In Vitro and In Vivo Tumor Targeted Photothermal Cancer Therapy

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In Vitro and In Vivo Tumor Targeted Photothermal Cancer Therapy Using Functionalized Graphene Nanoparticles Sung Han Kim,†,‡ Jung Eun Lee,†,§ Shazid Md. Sharker,∥ Ji Hoon Jeong,§ Insik In,*,‡,⊥ and Sung Young Park*,‡,# ‡

Department of IT Convergence, ⊥Department of Polymer Science and Engineering, and #Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea § School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea ∥ Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea S Supporting Information *

ABSTRACT: Despite the tremendous progress that photothermal therapy (PTT) has recently achieved, it still has a long way to go to gain the effective targeted photothermal ablation of tumor cells. Driven by this need, we describe a new class of targeted photothermal therapeutic agents for cancer cells with pH responsive bioimaging using near-infrared dye (NIR) IR825, conjugated poly(ethylene glycol)-g-poly(dimethylaminoethyl methacrylate) (PEG-g-PDMA, PgP), and hyaluronic acid (HA) anchored reduced graphene oxide (rGO) hybrid nanoparticles. The obtained rGO nanoparticles (PgP/HA-rGO) showed pHdependent fluorescence emission and excellent near-infrared (NIR) irradiation of cancer cells targeted in vitro to provide cytotoxicity. Using intravenously administered PTT agents, the time-dependent in vivo tumor target accumulation was exactly defined, presenting eminent photothermal conversion at 4 and 8 h post-injection, which was demonstrated from the ex vivo biodistribution of tumors. These tumor environment responsive hybrid nanoparticles generated photothermal heat, which caused dominant suppression of tumor growth. The histopathological studies obtained by H&E staining demonstrated complete healing from malignant tumor. In an area of limited successes in cancer therapy, our translation will pave the road to design stimulus environment responsive targeted PTT agents for the safe eradication of devastating cancer.

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INTRODUCTION The tremendous number of studies in cancer therapy have explored new avenues for the treatment of this devastating disease. Although astonishing progress has been achieved through gaining a better understanding of the growth promoting machinery of cancer, the mortality rates are still horrible.1 Cancer treatments, including chemotherapy, surgical intervention, and radiation, which often cause toxicity to patients, are the currently available weapons to fight cancer.2 It would therefore be desirable to reinforce such treatment options by exploring alternative therapeutic systems. As an alternative, photothermal therapy (PTT) has recently achieved promising success, providing new hope to cancer patients.3 In the past decades, near-infrared (NIR) active small organic fluorescent dyes, including indocyanine green (ICG), IR825, and BODIPY, have been extensively studied to design fluorescence nanomaterials for optical imaging.4 Instead of heat, the majority of these molecules have fluorescence © 2015 American Chemical Society

emission in response to NIR light. Engineered organic dyes with strong NIR absorption efficiency but low emission were proven as the best photothermal candidates.5 To explore these candidates, modification of the organic fluorescent dyes with composite materials has prevailed for the improvement of photothermal agents.6,7 As the efficacy of anticancer agents depends on targeting, targeted ligands including antibody, aptamer, RGD peptide, folic acid, and hyaluronic acid (HA) have been utilized in conjunction with delivery carriers to achieve this goal.8 Among them, the HA can be considered a preferable choice due to its easy fabrication and functionalization. The polysaccharide HA which is known as extracellular cell surface specific ligand, and confer critical roles during cells growth, healing process, and Received: July 15, 2015 Revised: September 30, 2015 Published: October 9, 2015 3519

