Mesoporous Silica Coated Polydopamine Functionalized Reduced

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Mesoporous Silica Coated Polydopamine Functionalized Reduced Graphene Oxide for Synergistic Targeted Chemo-Photothermal Therapy Leihou Shao, Ruirui Zhang, Jianqing Lu, Caiyan Zhao, Xiongwei Deng, and Yan Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11209 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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ACS Applied Materials & Interfaces

Mesoporous Silica Coated Polydopamine Functionalized Reduced Graphene Oxide for Synergistic Targeted Chemo-Photothermal Therapy

Leihou Shao, ‡a,c Ruirui Zhang, ‡b Jianqing Lu, ‡a,c Caiyan Zhao,a,c Xiongwei Deng,a,c and Yan Wu*a,c

a

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for

Excellence in Nanoscience，National Center for Nanoscience and Technology，Beijing 100190, P. R. China b

Beijing Key Laboratory of Ionic Liquids Clean Process, Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Zhongguancun, Beiertiao, Beijing 100190, P. R. China c

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

* Corresponding author E-mail: [email protected]; Fax: +86 10 62656765; Tel: +86 10 82545614 ‡These authors contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT The integration of different therapies into a single nanoplatform has shown great promise for synergistic tumor treatment. Herein, mesoporous silica (MS) coated polydopamine functionalized reduced graphene oxide (pRGO) and further modified with hyaluronic acid (HA) (pRGO@MS-HA) has been utilized as a versatile nanoplatform for synergistic targeted chemo-photothermal therapy against cancer. A facile and green chemical method is adopted for the simultaneous reduction and noncovalent functionalization of graphene oxide (GO) by using mussel inspired dopamine (DA) to enhance biocompatibility and photothermal effect. Then, it was coated with mesoporous silica (MS) (pRGO@MS) to enhance doxorubicin (DOX) loading and be further modified with the targeting moieties hyaluronic acid (HA). The pH-dependent and near-infrared (NIR) laser irradiation-triggered DOX release from pRGO@MS(DOX)-HA is observed, which could enhance the chemo-photothermal therapy effect. In vitro experimental results confirm that pRGO@MS(DOX)-HA exhibits good dispersibility, excellent photothermal property, remarkable tumor cell killing efficiency and specificity to target tumor cells. In vivo anti-tumor experiments further demonstrated that pRGO@MS(DOX)-HA could exhibit an excellent synergistic antitumor efficacy, which are much more distinct than any mono-therapy. This work presents a novel nanoplatform which could load chemotherapy drugs with high efficiency and be used as light-mediated photothermal cancer therapy agent.

KEYWORDS: polydopamine, mesoporous silica, reduced graphene oxide, drug delivery, chemo-photothermal therapy

1. INTRODUCTION sp2-Hybridized carbon nanomaterials including fullerene, single-walled carbon nanotubes, and graphene have been widely exploited for biomedicine application.1 Specially, graphene as the most well-known two-dimensional (2D) nanomaterial possesses interesting optical, electrical, and chemical properties, which exhibits potential as novel biosensor platform for biomolecules detection, contrast agent in different imaging modalities, and drug delivery vehicle for cancer


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treatment.2 Apart from the advantage of large surface area with both sides as drug carrier, GO can also serve as photothermal agent to induce hyperthermia for photothermal therapy (PTT)3-5 and augment the sensitivity of chemotherapy with improved therapeutic effects.6-8 However, GO’s usefulness as a nanocarrier is limited by its poor colloidal stability and biocompatibility,9,10 which necessitates surface chemistry modification with stabilizing agents.11-13 Moreover, unsatisfactory photothermal conversion efficiency greatly influenced the therapy effect, which hardly combining the merits in one nanoplatform and exerting their synergistic effect. To date, several modifications have been adopted to improve NIR absorption or enhance the photothermal conversion efficiency of GO. One of the pioneering works exploring the possibility of improving NIR absorption by reduced GO (RGO) was achieved.3 And on this basis, plasmonic nanoparticles anchored on the RGO surface was emerged, which could improve the photocurrent and surface enhanced Raman scattering performances thus achieved superior efficiency of the photothermal conversion.14 In particular, polydopamine functionalized RGO (pRGO) is more attractive for PTT application attributed to the strong absorption in the NIR region and high photothermal conversion efficiency (40%) of polydopamine (PDA).15,16 Furthermore, during the polymerization of dopamine (DA) it can simultaneous reduce of GO into RGO and endow it excellent dispersibility and biocompatibility.17-22 Nevertheless, some shortcomings including low drug loading efficiency, and difficulty for surface modification with functional groups still remarkably limit its applications in combination tumor therapy. Mesoporous silica (MS) has been recognized as a versatile solid support for fabricating multifunctional drug carrier due to its unique features such as well biocompatibility, uniform pore structure, and great diversity in surface functionalization.23-26 Coating of MS on GO could enhance the interfacial properties of GO and realize the combinational advantages which have attracted great attention as drug carrier.3-5 Moreover, modification of MS surface with active targeting moieties, such as specific ligands, peptides, and antibodies is relatively facile by covalent and non-covalent approaches.27,28 In fact, it is highly desirable to assemble delivery system with MS and targeting moieties that can be activated by external stimuli to release the drug molecules at the sites of tumor.29-33 Generally, nanocarrier with photothermal ability and targeting properties would be an efficient way to improve the accumulation of chemotherapy drugs, reduce side effects



