovalent) with PEG-DSPE. The physicochemical properties of O-GNR-PEG-DSPE have also been extensively characterized and previously reported. We have previously shown that OGNR-PEG-DSPEs are ∼300−500 nm in length and 80−125 nm in breadth and have a surface amenable for loading hydrophobic pharmaceuticals. Previous investigations have also established that functional groups (such as carboxyl and hydroxyl functional groups) mainly along the edges of OGNRs make them stable in an aqueous environment.19−21 We have previously established that O-GNR-PEG-DSPE activate EGFRs, and this activation leads to uptake via macropinocytosis of high amounts of the nanoparticle into cells expressing these receptors.21 This receptor activation is further enhanced by presence of human papilloma virus E5 proteins which helps recycle the already activated and internalized receptors to the cell surface. However, the mechanism of receptor activation was not determined.21 Direct EGFR binding,22 oxidative stress,23 or membrane depolarization could cause EGFR activation.24 Thus, in our initial in vitro experiments, we examined whether OGNR-DSPE-PEG induces EGFR activation via any of these three mechanisms. Treatment of HeLa cells at increasing concentrations of O-GNR-PEG-DSPE showed a concentration dependent increase in EGFR activation (Figure 1). To ascertain whether direct EGFR binding by the nanoparticles induces receptor activation, we treated HeLa cells with anti EGFR antibodies. These antibodies block the receptor’s ligand binding domain. We then performed receptor activation studies on the antibody treated cells. Figure 2A−F shows the results from this study that characterizes the activation of EGFR in HeLa cells (pretreated with anti EGFR antibody) in response to O-GNRPEG-DSPE. Figure 2A−C represents fluorescence, bright field, and merge images of HeLa cells treated with epidermal growth factor (EGF) after pretreatment with anti-EGFR antibody (that blocks ligand binding domain of EGFR). The confocal images show almost no fluorescence indicating very little EGFR activation due to the EGFR blocking antibodies. Figure 2D−F represents fluorescence, bright-field, and merge images of HeLa cells treated with O-GNR-PEG-DSPE after pretreatment with anti-EGFR antibody (that blocks ligand binding domain of EGFR). The images show high fluorescence, especially in the areas where O-GNR-PEG-DSPE is being internalized into the cell (red arrows) indicating that direct receptor by the nanoparticles binding is not the cause of the receptor activation observed. To determine whether induced oxidative stress was responsible for nanoparticle-mediated EGFR activation, we pretreated the HeLa cells with 2′,7′-dichlorofluorescin diacetate (DCFDA) before exposing the cells to different concentrations of O-GNR-PEG-DSPE. DCFDA is a cellular reactive oxygen species detection agent, and higher amounts of oxidative stress would lead to higher amounts of DCF fluorescence. Figure 3E (representative images in Figure 3A−D) shows that all nanoparticle treatment concentrations generated enhanced albeit similar amounts of oxidative stress. The lack of correlation between this result and the concentration dependent activation of receptors (Figure 1 and Figure S1) implied that oxidative stress due to the nanoparticles was an unlikely cause of EGFR activation.

Figure 3. Representative DCFDA fluorescence images of HeLa cells treated with (A) 10, (B) 15, (C) 25, and (D) 50 μg/mL O-GNR-PEGDSPE. Red arrows indicate individual cells. (E) Multiwell plate-based DCFDA fluorescence assay in HeLa cells exposed to 10−50 μg/mL OGNR-PEG-DSPE. Fifteen cells from each treatment group per field were analyzed for fluorescence quantification. A total of 6 fields were analyzed from 4 repeats. Data presented as mean + standard deviation. N = 4.