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tumor prognosis. It therefore has been widely used in tumor targeting owing to its binding surface CD44 receptor, which are overexpressed in various cancer cells. Due to this interesting affinity, the surface binding of HA by cancer cells has been popularly used in designing drug and gene carrier systems for tumor-specific delivery.9 Recently, the reduced form of graphene oxide (rGO) has attracted increasing interest or use in drug delivery, gene delivery, PTT, polymeric nanostructure development, and designing functional biomaterial for biomedical application.10,11 Interestingly, along with these properties, the quenching capability of graphene oxide has proven efficacy for the development of controllable fluorescence materials.12 More recently, composite polymers with rGO base design have also demonstrated an emerging possibility due to easy incorporation of environmentally sensitive desired cues inside these systems. For optical biosensing, graphene oxide was suggested as an optimal quencher, with a lattice of acceptor molecules exposed on the surface of its two-dimensional structure. Importantly, graphene oxide is considered the best FRET acceptor when compared to other carbon structures such as graphite, carbon nanotubes, and carbon nanofibers. It is also noteworthy that the high planar surface of rGO gained acceptance as a highly efficient universal long-range quencher.12 To construct composite material, the adhesive properties of catecholcontaining polymers on the rGO plane have shown promising results to design different functional biomaterials. The length of adhesion depends on available surface and quantity of catechol, which can be utilized to integrate different functional clues on the rGO plane.13,14 For the treatment of cancer, NIR-irradiated PTT agents appeal as one of the most promising tools, where treated photoactive agents convert NIR light into photothermal heat for the photothermolysis of the treated cells. As a result, PTT has gained exciting success, along with the typical mainstream cancer therapy. The deep tissue penetration with NIR light (690−900 nm) causes less photodamage to influence its superior photothermal activity.3 Besides this, the possibilities for early recovery from the disease and treatment of chemotherapy-resistant cancers have made PTT more acceptable compared with other existing therapies.3,15 However, due to design difficulties, most of the PTT agents currently available have adopted passive tumor targeting. Since passive targeting is much more complex than active targeting,16 it would be desirable to develop PTT agents that are responsive to the tumor environment and can actively target tumors to maximize the therapeutic benefits. In this study, we report NIR active IR825, and adhesive 2chloro-3′,4′-dihydroxyacetophenone (CA) quaternized with polyethylene glycol grafted poly(N,N-dimethylaminoethyl methacrylate) (I/C-PgP) and HA-conjugated dopamine (DHA) anchored to rGO, achieved by utilizing the adhesive properties of catechol to make stable NIR active hybrid nanocomposites. Herein, the pH-triggered regulation of fluorescence resonance energy transfer (FRET) was achieved by relief of the quenching effects of PDMA on IR825 on a single rGO sheet. With converted photothermal heat and cancer cell-targeted receptor−ligand (CD44-HA) interactions, depending on time, concomitant in vitro cellular uptake into cancer cells and targeted in vivo tumor ablation can be achieved.

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EXPERIMENTAL SECTION

Materials. Polyethylene glycol (PEG, Mn 3500), 2-(dimethylamino) ethyl methacrylate (DMA), toluene, hexane, 2-chloro-3′,4′dihydroxyacetophenone(CA), dichloromethane (MC), diethyl ether, ethanol, Trizma base, Trizma HCl, and [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma-Aldrich, Korea. Propidium iodide, calcein AM, and LysoTracker Blue DND-22 were purchased from Molecular Probe, Life Technologies (Invitrogen). Penicillin−streptomycin, fetal bovine serum (FBS), 0.25% (w/v) Trypsin, 0.03% (w/v) EDTA (ethylene diamine tetra acetic acid) solution, and RPMI-1640 medium were purchased from Gibco BRL (Carlsbad, CA, U.S.A.). Polyethylene glycol grafted poly(N,N-dimethylaminoethyl methacrylate) [PEG-gPDMA] was synthesized following the method in a report published elsewhere.17 Hyaluronic acid conjugated dopamine (D-HA) was also synthesized following a report published elsewhere.18 The heptamethine indocyanine dye IR825 was synthesized by adopting a recently published method.5 1 H NMR spectra were recorded using a Bruker Advance 400 MHz spectrometer with deuterium oxide (D2O) and deuterium dimethyl sulfoxide (DMSO-d6) as the solvent. The UV−vis spectra were recorded using an Optizen 2020UV; Mecasys Co. AFM imaging was carried out in tapping mode on a MultiMode8 (Bruker) with a silicon probe. For AFM images, samples were prepared on silicon wafers. Using an infrared camera (NEC Avio, Thermo Tracer TH9100), particle size was measured with dynamic light scattering (DLS; Zetasizer Nano, Malvern-Germany). XPS spectra were obtained using an Omicrometer ESCALAB (Omicrometer, Taunusstein, Germany) and transmission electron microscopy (FEI, Netherlands). Photoluminescence (PL) spectra were obtained on a L550B luminescence spectrometer from PerkinElmer. Zeta potential data were obtained using a particle size analyzer (ELS-Z) from Otsuka Electronics Corporation. The NIR laser was 808 nm (PSU-III-LRD, CNI Optoelectronics Tech. Co. LTD, China). Synthesis of IR825 and 2-Chloro-3′,4′-dihydroxyacetophenone (CA) Quaternized PEG-g-PDMA (I/C-PgP). PEG-g-PDMA (2 g), IR825 (0.517 g), and CA (0.115 g) were dissolved in 50 mL of anhydrous ethanol in a 250 mL flask. The mixture was stirred for 12 h at 70 °C under a nitrogen atmosphere. At the end of the reaction, the solvent was evaporated in a rotary evaporator. Diethyl ether was added to the polymer to induce precipitation. The resulting I/C-PgP was dried in a vacuum oven and analyzed. The conjugated composite materials (degrees of IR825 and CA units were 4 and 5, respectively) were characterized by 1H NMR spectroscopy (Supporting Information, Figure S2). 1H NMR (400 MHz, DMSO-d6): δ 0.81−1.25 (3H; −CH3 of DMA), 2.10−2.32 (2H; −O−CH2− of DMA), 3.63 (4H; −CH2−CH2−O− of PEG), 6.25 (1H; aromatic), 7.00 (1H; aromatic), 7.40−8.10 (2H; aromatic), and 7.90−8.12 ppm (2H; aromatic proton of IR825). Preparation of Reduced Graphene Oxide (rGO)-Anchored I/ C-PgP and D-HA (PgP/HA-rGO). The D-HA and I/C-PgP were anchored with reduced graphene oxide at the ratio of 20/100 to obtain functionalized GO nanoparticles as a theranostic agent for imagingguided photothermal therapy (Supporting Information, Figure S1). In detail, 20 mg of D-HA and 100 mg of I/C-PgP composites were dissolved in 9 mL of 2× PBS (pH 8.5) and then mixed with 1 mL (1 mg/mL) of GO for 24 h. The reaction was performed at room temperature, and the pH was maintained at a mildly basic level during mixing (pH 8.5). Finally, the unreacted composite polymer and HA were remove by centrifugation, and the solution was freeze-dried to obtain the PgP/HA-rGO nanoparticles. Confocal Imaging. The cellular uptakes of the PgP/HA-rGO photothermal materials were analyzed via confocal imaging. The MDAMB-231, A549, and MDCK cells were plated over a cover slide on an eight-well plate at a density of 2 × 105 cells/mL per well and incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. The cells were then treated with PgP/HA-rGO at 0.01 mg/mL for 30 min in fresh culture media with pH values of 5.0, 6.0, 6.8, and 7.4, respectively. HCl (0.1 N) and NaOH (0.1 N) were used to adjust the pH of the culture medium. The cells were then washed several times 3520