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and synergistically enhance the therapeutic effects, which will be a great significance in tumor treatment. Here we constructed a layer-by-layer engineered nanocomposite for synergistic actively targeted chemo-photothermal therapy. GO was reduced by DA and functionalized by the subsequently formed PDA to acquire enhanced solubility and stability. MS was then fully covered pRGO to improve its drug loading capacity. Hyaluronic acid (HA), a molecule with specific tumor-targeting property, was coated on the surface of DOX loaded pRGO@MS nanocomposite by electrostatic interactions to realize drug controlled release and actively targeted therapy. The detailed illustration of the pRGO@MS(DOX)-HA nanocomposite is shown in Scheme 1A. Compared with the other methods of reducing GO to RGO, the adopted method in this work was environment friendly (no toxic chemical reduction reagent), facile, efficient, and can obtain an enhancer NIR absorption ability. The results demonstrated that the pRGO@MS(DOX)-HA nanocomposite has an excellent cancer cell killing effect both in vitro and in vivo by combined photothermal and chemotherapy. Thus, PDA functionalized RGO with MS coating could be a promising nanoplatform for drug delivery and multimodal therapy of cancer.

2. EXPERIMENTAL SECTION 2.1. Materials Tetraethylorthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), (3-aminopropyl) triethoxysilane (APTES) were obtained from Sigma-Aldrich (St. Louis, MO). Doxorubicin hydrochloride salt (DOX) was bought from Beijing HuaFeng Co., Ltd (Beijing, China). Sodium hyaluronate (HA) (Mw≈6.4 kDa) was purchased from the Shandong Freda Biopharmaceutical Co., Ltd (Shandong, China). Graphene oxide (GO, 200nm) was purchased from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). Dopamine hydrochloride was bought from J&K Chemical Ltd (Beijing, China). Tris (hydroxymethyl)aminomethane (Tris) buffer were obtained from Beijing Leagene Biotech. Co., Ltd (Beijing, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin/streptomycin) and Dulbecco's phosphate buffered saline (DPBS) were bought from Gibco-BRL (Invitrogen Corp., Carlsbad, USA). LysoTracker Green DND-26 was purchased from Molecular Probes (Eugene, OR).


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Deionised water (H2O) was purified by a Millipore system (Milli-Q, 18.2 MΩ•cm). All other chemicals were purchased from commercial sources and used without further purification. 2.2. Synthesis of pRGO, pRGO@MS and pRGO@MS-HA For the synthesis of polydopamine modified reduced GO(pRGO), 10 mL GO (2 mg/mL), 10mg dopamine and 0.1 mL Tris buffer (pH 8.5) were mixed and then heated to 60 °C for 24 h under stirring. The reaction product was dialyzed against deionized water using a dialysis membrane (MWCO: 3 kDa) for 3 days to remove free PDA. For the synthesis of pRGO@MS, 0.8 mL CTAB (0.2 mol/L) was added into the prepared pRGO nanocomposites solution (0.2 mg/mL) under stirring. 0.18mL NaOH solution (0.1 mol/L) was subsequently added. Next, 0.3 mL 20% APTES and TEOS in ethanol were dropped slowly into the above mixture three times at a 60 min interval. After that, the mixture was reacted under room temperature for 24 h. This mixed solution were then centrifugated and washed with methanol to remove the CTAB molecules. Finally, the pRGO@MS nanocomposites were redispersed in deionized water for further use. For the preparation of HA modified pRGO@MS(pRGO@MS-HA), the obtained pRGO@MS dispersion was slowly added to HA solution with different concentrations under stirring. The mixed solution was incubated for 2 h and the resulting pRGO@MS-HA was subsequently purified via centrifugation. 2.3. Preparation of pRGO@MS(DOX)-HA To load the chemotherapy drug, the pRGO@MS nanocomposites were mixed with a DOX/water solution (2 mg/mL), and then the mixed solution were stirred for 4 h to enable DOX loading into the mesoporous. Subsequently, HA (4 mg) was added into the solutions and stirred for another 4 h. Finally, pRGO@MS(DOX)-HA nanocomposites were collected by centrifugation and washed with deionized water. Other nanocoposites, Cy5-labeled pRGO@MS-HA, were prepared by the same procedure. 2.4. Characterization of pRGO@MS-HA and pRGO@MS(DOX)-HA Zeta potentials of the GO, pRGO, pRGO@MS and pRGO@MS-HA were evaluated on a Malvern ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK). X-ray diffraction patterns (XRD) of GO, pRGO, pRGO@MS were obtained from a SAXS/WAXS SYSTEM (French, XENOCS SA). FT-IR was analyzed on a spectrophotometer (Perkin Elmer, USA). The fluorescence spectra of DOX, pRGO@MS-HA and pRGO@MS(DOX)-HA were measured by an