Membrane depolarization has been previously linked to aberrant EGFR phosphorylation and activation in several studies.24,25 The membrane depolarization of HeLa cells exposed to O-GNR-PEG-DSPE was investigated using the potential sensitive dye DiBAC4 (3). Entry of DiBAC4 (3) into cells depends on the depolarization potential status of its membrane i.e increasing depolarization potential leads to increased uptake of the dye into the cells.26 Once uptaken into cells, DiBAC4 (3) binds to intracellular proteins or cell organelle membranes to fluoresce.26 Figure 4F shows the fluorescence quantification of DiBAC4 (3) in HeLa cells treated with various concentrations of O-GNR-PEG-DSPE (Representative images in Figure 4A−E). The figure shows that increasing concentration of nanoparticle treatment produced increasing DiBAC4 (3) fluorescence in HeLa cells. The results provided evidence that O-GNR-PEG-DSPEs depolarize the cell membrane. Next, drug delivery studies using O-GNR-PEGDSPEs were performed to further corroborate above mechanism-of-action results. Previous in vitro studies had already established the anticancer drug loading and delivery capabilities of O-GNRPEG-DSPEs in drug resistant cancer cells that express EGFR.21 The choice of cells (drug resistant HeLa cells) and drug (doxorubicin) was based on those previous studies. Lactate dehydrogenase (LDH) assay, previously validated as a suitable for cytotoxicity studies without any interference from OGNRPEG-DSPE,19 was employed to characterize cytotoxicity due to cellular delivery of Dox.21,27 Figure 5A shows the drug D


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EGFR Activation Occurs via Influx of Ca2+ Ions. In excitable and nonexcitable cells, membrane depolarization results in entry of extracellular Ca2+ into the cells.28,29 This Ca2+ entry can potentially lead to growth and proliferation promoting events such as activation of EGFR signaling.30 Ca2+ transport into cells has been reported to play a crucial role in the EGFR activation process.24,25,31 Hence, we further investigated whether O-GNR-PEG-DSPE treatment induced membrane depolarization produced Ca2+ entry in to the cells. Fura 2- acetoxy methyl (Fura 2-AM) is a cell-permeable dye that can enter cells and get cleaved by intracellular esterases and bind to Ca2+ ions to form a complex.25,32With increasing intracellular Ca2+ concentrations (i.e more Ca2+ binding to the Fura-2AM), there is a change in the excitation maxima from 380 to 340 nm. This change implies, with increasing Ca2+ entry, the emission from the cells will also increase when excited at 340 nm. Although Fura2-AM is generally used as a ratiometric dye (emission measured at 510 nm after excitation at both 380 and 340 nm), we used only one wavelength since our interest was to only qualitatively analyze the increased fluorescence due to Ca2+ entry.18 Figure 5F shows the quantification of the fluorescence in HeLa cells loaded with Fura 2-AM that were treated with increasing concentrations of O-GNR-PEG-DSPE (Representative images in Figure 5C−E). Increases in fluorescence of HeLa cells were noted with increases in OGNR-PEG-DSPE treatment concentrations. Fluorescence intensity of cells treated with 50 and 100 μg/mL O-GNRPEG-DSPE were ∼5 times and ∼8 times greater, respectively, compared to untreated cells. The results (Figures 1−5) taken together provided evidence of O-GNR-PEG-DSPE-mediated membrane depolarization and clues on the mechanism of EGFR activation due to the depolarization. Because membrane depolarization leads to cellular entry of Ca2+, we investigated whether this entry was necessary for the EGFR activation post membrane depolarization by the nanoparticles. Drug delivery experiments in normal media (DMEM) containing Ca2+ showed ∼100% more cell death in HeLa cells in case of in Dox-loaded O-GNR-PEG-DSPE compared to free drug (Figure 5G). In contrast, repeating the same experiment in cell media without Ca2+ resulted in a decrease of the drug delivery efficiency of Dox-loaded O-GNRPEG-DSPE (i.e., cell death) to ∼35% of free drug in PEGDSPE (Figure 5H). This result hinted that extracellular Ca2+ entry maybe essential for the uptake of drug loaded O-GNRPEG-DSPE. However, completely eradicating the increased OGNR-PEG-DSPE-mediated drug delivery and resulting cell death could not be possible due because of the following reasons: (1) there were some remnant Ca2+ from plating the cells that could not be removed or (2) down regulated, there could be other minor pathways at play contributing to the increased uptake of the nanoparticles. In Vivo Drug Delivery into Drug Resistant Tumors. In vivo proof-of-principle studies were performed to investigate whether the uptake phenomenon could be exploited to deliver drugs into drug-resistant cells for improved chemotherapeutic effect. A growing body of in vitro studies show that drug resistance in cancer cells could be overcome by efficient delivery and release of the drug inside the cancer cells.21,33−35 However, demonstrating the in vivo potential of a drug delivery system is significantly more challenging. Once drug-loaded nanoparticles accumulate inside the tumor interstitium, their uptake into cells and release of the drug determines their drug delivery efficiency.36 The tumor microenvironment is a