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Scheme 1. Schematic Illustration of the Preparation of PgP/HA-rGO Nanoparticles and Active Target Specific Endosomal Releasea

a

The prepared nanoparticles carry near-infrared (NIR) active IR825 agent, which released photothermal heat in response to NIR irradiation.

with PBS to remove the unbound PgP/HA-rGO materials. Finally, the cells were examined at 20× magnification using an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany). To quantify the cellular uptake of PgP/HA-rGO, cancerous MDAMB-231, A549, and normal MDCK cells were selected and seeded in a 96-well plate at a concentration of 0.5 × 105 cells/mL. After 24 h of incubation, the media were removed, and the cells were treated with 0.2 mg/mL of PgP/HA-rGO -containing media. Cells were then allowed to incubate for another 4 h. At the end of the period, the sample containing media was removed, and the cells were washed several times with PBS (1×, pH 7.4). Triton X-100 (1%) was then used to lysis the cells. The relative amounts of accumulated PgP/ HA-rGO within the cell interior were determined by measuring the fluorescence intensity at excitation wavelengths of 365 nm and emission wavelengths of 465 nm, respectively, in a multimode microplate reader (Filter MaxF3, Molecular Devices, LLC.) MTT Assay. Cytotoxicity was measured using the [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay method. Here, 200 μL aliquots of MDAMB (MDAMB-231), A549, and MDCK cells, at a density of 2 × 105 cells/mL, were placed in each well of a 96-well plate. The cells were then incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. To determine the cellular viability, a stock solution of PgP/HA-rGO was dissolved in RPMI medium at the concentration of 1 mg/mL, after which the stock solution was diluted to 0.01 mg/mL. The media was removed and the cells were treated with different concentrations of the PgP/HA-rGO composite material. The cells were then incubated as described above for another 24 h. The media containing PgP/HA-rGO was then replaced with 180 μL of fresh medium and 20 μL of a stock solution containing 15 mg of MTT in 3 mL of PBS, after which the cells were incubated for another 4 h. Finally, the medium was removed and 200 μL of MTT solubilizing agents was added to the cells. Shaking was performed for 15 min, and then the absorbance was measured at 570 nm using a microplate reader (Varioskan Flash, Thermo Electron Corporation). Relative cell viability was measured by comparison with the control wells containing only cells. In Vitro Photothermal Cytotoxic Assay. The photothermal cytotoxicity of PgP/HA-rGO was evaluated on MDAMB-231, A549, and MDCK cells. First, 200 μL of the cells at a density of 2 × 105