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LS 55 fluorescence spectrometer (Perkin Elmer, Fremont, CA, USA). Raman spectra were detected by a Renishaw Invia Plus laser Raman spectrometer (Renishaw, UK). Morphological examination of the GO, pRGO and pRGO@MS was performed by transmission electron microscopy (TEM) (Tecnai G2 20 S-TWIN, FEI Company, Philips, Netherlands). Nitrogen adsorption−desorption isotherms and pore size distribution curves were detected by using Micromeritics Tristar II 3020. 2.5. Loading Efficiency Measurement The loading content (LC) of DOX was measured using an ultraviolet-visible (UV-Vis) spectrophotometer (Puxi TU-1810 visible spectrophotometer, Beijing, China) at 480 nm. The LC was defined as following formula: LC = (weight of loaded drug)/ (total weight of nanocomposites)×100%. 2.6. Photothermal Heating Effect of pRGO and pRGO@MS-HA To determine the photothermal heating effects of pRGO@MS-HA nanocomposites, various solutions of pRGO@MS-HA in PBS (pH 7.4) containing various concentrations of GO (0.1-50 µg/mL) were irradiated by an 808 nm-laser (1.5 W/cm2, Beijing Laserwave Optoelectronics Technology Co., Ltd.). To investigate the influence of laser power density on photothermal effect, pRGO and pRGO@MS-HA solutions (with the same GO concentration) were irradiated under different power densities and measured the changes of temperature. GO and PBS was applied as control groups with the same procedure. The temperatures of solutions were monitored by an infrared thermal imaging camera (Ti27, Fluke). 2.7. In Vitro Release Next, we detected the DOX release behaviour by a dialysis method. Three milliliters dispersion of pRGO@MS(DOX)-HA was introduced into a dialysis bag (molecular weight cutoff = 3500 Da, Mym Biological Technology Company Limited, USA), and then dialyzed against 40 mL different PBS solutions (pH 7.4 or pH 5.4) with or without subjecting to 808 nm-laser irradiation (1.5 W/cm2) for 5 min and vibrated at 100 rpm. One milliliter of the supernatant was collected at different time intervals and the equal volume of fresh buffers was added again. The concentration of DOX released from nanocomposites was determined by using a UV-Vis spectrophotometer (480 nm). The cumulative amount of released DOX from pRGO@MS(DOX)-HA was calculated. 2.8. Cell culture and cytotoxicity assay


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Human cervical carcinoma (HeLa) cells were cultured in DMEM medium with 10% FBS, 1% penicillin, and 1% streptomycin, in a 5% CO2 incubator at 37 °C. To evaluate the cytotoxicity of nanocomposites, HeLa cells were seeded in 96-well plate with a density of 5 × 103 cells per well for 12 h. Then, the original culture medium were replaced by 200 µL fresh culture medium containing various concentrations of PBS, DOX, pRGO@MS-HA and pRGO@MS(DOX)-HA (the equivalent DOX concentrations are 0.1, 1, 5, 10, 25 and 50 µM, respectively) for 24 or 48 h, respectively. Cell viability was measured by CCK-8 assay, carrying out in accordance with the manufacturer's

instructions

(Dojindo, Japan).

To investigate

the

biocompatibility of

pRGO@MS-HA, HeLa cells were seeded in 96-well plates with a density of 5 × 103 cells per well and cultured for 12 h. Then, the original culture medium were replaced by 200 µL fresh culture medium containing various concentrations of GO, pRGO, pRGO@MS and pRGO@MS-HA (the equivalent GO concentrations are 0.1, 1, 5, 10, 25 and 50 µg/mL, respectively) for 48 h, respectively. Then the viability of cells was measured by CCK-8 assay. Subsequently, we investigate the PTT antitumor effect of pRGO@MS-HA and pRGO@MS(DOX)-HA. HeLa cells were seeded in 96-well plates with a density of 5 × 103 cells per well and cultured for 12 h. Then, the original culture medium were replaced by 200 µL fresh culture medium containing same concentrations of pRGO@MS-HA and pRGO@MS(DOX)-HA (the total drug content was kept at 5 µM). After 12 h incubation, the cells were subjected to 808 nm laser irradiation (1.5 W/cm2, 5 min). The treated cells were cultured for additional 24 h or 48h, cell viability was measured by CCK-8 assay. 2.9. In Vitro Cellular Uptake HeLa cells were cultured in DMEM media containing 10% FBS and incubated at 37°C incubator with 5% CO2 for 24h. The original culture medium was then replaced by fresh culture medium containing pRGO@MS(DOX)-HA at the same concentration of DOX (5 µM ) and the cells were cultured for another 2, 4, or 6 h respectively. Then the cells were washed with pH 7.4 PBS three times. Finally, the cellular lysosomes were stained by LysoTracker Green and confocal laser scan microscopy (CLSM, Carl Zeiss, Boston, MA, USA) was used to evaluate the cellar uptake of pRGO@MS(DOX)-HA. 2.10. Targeting Ability Evaluation



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HeLa cells were respectively seeded in 96-well microplates with a density of 5 × 103 cells per well and one group were pretreated with free HA (10 mg/mL) to block CD44 prior to HA in pRGO@MS(DOX)-HA. The two groups incubated at 37 °C with 5% CO2 for 12 h. After cells were washed, fresh culture medium containing pRGO@MS(DOX)-HA were added into the cell wells. After 48 h, CCK-8 assay was carried out for quantitative evaluation. To quantitively measure the uptake of pRGO@MS(DOX)-HA nanocomposites, HeLa cells with and without pretreated with free HA (10 mg/mL) were incubated with pRGO@MS(DOX)-HA for 4 h at 37 °C, respectively. Then the cells were washed with PBS three times and resuspended in PBS for the flow cytometric analysis (Attune® acoustic focusing cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA). A total of 10, 000 events were collected for each sample. 2.11. Animals and Tumor Model Healthy female BALB/c nude mice (18-20 g, 6-7 weeks old) were obtained from Beijing Vital River Laboratories (Beijing, China). HeLa cells (5.0 ×106 cells in 100 µL PBS) were implanted subcutaneously into the lateral thigh of nude mice and allowed to grow to a tumor size of ∼100 mm3 before initiating treatment. Tumor volumes were measured and calculated every two days according to the following formula: Volume = (Tumor length) × (Tumor width)2 × 1/2. The tumor-bearing mice were then randomly divided into 6 groups with 5 mice in each group. All the animal studies were performed strictly in accordance with the protocols approved by the ethics committee of Peking University. 2.12. In Vivo Imaging and Biodistribution Analysis In vivo images of nude mice after intravenous injection of Cy5-labeled pRGO@MS-HA nanocomposites were carried out by the ex/in vivo imaging system (CRi, Woburn, MA). After tail vein injection with 100 µL of saline, 100 µL of Cy5 (1 mg/kg, Cy5) or 100 µL of Cy5-labeled pRGO@MS-HA nanocomposites (1 mg/kg, Cy5), the fluorescent signals of Cy5 were collected at 1, 4, 8, 12 and 24 h post-injection. The mice were sacrificed after in vivo imaging, and the tumors and other major organs (heart, liver, spleen, lung, kidney) were collected for imaging. 2.13. In Vivo Treatments 100 µL of saline, pRGO@MS-HA, DOX (5mg/kg), or pRGO@MS(DOX)-HA(DOX, 5mg/kg) were intravenously injected into the mice. For the NIR laser treatment groups, 4 h after injection, the tumors of mice were subjected to the 808 nm laser irradiation (1.5 W/cm2, 5 min) and an