Figure 4. Representative DiBAC4(3) fluorescence images of HeLa cells treated with (A) 0, (B) 10, (C) 25, (D) 50, and (E) 100 μg/mL O-GNR-PEG-DSPE. Red arrows indicate individual cells. (F) Quantification of DiBAC4(3) fluorescence in HeLa cells treated with 0−100 μg/mL O-GNR-PEG-DSPE. Fifteen cells from each treatment group per field were analyzed for fluorescence quantification. A total of 6 fields were analyzed from 4 repeats. Data presented as mean + standard deviation * indicates significant increase (p < 0.05) in fluorescence intensity compared to untreated cells. N = 4. Data presented as mean + standard deviation.

delivery efficiency of different concentrations of O-GNR-PEGDSPE. The results show that LDH release increased in a concentration dependent manner implying increasing cell death. Treatment of the HeLa cells with 10 μg/mL Doxorubicin (Dox) loaded O-GNR-PEG-DSPE resulted in ∼36% LDH release compared to lysed control cells. Treatment of the cells with 25, 50, and 75 μg/mL Dox-loaded O-GNRPEG-DSPE resulted in ∼47, ∼65, and ∼75% LDH release compared to lysed control cells, respectively. We further confirmed that this O-GNR-PEG-DSPE-facilitated drug delivery was not due to the binding of the nanoparticles to EGFRs by repeating the drug delivery studies at a fixed O-GNR-PEGDSPE concentration (50 μg/mL) in drug resistant HeLa cells with blocked EGFR ligand binding domains (with anti-EGFR antibodies). Results (Figure 5B) showed that LDH release from HeLa cells without antibody treatment was ∼68% of lysed control cells. In comparison, LDH release in 65 nM, 130 nM and 200 nM antibody treated cells was ∼63, ∼64, and ∼66% of lysed control cells, respectively. The results indicated that LDH release from HeLa cells did not change with or without antibody treatment. They further corroborated the results presented in Figures 2A−F, where EGFR activation was noted even after blocking the EGFR ligand binding domain with antibodies. E