cells/mL was placed in each well of a 96-well plate. The cells were then incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. To assess the in vitro photothermal effects of PgP/HA-rGO, the culture media were replaced with media containing PgP/HA-rGO at concentrations of 1 mg/mL to 0.01 mg/mL. HCl (0.1 N) and NaOH (0.1 N) were used to adjust the pH (5.0, 6.0, and 7.4). After replacement of the media, the cells were incubated in 96-well plates at 37 °C for 30 min and then irradiated with an 808 nm laser at the power density of 2 W/cm2 for 5 min. The cells were then incubated for another 24 h. The viability and proliferation of MDAMB, A549, and MDCK cells were evaluated by the MTT assay method. Calcein AM and Propidium Iodide Cell Staining Assay. Imaging of the photothermal cytotoxicity was carried out through a calcein AM and propidium iodide (PI) staining method, following that described in the literature.19 To assess the photothermal effects, the MDAMB-231, A549, and MDCK cells were incubated for 30 min at 37 °C in 8-well plates containing PgP/HA-rGO at a concentration of 0.2 mg/mL, with different pH values (5.0, 6.0, 6.8, and 7.8) of the cell culture media. HCl (0.1 N) and NaOH (0.1 N) were used to adjust the pH of the cell culture medium. At the end of the incubation time, the cells were irradiated with a laser at 808 nm with the power density of 2 W/cm2 for 5 min. The cells were then stained with both calcein AM (calcein acetoxymethyl ester) and PI (propidium iodide). Finally, an LSM510 confocal laser scan microscope (Carl Zeiss, Germany) was used at 10× magnification for imaging of the stained (live/dead) cells. In Vivo Thermographic Imaging. Six-week-old balb/c mice were used to carry the investigation. All animal studies were approved by the SKKU School of Pharmacy Institutional Animal Care. To evaluate the in vivo PTT efficiency, PgP/HA-rGO and free IR825 were injected into the tail vein at a concentration of 30 mg/kg of body weight. The treated mice were then observed at 0, 4, 8, 12, and 24 h post-injection. Thermographic images were captured using an OPTIX MX3 imaging system (ART Inc., Montreal, QC) at 0, 1, 2, 3, 4, and 5 min intervals, and the average temperatures were plotted as a function of NIR irradiation time. In Vivo Biodistribution. To observe biodistribution of PgP/HArGO separately in an in vivo model, the athymic nude mice were considered for investigation when they reached 6 weeks of age, and 200 μL of PBS containing the PgP/HA-rGO (5 mg kg −1), 3521

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Figure 1. (a) UV−vis absorption spectra of the prepared I/C-PgP and PgP/HA-rGO nanoparticles, respectively, at 0.1 mg/mL. (b) The fluorescence emission intensity of PgP/HA-rGO nanoparticles (0.1 mg/mL) at different pH values (5.0−7.4) upon excitation at 480 nm. (c) Concentration-dependent (1.0−0.001 mg/mL) photothermal heat (ΔT = Tn − T0) generation curve of PgP/HA-rGO nanoparticles as a function of irradiation time. The NIR laser was at 808 nm with a power density of 2 W/cm2. respectively, was injected into the tail veins of the mice. They were then sacrificed at 24 h post-injection, and the major organs were excised and observed using the Kodak image station (Kodak Image Station 4000MM, New Haven, CT, U.S.A.). All the near-infrared fluorescence (NIRF) intensities were calculated using the Analysis Workstation software (ART Advance Research Technologies Inc., Montreal, Canada). In Vivo Photothermal Therapy and Histology. Tumor-bearing mice models were prepared in an identical manner, as described in a published report.20 To observe the in vivo photothermal effects of PgP/HA-rGO, athymic nude mice were prepared by inoculation of the left flank of the mice with a suspension of MDAMB cells at 1 × 107 cells/mL in saline (60 μL). When the tumor reached about 255 ± 10 mm3 in volume, 200 μL of saline or PBS containing free IR825 or PgP/HA-rGO were injected into the mice (n = 5 per group). At 2 h post-injection, the tumor tissues were irradiated with the 808 nm laser (2 W/cm2) for 5 min. In the next step, the therapeutic results of each group were evaluated by measuring the tumor volumes for 18 days. Tumor tissues were excised from the mice at days 1 and 10 postinjection for histological analysis and were fixed with 2% paraformaldehyde solution and embedded in paraffin. The sliced tumor tissues (6 μm) were stained with Haematoxylin and Eosin (H&E) and observed via an optical microscope. All in vivo studies have been performed by taking five mice in each experimental group.