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infrared camera was used to capture the temperature changes of mice. All the mice were sacrificed at the end of experiment and the tumors were collected and stained with hematoxylin and eosin (H&E) for analysis the therapeutic efficacy. 2.14. Blood Biochemistry and Pathology The tissues (heart, lung, liver, spleen, and kidneys) from these mice were collected and stained with hematoxylin and eosin (H&E). The bright field images of tissues were obtained by a digital microscope. Blood samples were collected from the eye vein by removing the eyeball quickly. After 3 h standing in 4oC, the collected blood samples were centrifuged at 1000 rpm for 3 min to obtain serum. The blood biochemistry analysis was determined by biochemical analyzer. 2.15. Statistical analysis All the values are expressed as means ± standard deviation (SD). P values were alculated by the twotailed Student’s t-test. p < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Nanocomposites The synthesis procedure of PDA-capped RGO (pRGO) was shown in Scheme 1A. pRGO was prepared via the simultaneous reduction of GO by DA and self-polymerization to form PDA layer. The morphology of GO and pRGO nanosheets were illustrated by transmission electron microscope (TEM). The representative TEM image (Figure S1Ab) of pRGO shows a monolayer RGO nanosheet capped with a PDA layer and many dispersed protuberances can be seen on the surface. Accordingly, rarely protuberances can be seen on the pristine GO (Figure S1Aa). The reduction process of GO by DA polymerization in Tris buffer was monitored by time dependent UV-vis absorption spectroscopy. Figure 1B showes two characteristic peaks, 230 nm and 300 nm, which corresponded to the characteristic peaks of GO (π–π* transitions of aromatic C=C bonds and n–π* transitions of C=O bonds), have shifted to ~270 nm, which indicated that the GO was reduced by DA and the aromatic structure within the GO nanosheets was restored.34-36 The structures of GO and pRGO were also evaluated by X-ray diffraction (XRD) (Supporting Information, Figure S3). The sharp diffraction peak in GO (2θ=10.8°) has decreased apparently after DA reduction and PDA modification, and a new diffraction peak (2θ=25°) has appeared in the pRGO, which is closer to a typical characteristic peak of PDA,37 indicating the successful



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reduction of GO by DA and modification by PDA. In addition, it was also confirmed by Fourier transform infrared (FT-IR) spectroscopy analysis. As shown in Figure S4, the graphene nanosheet in pRGO were deoxidized since the stretching vibration of C-O bonds (1382 cm-1) and C=O bonds (1730 cm-1) of GO disappeared due to the most oxygen functional groups of GO were removed during DA reduction. To synthesize pRGO@mesoporous silica (pRGO@MS) nanocomposite with a sandwich structure, a surfactant-assisted approach was adopted to realize pRGO coated with an ordered mesoporous silica layer. The surfaces of the pRGO were negatively charged (-29.6 mV) since the isoelectric point of PDA is determined to be around.20 Therefore, a cationic surfactant, cetyltrimethyl ammonium bromide (CTAB), could serve as the positively charged coating monolayer on the surface of pRGO, which subsequently acted as nucleation sites for the growth of the mesoporous shell. The CTAB template molecules were removed by washing with methanol and ethanol. Figure 1A shows a typical TEM image of pRGO@MS, which clearly showed mesoporous silica layers on pRGO nanosheets. The XRD pattern (Figure 1C) revealed that pRGO@MS had an ordered pores and structure. The results of N2 adsorption-desorption isotherm and pore size distribution curve (Figure 1D) indicated that the surface area, pore volume, and average pore diameter of pRGO@MS were measured to be 1154 m2/g, 1.10 cm3/g, and 3.82 nm, respectively. These results indicated that pRGO@MS can serve as an excellent drug delivery platform for high drug loading. Furthermore, the structure of silica–pRGO–silica (pRGO@MS) nanocomposites was also proved by the Raman spectra (Figure 1E). The Raman spectrum indicates two characteristic peaks of GO in pRGO@MS at 1593 cm −1 (G band) and 1332 cm −1 (D band), confirming the existence of GO between the mesoporous silica layers and supporting the formation of pRGO@MS nanocomposites.38 FT-IR spectra (Figure S4) also verified the pRGO@MS was successfully prepared. pRGO@MS was further modified with hydrophilic acid (HA) (pRGO@MS-HA) to endow this nanocomposite with targeting function. The successful HA modification for pRGO@MS was characterized by FT-IR spectra (Figure S4). Zeta (ζ) potential analysis was performed to investigate the changes in surface charge of the prepared nanocomposites (Figure 1F). pRGO (-29.6 mV) showed a much smaller ζ potential than GO (-47.4 mV), which was ascribed to the deoxidization of hydroxyl and carboxyl groups on GO during DA reduction and PDA coating. After MS coating, the ζ potential increased to -10.9 mV may be due to the modification of