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Figure 5. (A) Lactate dehydrogenase release after treatment of HeLa cells with different concentrations of doxorubicin-loaded O-GNR-PEG-DSPEs. (B) Lactate dehydrogenase release after treatment of doxorubicin (Dox)-loaded O-GNR-PEG-DSPEs to untreated or anti-EGFR antibody treated HeLa cells. N = 4. * indicates significant increase (p < 0.05) in LDH release compared to untreated cells. Data presented as mean + standard deviation. Representative Fura2-AM-Ca2+ fluorescence images of HeLa cells treated with (C) 0, (D) 50, and (E) 100 μg/mL O-GNR-PEG-DSPE. Red arrows indicate individual cells. (F) Quantification of Fura-2AM fluorescence in HeLa cells treated with 0−100 μg/mL O-GNR-PEG-DSPE. Fifteen cells from each treatment group were analyzed for fluorescence quantification. Data presented as mean + standard deviation * indicates significant increase (p < 0.05) in fluorescence intensity compared to untreated cells. N = 4. Data presented as mean+standard deviation. (G and H) Lactic dehydrogenase (LDH) release after treatment with Dox-loaded O-GNR-PEG-DSPEs in HeLa cells grown in (G) normal DMEM (H) DMEM without Ca2+. Untreated cells, cells treated with free doxorubicin in PEG-DSPE, and lysed cells were additional controls. All data are normalized to LDH released from lysed control cells. Data are presented as mean + standard deviation (n = 6 per group). * indicates significant increase (p < 0.05) in LDH release compared to cells exposed to Dox in PEG-DSPE at the same concentration as loaded onto O-GNR-PEG-DSPEs. 1 = untreated control, 2 = O-GNR-PEG-DSPE (50 μg/mL), 3 = O-GNR-PEG-DSPE-Do,x 4 = Dox in PEG-DSPE (same concentration as loaded), 5 = Dox in PEG-DSPE (double concentration as loaded).

MCF cells do not express EGFR.39,40 Stalled or decreased tumor growth is a good indicator of efficacious nanoparticle cellular uptake and drug delivery.38 Efficiency of drug delivery by O-GNR-PEG-DSPE in subcutaneous xenograft tumors was evaluated by measuring the change in tumor volume and morphological analysis of the tumors. Figure 6A, B shows the change in volume of HeLa and MCF7 cell xenograft tumors in immune-compromised mice injected with 50 μL of drug-loaded O-GNR-PEG-DSPE and

complex milieu which may present barriers in the intracellular uptake of nanoparticle drug delivery systems.37 Thus, for the in vivo study, the Dox-loaded O-GNR-PEG-DSPE or the controls were injected directly into tumors. A well-established subcutaneous xenograft tumor model was employed for these studies.38 Doxorubicin-resistant HeLa or MCF7 cells were chosen as the cell lines for inducing xenograft tumors as these cell lines have significantly different EGFR expression. HeLa cells express high (∼5 × 104) EGFR receptors per cell, whereas F


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Figure 6. (A, B) Change in tumor volumes of HeLa and MCF7 cell xenografts injected thrice with 50 μL of O-GNR, O-GNR-PEG-DSPE, PEGDSPE, and Dox. N = 3 tumors per group. Hematoxylin and Eosin stained histology sections of subcutaneous (C, D) HeLa tumors and (E, F) MCF-7 tumors injected thrice with 50 μL of Dox-loaded onto O-GNR-PEG-DSPE. Hematoxylin and Eosin stained histology sections of untreated (G, H) HeLa xenograft tumors and (I, J) MCF7 xenograft tumors. White arrows indicate necrotic cells. (K) Schematic representation of probable mechanism of membrane depolarization and EGFR activation by O-GNR-PEG-DSPE.

other controls (50 μL of O-GNR-PEG-DSPE, 50 μL of free Dox at the identical concentration as loaded in O-GNR-PEGDSPE, and 50 μL of PEG-DSPE) 3 times over 12 days (injections on days 0, 4, and 8). HeLa tumors treated with PEG-DSPE and O-GNRs showed an increase in tumor volume by ∼80 and ∼33%, respectively. Free Dox in PEG-DSPE showed a tumor volume increase of ∼120%. A statistically insignificant increase in tumor volume (∼8%) was noted in animals treated with Dox-loaded O-GNR-PEG-DSPE. Rodents with MCF7 xenograft tumors showed significant increase in size across all groups. MCF7 tumors treated with PEG-DSPE and O-GNR-PEG-DSPEs showed an increase in tumor volume by ∼200 and ∼150%, respectively. In these tumors, both free drug in PEG-DSPE and drug-loaded O-GNR-PEG-DSPE groups also showed ∼200% increase in tumor volume. Figure 6C−J show bright-field images of histological sections from the HeLa and MCF7 xenograft tumors stained with hematoxylin and eosin. The histology specimens of treated HeLa tumors (Figure 6C, D) showed higher number of necrotic cells (white arrows) compared other treatment groups (Figure 6G, H) untreated cells shown as reference, other treatments showed a similar number of necrotic cells as untreated cells). Additionally, fibrosis was observed in the HeLa tumors treated with Dox-loaded O-GNR-PEG-DSPE (Figure 6C, D, red arrows).