way, it becomes apparent that passive targeting strategies might not capitalize the full potential benefits of therapeutic agents.8 Herein, we developed GO hybrid nanoparticles for tumor targeting of a PTT agent combined with pH-responsive bioimaging effects, where incorporated HA-Dopa (D-HA) facilitates target localization of the graphene sheets.18 The NIRirradiated target-localized PTT agent can release its maximum beneficial photothermal heat due to localization at the tumor environment using HA as a target molecule for delivery of the PgP/HA-rGO nanoparticles. The tertiary amine of PDMA in the rGO sheet leads to dissociation of IR825 on the composite polymer from its quencher on the rGO plate, allowing for a pHtunable bioimaging agent.19 The overall plan to achieve this goal is illustrated in Scheme 1. To enable the use of photothermal cytotoxic cancer agents, it is essential to design materials that have NIR windows (650− 900 nm wavelength).3 The characteristic intense UV−vis absorption of PgP/HA-rGO nanoparticles demonstrated NIR sensitivity, wherein IR825 absorption peaked at ∼800 nm (Figure 1a).5 Moreover, the heptamethine indocyanine derivative IR825 has strong emission near the 500−600 nm area when excited at 480 nm. This emission can be controlled by engineering the IR825 carrier system to obtain integration of diagnostic and therapeutic functionalities.5 The PgP/HA-rGO nanoparticles resulted in pH-dependent fluorescence emission, where the intensity increased with decreasing pH (Figure 1b).

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RESULTS AND DISCUSSION Like other cancer therapies, the challenge for success of PTT is to come up with an efficient delivery to the desired site. In this 3522

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Figure 2. (a) Transmission electron microscopy (TEM) image of PgP/HA-rGO nanoparticles. (b) Atomic force microscopic (AFM) imaging of a droplet of PgP/HA-rGO nanoparticles on silicon wafers and corresponding particle height profile of the respective AFM image. (c) Dynamic light scattering (DLS) measurement of PgP/HA-rGO nanoparticles in an aqueous medium (pH 7.4).

Figure 3. (a) Confocal laser scan microscopic (CLSM) imaging of A549, MDAMB (MDAMB-231), and MDCK cells treated with PgP/HA-rGO nanoparticles in different pH (5.0, 6.0, 6.8, and 7.4, respectively). All scale bars are 20 μm. (b) Quantitative cellular accumulation (%) of PgP/HArGO nanoparticles in A549, MDAMB, and MDCK cells. This study was performed in neutral pH.

The increasing acidity below the pKa of the DMA units resulted in increased hydrophilicity through the protonation of DMA on a single rGO sheet, which generates a gap between IR825 and rGO, finally relieving the fluorescence quenching.17 This leads to collapse or swelling of the aggregate and enhanced or decreased fluorescence resonance energy transfer (FRET) efficiency. The efficient NIR light absorption properties of the PgP/ HA-rGO nanoparticles demonstrate promising potential PTT

candidate. To determine the heat generation capacity upon irradiation with NIR light, different ratio (Supporting Information, Figure S3) and concentrations of the PgP/HArGO nanoparticles were examined, showing a sharp rise in the photothermal heat with increasing concentration. Interestingly, 1 mg/mL of the PgP/HA-rGO generated a 50 °C higher temperature (Figure 1c) where the polymeric carrier C-PgP with rGO did not show any significant photothermal response. This is because C-PgP is non-NIR-responsive polymer and the 3523

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Figure 4. (a) MTT assay for concentration-dependent in vitro biocompatibility study of MDAMB, A549, and MDCK cells treated with PgP/HArGO nanoparticles. (b) In vitro photothermal cytotoxicity of PgP/HA-rGO nanoparticles in different concentration (1.0−0.001 mg/mL) after NIR irradiation. (c) Calcein AM and propedium iodide stained live/dead cells fluorescence imaging of PgP/HA-rGO-treated A549, MDAMB-231, and MDCK cells. All scale bars were 50 μm, and the NIR irradiation was carried out for 5 min with an 808 nm laser at the power density of 2 W/cm2. All studies were performed in normal pH.

contents of rGO in C-PgP with rGO system are too little to generate significant photothermal heat. Furthermore, free IR825 and graphene oxide are not aqueous stable, which require functionalization for biological application (Supporting Information, Figures S4 and S5).6,20 In compositional structural evaluation, the X-ray photoelectron spectroscopy (XPS) spectra of PgP/HA-rGO showed characteristic C 1s core level peaks which can be curve-fitted by three components with the binding energies of about 283.6, 284.5, and 287 eV, attributable to the CC (sp2), C−C (sp3), and CO/C−N species, respectively (Supporting Information, Figure S6).19 The nitrogen core-line spectrum revealed two components at 399 and 401 eV, which are characteristic of the neutral amine (N0) and cationic amine (N+) functions, respectively, which confirmed the quaternization of CA and IR825. These results ensured the structural integrity of the rGO-anchored polymer composites (PgP/HA). Furthermore, the thermogravimetric analysis (TGA) demonstrated 35% weight of PgP/HA composite on PgP/HA-rGO nanoparticles (Supporting Information, Figure S7). At the same time XRD patter and Raman D/G band at ∼1330 and 1580 cm−1