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electropositive amine during MS coating. Coating with HA could decrease the surface charges of nanocomposite. It was measured that the ζ potential decreased with the increasing amount of added HA (Figure 1F), and reached to -34.7mv when the mass ratio of added HA to pRGO@MS was 3:1. In addition, the ζ potential showed no difference with the mass ratio further increasing. In addition, the hydrodynamic diameters the nanocomposites were examined by dynamic light scattering (DLS). The hydrodynamic size of all these nanocomposites in aqueous solutions have similar particles distribution and the size were approximately 200 nm as shown in Figure S1B. It should be noted that both the PDA coating and the postmodification of mesoporous silica (MS) and HA would improve the water dispersity of nanocomposites (Figure S2). 3.2. Photothermal Heating Effect To explore the photothermal effect of pRGO@MS-HA, the changes of temperature were detected under NIR laser irradiation in vitro. As shown in Figure 2A and D, the solution temperatures of pRGO and pRGO@MS-HA both increased rapidly under the 808 nm laser irradiation (1.5 W/cm2) and exceeded 61.8 °C and 57.8 °C within 10 min at the same GO concentration, respectively. In comparision, the GO and PBS solution remained below 37 °C only increased to 35.6 °C and 32.5 °C under the same laser irradiation (Figure 2A,D). Such excellent photothermal conversion property of pRGO@MS-HA may be explained in two aspects. Firstly, RGO shows much higher NIR absorbance as compared to GO, leading to a stronger photothermal effect (Figure S5). In addition, as revealed by many previous studies, PDA could also be used as photothermal agents, since they have obvious NIR absorption and excellent photothermal conversion efficiency. 15,16 In additon, the influences of concentration (with GO concentration from 0.1 to 50 µg/mL) and power intensity (from 0.5 to 2.0 W/cm2) on photothermal efficiency of pRGO@MS-HA were detected by monitoring the temperature changes under 808 nm laser irradiation. Figure 2B and 2C indicate that the photothermal efficiency of pRGO@MS-HA showed a concentration- and laser power intensity-dependent manner. Thus, the excellent photothermal efficiency of pRGO@MS-HA demonstrated above showed great potential using pRGO@MS-HA as thermal therapy agent. 3.3. Drug Location and Release In Vitro To evaluate the potential of using pRGO@MS-HA as a drug delivery system, DOX was loaded into pRGO@MS-HA nanocomposite. In many previous reports, GO could directly load several aromatic drugs including DOX through π–π stacking and/or hydrophobic interaction.39 In this



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work, the “sandwich” structure with mesopores of pRGO@MS-HA is considered to be more efficient for drug adsorption by various special interactions including π–π stacking, pore adsorption, and/or van der Waals forces.3 The result showed that the drug loading capacity of pRGO@MS-HA is as high as 145 ± 25% for loading aromatic DOX molecules (GO, RGO, pRGO, was 40%, 22%, 58%, respectively, Figure S6). The successful loading of DOX on the pRGO@MS-HA nanocomposites was confirmed by fluorescence spectra. Figure 2E show that the fluorescence of DOX after loading on the pRGO@MS-HA was much weaker than that of free DOX with the same DOX concentration, which could be attributed to the fluorescence quenching effect. All of above results illustrated that pRGO@MS-HA nanocomposites could successfully load DOX molecules. Next, we detected the DOX release behaviour from the pRGO@MS(DOX)-HA nanocomposites in pH 7.4 and pH 5.4, which treated with or without the NIR laser irradiation, respectively. Figure 2F showed that the amount of DOX released from the pRGO@MS(DOX)-HA exhibited a pHand laser irradiation-dependent release behavior. The enhanced DOX release under NIR laser irradiation may be attributed to the photothermal effect of pRGO@MS(DOX)-HA, which could weak the interactions between DOX and nanocomposites. These results demonstrated that the sensitivity of chemotherapy could be significantly increased when coupled with photothermal therapy. Furthermore, the release efficacy of DOX also indicated a pH-responsive manner (Figure 2F). Under acidic condition, the reduction of the electrostatic interaction between HA and the amine groups of nanocomposites could lead to the swelling and dissociation of the HA layer from the pRGO@MS(DOX)-HA surfaces. Meanwhile, the weakened electrostatic attraction between nanocomposites and DOX attributed to the protonation effect of more surface silanols with the decrease of pH, resulting in the pH triggered release pattern of DOX. Therefore, the pH-dependent and NIR laser irradiation-triggered release of DOX could effectively enhance the synergistic chemo-photothermal therapy effect. 3.4. In Vitro Synergistic Therapeutic Efficacy In general, graphene oxide could generate reactive oxygen species (ROS) leading to cell death without any functionalization.13 Nevertheless, it has been found that appropriate surface modification including amination and PEGylation could significantly reduce these untoward effects.11-13 In this study, GO was reduced by DA before coating of biopolymer PDA, and further