MCF7 tumors did not show any morphological change in the cells for all four treatment groups (Figure 6E, F, I, J). The in vivo results clearly indicate than uptake and delivery of Dox by O-GNR-PEG-DSPE is the main reason for the observed tumor growth inhibition. On the basis of the in vitro and in vivo results, the following model is proposed to explain the cellular uptake and accumulation of O-GNR-PEG-DSPEs. The O-GNR-PEGDSPEs cause transient depolarization of cell membranes resulting in influx of Ca2+ from the extracellular space. The Ca2+ influx leads to EGFR activation. In cells with high EGFRs, this activation leads to a macropinocytotic response and uptake of the drug loaded nanoparticles into the vicinity of the cells (Figure 6K). Recent in vitro studies indicate certain nanoparticles activate cell surface proteins;41 however, the mechanism of activation still needs to be determined. The above results add O-GNRPEG-DSPEs to this growing list of nanoparticles that activate cell surface receptors and elucidates the mechanism of activation. However, we note that this study serves as the first step for additional future studies that are needed to examine the molecular basis in finer detail and leverage those findings for drug delivery in other model systems. Nanoparticles have been shown to induce cell membrane G


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1DP2OD007394-01), and the Wallace H. Coulter Foundation Translational Grant.

depolarization by interacting with the membrane and altering their resting membrane potential.30,42,43 The extent of depolarization induced depends on the surface chemistry of the nanoparticle, especially on the type of material, shape, size, and charge.30 Thus, other nanoparticles could be tested and potentially elicit membrane depolarization induced EGFR activation. Taken together, the results could allow development of alternate strategies for cellular uptake of nanoparticles targeting for drug delivery and imaging applications. EGFRs, often overexpressed in large number of cancers, are a highly attractive targets for anticancer therapeutics.24 However, resistance associated with antibody therapies targeting EGF receptor have severely limited treatment options.44 Exploiting membrane depolarization to facilitate the activation of EGFRs and induction of micropinocytosis could serve as alternative strategy to increase uptake of drugs into EGFR overexpressing cells and improving therapeutic outcomes.

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CONCLUSIONS We have previously shown in our published studies that OGNR-PEG-DSPE is internalized in EGFR overexpressing cells within seconds of coming in contact with cell and the macropinocytotic event results in large nanoparticle filled vesicles in the cytoplasm of the cells. However, until now the exact series of events that leads to this macropinocytotic event is unexplored. Our results indicate that O-GNR-PEG-DSPEs are uptaken into HeLa cells via micropinocytosis uptake mechanism that is induced through transient cell membrane depolarization, and the consequent activation of EGFR receptors by calcium ions that enter these cells. This phenomenon could be exploited to facilitate efficacious drug delivery efficiency even in the complex milieu of EGFR overexpressing tumors. Taken together, the results suggest cellular uptake mechanism such as endocytosis or micropinocytosis induced via cell surface receptor activation through transient membrane depolarization could serve as an alternate strategy for uptake and delivery of therapeutic or imaging agents into specific cells.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00338.

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REFERENCES

Figures S1−S3 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 631-6321810. Present Address †

S.M.C. is currently at Stanford University School of Medicine, 3155 Porter Drive, Palo Alto, CA 94305, USA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Guo-Wei Tian (Central Microscopy, Stony Brook University) for his help in Confocal Microscopy. This work was supported by the National Institutes of Health (Grant H


Article

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