demonstrate PEG functionalized rGO base nanoparticles where the FTIR peak at around 1730 cm−1 confirm carbonyl group originate from reduce graphene oxide (Supporting Information, Figure S8).14,21,22 The advantage of using nanostructures in therapeutic systems is that the small size allows biological barriers to be overcome, achieving cellular uptake.23 To verify the PgP/HA-rGO formation, the TEM images reveal that nanoparticle comprised of small spherical shape (Figure 2a). The size of the PgP/HArGO composites was studied via atomic force microscopy (AFM) of an aqueous drop of nanocomposite on a silicon wafer. The nanoparticles exhibited sizes of 157.8−149 nm in diameter with the depth of 15.97−16.8 nm (Figure 2b). Furthermore, the particle size measured by dynamic light scattering (DLS) showed the size distributions of 120−190 nm in aqueous medium (pH 7.4; Figure 2c).24 At the same time, colloidal stability study against serum and saline solution shows uniform dispersion to retain required stability in physiologic medium (Supporting Information, Figure S9). The selection and incorporation of smart functionalities can make theranostic agents targetable with controllable release of 3524

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Figure 5. In vivo thermographic image (a) and temperature elevation curve (b) at 0 (I), 4 (II), 8 (III), 12 (IV), and 24 h (V) time intervals from intravenous (i.v.) administrated of PBS, free IR825, and PgP/HA-rGO nanoparticles in tumor bearing nude mice. The plots of in vivo average temperature generation at i.v. injection of tumor bearing mice from the thermographic images. The NIR laser was 808 nm, applied for 5 min at the power density of 2 W/cm2.

quantified by comparing the percentage (%) of fluorescence emission from the A549, MDAMB, and MDCK cells examined (Figure 3d). After 4 h incubation with 0.1 mg/mL, followed by cells lysis, the A549 and MDAMB cells exhibited increased levels of internalization (38−40%), whereas the MDCK cells showed considerably lower uptake (18%), resulting in low levels of fluorescence from the internalized particles. The cellspecific internalization and pH-dependent relief of fluorescence quenching due to cell uptake reasonably demonstrated the target specificity and environmental responsiveness of the PgP/ HA-rGO nanoparticles. The biocompatibility and target ability are two prime obstacles for the development of smart targeted nanotheranostic agents, which can precisely deliver therapeutic ingredients directly to cancer cells.28,29 To ensure better efficacy and lower toxicity in the treatment of cancer, surface modification with a target ligand was previously proven to be the most promising strategy. In an MTT-based cell viability study, the PgP/HA-rGO nanoparticles showed concentrationindependent biocompatibility, even at higher concentrations (1 mg/mL), with more than 98% cell viability remaining, irrespective to the type of cells tested (MDAMB-231, A549, and MDCK; Figure 4a). However, the viability drastically changed after exposure to NIR light. The NIR-irradiated cancerous MDAMB-231 and A549 cells treated with PgP/HArGO nanoparticles showed sharp decreases of cell viability from 65 to 35% with increasing concentrations of the nanoparticle. At the same time, normal MDCK cells showed a slow decline of cell viability (90 to 65%), depending on the concentration

therapeutic activity in desired ways. Typically, the therapeutic activity of functionalized nanoparticles depends on efficient cellular uptake and endosomal release from endocytosis.25 In Figure 3, the in vitro cellular uptake of PgP/HA-rGO nanoparticles showed pH dependent fluorescence intensity upon confocal laser scan microscopic (CLSM) imaging. The A549 and MDAMB-231 cells treated with nanoparticles showed increasing fluorescence with decreasing pH (Figure 3a,b). However, the MDCK cells showed considerably lower rates of emission with decreasing pH of the medium (Figure 3c). One major difference between cancer and normal cells is the overexpressed receptor and acidic microenvironment, leading to control cellular uptake and higher fluorescence emission from uptake PgP/HA-rGO nanoparticles.21,26 The merged Lysotracker-labeled cells also support the above cellular internalization. The surface lattice acceptor molecule on the graphene oxide (GO) participated in quenching and, in turn, tuning of the desired pH-dependent fluorescence emission. In neutral pH, the cellular uptake followed modification of PgP/ HA-rGO in endosome acidic environment required sufficient incubation time to release fluorescence. Interestingly, acidic pH-induced in vitro internalized nanoparticles require protonation and a resulting increased fluorescence intensity in the early stage of lysosome (Supporting Information, Figure S10).27 As a result, increasing acidity resulted in increased fluorescence intensity through interactions between rGO and the pH-sensitive tertiary amino group of PDMA, which caused exposure of the IR825 fluorophores from the quencher (rGO). The internalized PgP/HA-rGO nanoparticles were also 3525