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coated with MS. Moreover, modification of HA not only could be used as targeted moiety but also would further reduce the toxicity and enhance the biocompatibility of nanocomposites. The potential cytotoxicity of pRGO@MS-HA nanocomposite in vitro was tested. Human cervical cancer HeLa cells, which have high expression of cell surface CD-44 receptor, were selected as a representative cells.41 As shown in Figure 3A, cells treated with various concentrations of GO showed a dose-dependent cytotoxicity. In contrast, when the GO was coated with PDA and MS, the cell viability increased significantly. In Figure 3A, both the pRGO and pRGO@MS nanocomposites showed no obviously cytotoxicity on HeLa cells, with cell viability remained over 80% in the concentration range from 0.1µg/mL to 50 µg/mL. In particular, the pRGO@MS-HA exhibits excellent cell compatibility even with a high concentration of 50 µg/mL. It’s important to note that free DOX and pRGO@MS(DOX)-HA nanocomposites with equivalent dose of DOX exhibited similar cytotoxic on HeLa cells and the cytotoxic showed a dose-dependent manner (Figure 3B). To investigate the combinational effects of photothermal with chemotherapy, different drug formulations were carried out to treat with HeLa cells and after incubation for 24h or 48h, following by the CCK-8 assay test. Schematic illustration of the proposed process of combination therapy against HeLa cells by pRGO@MS(DOX)-HA was shown in Scheme 1B. The results showed that the viability of cells treated with different drug formulations significantly decreased after 24 h (Figure 3C) or 48 h (Figure 3D) treatment, while the cell viability of control groups (PBS, pRGO@MS-HA) had no obviously decrease. The mono-therapy groups，DOX and pRGO@MS(DOX)-HA with equivalent dose of DOX，exhibited apparent cytotoxicity to HeLa cells (Figure 3C,D), illustrating the chemotherapy effect of pRGO@MS(DOX)-HA nanocomposites. In addition, the cell viability in radiated groups were much lower than that of the cells in nonradiated groups, illustrating the PTT effect of pRGO@MS(DOX)-HA nanocomposites. The dual-therapy group exhibited a remarkably combinational therapy effect of chemotherapy & PTT and a excellent theraputic efficiency than the mono-therapy groups (chemotherapy or PTT). In this study, NIR laser irradiation not only induced PTT for cancer therapy but also could accelerate the release of DOX from pRGO@MS(DOX)-HA leading to an enhanced chemotherapy. Furthermore, moderate heating could enhance the intracellular transfer of nanomaterials by



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increasing the permeability of cell membrane.42,43 In conclusion, pRGO@MS(DOX)-HA with NIR laser irradiation could realize combinational chemo-photothermal synergistic therapy efficacy. 3.5. Endocytosis and Subcellular Localization of the Nanocmposites Confocal laser scanning microscopy was performed to measure the cellular uptake and intracellular localization of the pRGO@MS(DOX)-HA nanocomposite in HeLa cells. For cells treated with pRGO@MS(DOX)-HA, red fluorescence was visible inside cells with colocalization in lysosome when the incubation time was 2 h (Figure 4A). After 4 h incubation, red fluorescence signals inside HeLa cells with colocalization in lysosome and partly in the cellular nucleus could be visualized, illustrating that the pRGO@MS(DOX)-HA could be effectively deliveried into HeLa cells. When the incubation time was extended to 6 h, most red fluorescence signals were observed in cellular nuclei. The results confirm that the pRGO@MS(DOX)-HA nanocomposite could be effectively internalized into living cells. 3.6. The targeting ability of pRGO@MS(DOX)-HA The targeted delivery ability of the pRGO@MS(DOX)-HA nanocomposites was evaluated. Several tumor cells over-express HA-binding receptors such as CD-44, and consequently, HA modification on the surface of pRGO@MS may result in active targeting ability. Here, HeLa cells and HeLa cells pretreatmented of HA (10 mg/mL) were used as control. Measurements of cell viability via CCK-8 analysis and cellular uptake via flow cytometry were carried out for quantitative

evaluation.

From

flow

cytometry

results

in

Figure

4B,

HA-modified

pRGO@MS(DOX)-HA nanocomposites exhibited remarkable drug delivery ability for deliver DOX to HeLa cells than control group. Cell viability experiments revealed that HA-modified pRGO@MS(DOX)-HA shows higher cytotoxicity. To some extent, the higher cytotoxicity in accordance with higher cellular uptake. These results confirmed that the pRGO@MS(DOX)-HA nanocomposites could target to CD-44-positive HeLa cells. Furthermore, the DOX uptake of pRGO@MS(DOX)-HA in control HeLa cell groups was significantly inhibited by the addition of a great deal of competing HA molecules (Figure 4B). As HA is a binding molecule to CD-44 receptor on HeLa cells, this is the typical phenomenon of receptor-mediated competitive inhibition. In addition, as shown in Figure S7, FITC labelled pRGO@MS-FITC/HA indicated more fluorescence signals in HeLa cells compared to pRGO@MS-FITC group. Therefore, we can


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speculate that one of the main mechanisms for cellular uptake of the pRGO@MS(DOX)-HA nanocomposites might be the HA-receptor-mediated endocytosis process. 3.7. In Vivo Distribution of the Nanocomposites To investigate the in vivo distribution of the nanocomposites, the free fluorescent dye Cy5 or pRGO@MS-HA loaded with Cy5(pRGO@MS(Cy5)-HA) nanocomposites were intravenously injected into Hela tumor bearing mice, and fluorescence distribution in vivo were captured at 1, 4, 8, 12 and 24 h post-injection. As shown in Figure 5A, strong fluorescence signal from liver was observed in both Cy5 and pRGO@MS(Cy5)-HA treated group 1 h after the injection, which may be attributed to the high macrophage uptake nature of liver. Along with prolonged circulation time, the fluorescence intensity in the tumor site of pRGO@MS(Cy5)-HA treated group gradually increased up to 12 h post-injection, while much weaker signal from tumor was observed in Cy5 treated group, indicating that the pRGO@MS(Cy5)-HA nanocomposites can gradually accumulate at the tumor site. At 24 h post-injection, the tumor in pRGO@MS(Cy5)-HA treated group still maintains strong fluorescence signal, suggesting good targeting and retention ability of the pRGO@MS(Cy5)-HA nanocomposites. Figure 5B shows the ex vivo fluorescence images of excised tissues obtained at 24 h post-injection, much higher fluorescence can be observed from the tumor in pRGO@MS(Cy5)-HA treated group compared to Cy5 group, confirming that the nanocomposites can effective accumulate in the tumor site. In addition, no obvious fluorescence signals were observed in the heart, spleen, lung in two groups, while a weak fluorescence signals in the liver and kidney organs can be seen in both groups, which may be due to the efficient renal clearance. 3.8. In Vivo Antitumor Activity of Nanocomposite Inspired by the excellent in vitro chemo-photothermal synergistic therapeutic effect of pRGO@MS(DOX)-HA, we then evaluated the in vivo anticancer potentials of these nanocomposites. The nanocomposites (pRGO@MS-HA) with and without DOX, free DOX, and PBS were intravenously injected into the HeLa tumor (∼100 mm3) bearing mice. The tumor regions of the half of the mice in nanocomposites (pRGO@MS-HA with and without DOX) groups were subjected to the NIR laser irradiation for 5 min and the other half mice of these groups as unheated controls. The temperature increase of tumor regions were monitored by an IR camera. As shown in Figure 6A and 6C, the pRGO@MS(DOX)-HA group exhibited a rapid



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temperature increase curve similar to the pRGO@MS-HA group after laser irradiation, and both the two groups temperature could reach over 50 oC, which is sufficient to PTT. Remarkably, all these tumors became scar within 10 days and the tumors of pRGO@MS(DOX)-HA group regressed almost completely within 12 days (Figure 6B). By contrast, the temperature of control groups injected with PBS or DOX showed a slow increase and only reached about 40 oC after laser irradiation (Figure 6A,C). All mice were sacrificed when the tumor volume of the mice injected with PBS reached about 1300 mm3. As shown in Figure 6D, a significant inhibition of tumor growth was observed in the mice injected with pRGO@MS(DOX)-HA coupled with NIR laser irradiation leading to the most efficient anti-tumor effect of the combined chemotherapy and PTT. In addition, chemotherapy (pRGO@MS(DOX)-HA or DOX) or photothermal therapy (pRGO@MS-HA (NIR+)) alone also showed some suppression of tumor growth, but not as distinct as that in the combinational therapy group. Such excellent synergistic theraputic effect could be attributed to that photothermal heating could induce drug release from pRGO@MS(DOX)-HA leading to an enhanced chemotherapy effect. On the other hand, as revealed by many previous studies, photothermal heating could significantly promote internalization of nanomaterials by improving the permeability of cell membrane.42,43 These results illustrated that pRGO@MS(DOX)-HA combined with NIR laser irradiation could realize efficient synergistic chemo-photothermal therapy effect for anti-tumor therapy. Furthermore, body weights of the mice were also recorded. As shown in Figure 6E, the body weight of mice in all the experimental groups did not show any noticeable loss, demonstrating satisfactory biocompatibility and biosafety of pRGO@MS-HA in vivo application. In addition, Hematoxylin and eosin (H&E) staining of tumor slices was performed to evaluate the anti-tumor

efficiency

of

the

combined

chemotherapy

and

PTT

based

on

the

pRGO@MS(DOX)-HA nanocomposites. As shown in Figure 6F, a severe tumor cellular damage was obseved in the group treated with pRGO@MS(DOX)-HA injection and NIR laser irradiation (dual-modal therapy), while other groups that received mono-therapy (chemotherapy or PTT) treatment only showed limited cellular destruction. By contrast, tumor cells in control group (saline or pRGO@MS-HA) retained their normal morphology. Those results also indicated the highly efficient of the nanocomposite for tumor therapy.


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Subsequently, we evaluated the potential side effects of pRGO@MS(DOX)-HA on major organs. The histopathological analysis of the major organs (heart, liver, spleen, lung, and kidneys) of tumor-bearing mice after receiving various treatments were carried out by standard histological techniques with H&E staining. Notably, no noticeable morphological changes of above organs were observed among these groups (Figure 7). These results confirmed that the pRGO@MS(DOX)-HA nanocomposites exhibited no noticeable side effect. At last, to further detect the anti-tumor efficacy of the pRGO@MS(DOX)-HA, several serum biochemical indicators were measured. As shown in Figure S8, the tumor-bearing mice and the normal mice exhibited significant differences in some serum biochemical indicators, which could be attributed to the tumor burden. After treatment with pRGO@MS(DOX)-HA coupled with NIR laser irradiation, the indicators returned to the levels of normal mice, in accordance with the result of in vivo inhibition of tumor growth. In addition, no additional serum biochemical abnormality was observed when treated with nanocomposite, indicating the safe application of pRGO@MS(DOX)-HA in tumor therapy.