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Figure 6. (a) In vivo biodistribution of intravenously (i.v.) administrated nanoparticles (5 mg/kg): control group, IR825, PgP/rGO, and PgP/HArGO at (a) 8 and (c) 12 h post-injection upon dissection of tumor, kidney, liver, heart, lung, and spleen. The corresponding normalized intensity from the dissected organs of IR825, PgP/rGO, and PgP/HA-rGO at (b) 8 and (d) 12 h, respectively.

the photothermal conversion effects on the surface of the tumor at various times of 0, 4, 8, 12, and 24 h. A sharp increase of the surface temperature of the tumor after NIR irradiation was found at 4 h (Figure 5a-II and b-II) and 8 h (Figure 5a-III and b-III) post-injection, which then decreased over time. This might be due to active targeting of the overexpressed CD44 receptor on the tumor surface, facilitated by the receptor− ligand (CD44−HA) interaction.21 However, an increase of tumor surface temperature totally disappeared at 24 h postinjection (Figure 5a-V and b-V). It is possible that the improper lymphatic drainage system retained the nanoparticle for a long enough period (4−8 h) of time, after which it disappeared (24 h). Besides, the surrounding tissue near the tumor showed very little photothermal effects, where the group treated with free IR825 did not show any potential temperature effects during the observation periods. The time-dependent in vivo thermographic images demonstrated the site-specific accumulation of PgP/HA-rGO nanoparticles in the tumor, providing targeted photothermal effects which can therefore be used wisely for tumor destruction.20,31 The biodistribution and tumor targeting efficiency of the PgP/HA-rGO nanoparticle were monitored using a real time near-infrared fluorescence (NIRF) imaging technique. For this purpose, the nanoparticles were administrated into the tail vein of the tumor bearing mice at the dose of 5 mg/kg, followed by sacrifice at 8 and 12 h later (Figure 6).32 Characteristic fluorescence signals were observed after 8 h examination, indicating maximum accumulation, whereas a comparatively low signal recorded at 12 h was due to the effect of accumulation time (Figure 6b,d). However, the PgP/HA-rGO nanoparticles performed simultaneous active targeting (CD44 receptor on cancer cells), with increasing fluorescence signals at

(Figure 4b). These results suggest that PgP/HA-rGO, which was specially designed for the tumor environment, and the tumor target (HA ligand) performed simultaneously, allowing the nanoparticles to exhibit outstanding results. Therefore, upon NIR irradiation, the PTT agent released the maximum photothermal heat within the tumor environment, resulting in the highest photothermal cytotoxicity to MDAMD and A549 cells. On the other hand, with lack of a responsive environment, the PTT agent showed lower photothermal cytotoxicity on normal MDCK cells. The characteristic enhanced uptake observed in cancer cells owing to cell surface receptor, which preferentially concentrates (PgP/HA-rGO) to be converted into photothermal heat in an efficient way.9,26 The cancertargeted and environmentally responsive photothermal cytotoxicity was further ascertained through examination of the live and dead cells via fluorescence imaging. As can be seen, the NIR-irradiated PgP/HA-rGO (0.2 mg/mL) nanoparticles to selectively increase in the number of dead cells (red color) for MDAMB and A549, while increased numbers of live cells (green color) were observed for the treated noncancer MDCK cells (Figure 4c). Overall, the above observations confirmed the pH-responsiveness of the PgP/HA-rGO nanoparticles as a targeted photothermal therapeutic (PTT) agent. Encouraged by the promising in vitro PTT results of the PgP/HA-rGO nanoparticles, we then evaluated the in vivo photothermal conversion efficacy using tumor mice models. Mice underwent i.v. injection of the samples at the dose of 30 mg/kg, after which they were subjected to NIR irradiation for 5 min. During this study, whole body thermographic images were captured using an infrared (IR) thermal camera.30 It is obvious that bloodstreaming facilitates the circulation of the therapeutic ingredient, which is dependent on time; we therefore measured 3526

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Biomacromolecules

Figure 7. In vivo photothermal therapy of PgP/HA-rGO nanoparticles. (a) Digital photo images of MDAMB tumor-bearing mice from 0 min to the 20th day of observation, after treatment with PBS, free IR825, PgP/rGO, and PgP/HA-rGO nanoparticles. The laser was 808 nm, irradiated at 2 W/ cm2 for 5 min. (b) Time-dependent tumor growth rate of (a). (c) The histological tumor tissue images of (a) after Haematoxylin and Eosin (H&E) staining.

tumor sizes reached 200−250 mm3 (day 0), the tumor bearing mice were divided into 4 groups, each of which received a different treated sample, including PBS, free IR825, PgP/rGO, and PgP/HA-rGO nanoparticles (Figure 7). After treatment, the mean tumor size in each group was measured up to the 20th day of examination. The sizes of tumors treated with PgP/ HA-rGO nanoparticles were observed to be remarkably reduced over time, where the tumors showed a mass of 50 mm3 after 10 days (Figure 7b). At the same time, the free IR825-treated group showed slow diminishment of the tumor mass (145 mm3), while no apparent tumor mass reduction was found in the PBS and PgP/rGO treated groups at the 10 day evaluation. Moreover, at the end of the 20th day, the mice treated with PgP/HA-rGO nanoparticles had totally recovered from malignancy, whereas the free IR825 treated groups still showed existence of tumor mass (Figure 7a). The histopathological image obtained from the sacrificed mice by Haematoxylin and Eosin (H&E) staining methods were used to additionally rectify the in vivo PTT of the targeted PgP/HArGO nanoparticles (Figure 7c).42 The H&E staining observations more clearly distinguished the recovery process, wherein the PgP/HA-rGO nanoparticle-treated tumor histology exhibited the shrinkage of cells, loss of contact, and eruption of the tumor extracellular matrix.41 However, the free IR825treated tumor histogram showed the presence of extracellular matrix and slow recovery process with negligible damage 10 days after therapy. Overall, these results demonstrated that the

8 h, while regular bloodstreaming facilitated continuous accumulation, resulting in strong signals after 12 h postinjection.33,34 This fast washing out from the body can significantly limits the post-treatment toxic effects. Meantime, in vivo photothermal conversion after NIR irradiation sharply increased at 4−8 h post-injection, which was comparable with a previous report optimized 4 h for a graphene-based therapeutics process.35−38 Moreover, the localization impact of tumor environment have retained stable detectable fluorescence signal, however, inherent photobleaching, metabolic alteration due to long time in living sample resulted weaker fluorescence signals. The anchoring polymer (D-HA with I/C-PgP) and rGO ratio was 120/1 which kept minimum toxic effects originate from graphene. Furthermore, functionalized graphene oxide maintains biocompatibility that could improve by adjusting dose of administration and NIR irradiation time. As the incident NIR laser power is an important regulator to generate photothermal heat, low concentration with required laser intensity can maintain more safe application.39,40 To summarize, the above ex vivo biodistribution of the PgP/HA-rGO nanoparticles clearly demonstrated tumor target ability, through which the nanoparticles precisely recognized and reached their destination. Finally, the stimulus environment for triggering the targeted hyperthermic cancer therapy of PgP/HA-rGO nanoparticles was studied by measuring tumor growth rates.41 When the 3527

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Biomacromolecules integrated tumor environment responsiveness and target delivery of PTT agents can make a significant advance in cancer therapy.

CONCLUSIONS We synthesized multifunctional hybrid graphene oxide nanoparticles for integrated pH tunable diagnosis and target therapy with PEG-g-PDMA quaternized IR825 and HA, anchored with rGO to control fluorescence quenching for regulation of the triggered photothermal heat. The cancer cell-sensitive in vitro cellular uptake and biocompatibility implies promising clinical utility, and the NIR-irradiated cancer cells treated with PgP/ HA-rGO nanoparticles showed specific in vitro photothermolysis-based cytotoxicity, proving the potential of PTT agents. The specificity influenced accumulation of the PTT agent selectively in malignant tumor cells, as demonstrated by the ex vivo biodistribution, while the in vivo thermographic image showed accumulation of the PTT agent generated the highest photothermal heat on the tumor surface in response to the extracellular tumor environment. This tuning of the photothermal elevation resulted in ablation of the tumor size from 225 to 50 mm3 within 10 days post-therapy. Moreover, the time-dependent in vivo accumulation of tumor-targeted photothermal agents may pave the road to in vivo clinical access. Therefore, precise in vivo delivery of PTT agents can be performed for safe treatment and early recover from malignant tumors. Toward the goal of cancer therapy, this translation would promise tumor environment-responsive targeted PTT for the safer and more effective treatment of cancer patients.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00944.

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NIR, near-infrared; I/C-PgP, IR825/2-chloro-3′,4′-dihydroxyacetophenone (CA) conjugated PEG-g-PDMA; D-HA, dopamine conjugated hyaluronic acid; PgP/HA-rGO, I/C-PgP and D-HA with reduce graphene oxide (rGO)

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Article

The polymer synthesis scheme, 1H NMR spectra, XPS, TGA, XRD, Raman, FT-IR, and additional experimental details mentioned in the text (PDF).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work (S.H.K. and J.E.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant Nos. 10046506 and 10048377 from the Ministry of Trade, Industry and Energy (MOTIE), and Fusion Research R&D Program from the Korea Research Council for Industrial Science and Technology (No. G02054) and was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2014055946). 3528

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