4. CONCLUSIONS In conclusion, we constructed a multifunctional drug delivery and multimodal therapy system based on pRGO@MS(DOX)-HA nanocomposites, which could realize efficient synergistic targeted chemo-photothermal therapy. Biocompatible biopolymer coated RGO nanosheets were prepared by using mussel inspired dopamine (DA) as the reducing reagent and the functionalized biomolecule in one step. Mesoporous silica (MS) was then coated on pRGO to enhance doxorubicin (DOX) loading and provide active interface for modifying with targeting moieties hyaluronic acid (HA). The as-prepared pRGO@MS(DOX)-HA exhibits good dispersibility and excellent photothermal property under NIR laser irradiation. The in vitro experimental results revealed that pRGO@MS-HA nanocomposites have minimal cytotoxicity, good specificity to target tumor cells and high biocompatibility. In contrast, the DOX-loaded pRGO@MS-HA nanocomposites presented excellent synergistic chemo-photothermal therapy effect under NIR laser irradiation, which could be attributed to its high drug loading capacity and inherent excellent photothermal conversion property. In vivo combination therapy was then performed, resulting in an outstanding synergistic suppression of tumor growth in the chemo-photothermal therapy



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compared with other mono-therapy, which is promising to reduce the inevitably tumor recurrence due to the inhomogeneous treatment of mono-therapy. Therefore, this powerful nanoplatform based on pRGO@MS-HA nanocomposite hold great promise for multimodal anti-tumor therapy.

ASSOCIATED CONTENT Supporting Information Available: Detailed information including photographs of GO, pRGO, pRGO@MS and pRGO@MS-HA, XRD patterns of GO and pRGO, FT-IR spectra of GO, pRGO, pRGO@MS

and

pRGO@MS-HA

，

Serum

biochemical

examination

of

pRGO@MS(DOX)-HA-treated mice are provided in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Yan Wu, Email: [email protected]. Author Contributions ‡Leihou Shao, Ruirui Zhang and Jianqing Lu contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was supported by National Natural Science Foundation of China (81272453, 81472850) and supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA09030301).

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Scheme 1. Schematic illustration of (A) the synthesis route of pRGO@MS(DOX)-HA nanocomposite and (B) the combined chemo-photothermal targeted therapy of tumors.



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Figure 1. (A) Transmission electronic microscopic (TEM) image of pRGO@MS. The scale bar: 100 nm (a) and 20 nm (b). (B) UV-vis spectra absorption of GO before and after being reduced by dopamine for 4, 8, 12, and 24 h. The insets show the photographs of aqueous dispersions GO and pRGO. (C) Small-angle XRD patterns of pRGO@MS powders. (D) N2 adsorption/desorption isotherms and corresponding pore-size distribution curve (inset) of the pRGO@MS sample. (E) Raman spectra of GO, pRGO, and pRGO@MS. (F) Zeta potential of GO, pRGO, pRGO@MS and the value changes of nanocomposite after modification with different amounts of HA.


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Figure 2. (A) Temperature variation curves of the aqueous dispersions of PBS, GO, pRGO, and pRGO@MS-HA exposed to the 808 nm laser at a power density of 1.5 W/cm2 for 10min. The GO solution has the same GO concentration to pRGO and pRGO@MS-HA solution. (B) Temperature variation curves of the pRGO@MS-HA solution with different concentrations. (C) Temperature variation curves of the pRGO@MS-HA solution under various power intensities. (D) IR thermal image of PBS, GO, pRGO, and pRGO@MS-HA solution under continuous NIR laser irradiation (1.5 W/cm2) for 10 min. (E) Fluorescence spectra of pRGO@MS-HA, DOX, and pRGO@MS(DOX)-HA (with the same DOX concentration of free DOX). (F) DOX release profiles from pRGO@MS(DOX)-HA nanocomposites at different pHs with or without NIR laser irradiation (808nm, 1.5 W/cm2).



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Figure 3. In vitro anti-tumor activity against HeLa cells. (A) Cytotoxicity assay of HeLa cells in the presence of GO, pRGO, pRGO@MS, and pRGO@MS-HA at various concentrations. (B) Cell viability of HeLa cells incubated with different concentration DOX and pRGO@MS(DOX)-HA (equivalent concentration of DOX). Cell viability of HeLa cells treated with different forms of nanocomposite with or without NIR laser irradiation at 37 °C for 24 h (C) or 48 h (D). The total drug content in all groups was kept at 5 µM.


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Figure 4. (A) Laser confocal scanning microscope images of HeLa cells incubated with pRGO@MS(DOX)-HA nanocomposites for 2 h, 4 h, and 6 h. The endosomes/lysosomes were stained with LysoTracker green (green). (B) Cell viability and flow cytometry results of HeLa cells incubated with pRGO@MS(DOX)-HA and pRGO@MS(DOX)-HA with the pretreatment of 10 mg/mL of HA.



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Figure 5. (A) In vivo fluorescence images of mice at different time points after administration of saline, Cy5 or pRGO@MS(Cy5)-HA (tumor pointed out with white circle). (B) Ex vivo fluorescence images of major organs and tumors at 24 h post-injection. H, heart; Li, liver; S, spleen; Lu, lung; K, kidney.


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Figure 6. In vivo anti-tumor activity of pRGO@MS(DOX)-HA nanocomposite. (A) IR thermal images of HeLa tumor-bearing mice upon 808 nm-laser irradiation for different periods of time. (B) Representative images of mice bearing HeLa tumor after different treatments for varied time periods. (C) Temperature variation curves of tumor region recorded by the IR thermal camera during NIR laser irradiation. (D) Tumor growth curves of mice after various treatments (five mice for each group). (E) The average body weights of mice after various treatments. (F) Representative H&E sections of tumors after treatment with saline, DOX, pRGO@MS-HA, pRGO@MS-HA+NIR, pRGO@MS(DOX)-HA, pRGO@MS(DOX)-HA+NIR. Data were presented as mean ± SD ( n = 5). P values in (D) were calculated by the twotailed Student’s t-test (*** p

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