porous-SiO2 Nanocomposites as Near

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Rattle-type Gold Nanorods/porous-SiO2 Nanocomposites as Near-infrared Light Activated Drug Delivery Systems for Cancer Combined Chemo-photothermal Therapy Yanyan Yu, Min Zhou, Wei Zhang, Lei Huang, Dandan Miao, Hongyan Zhu, and Gaoxing Su Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01298 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Molecular Pharmaceutics

Rattle-type Gold Nanorods/porous-SiO2 Nanocomposites as Near-infrared Light Activated Drug Delivery Systems for Cancer Combined Chemo-photothermal Therapy Yanyan Yu,†,# Min Zhou,†,# Wei Zhang,† Lei Huang,‡ Dandan Miao,† Hongyan Zhu,*,† Gaoxing Su*,†

†School ‡Phase

of Pharmacy, Nantong University, Nantong226001, China I Clinical Laboratory of Nanjing Drum Tower Hospital,

Nanjing210008, China

Corresponding Authors: [email protected] (Hongyan Zhu) [email protected] (Gaoxing Su)

#These

authors contributed equally to this work.

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Rattle-type nanostructures with movable cores, porous shells and hollow interiors, have become attractive nanoplatforms in the field of nanomedicine, especially for targeted and stimuli-responsive drug delivery. In this work, rattle-type gold nanorods@void@porous-SiO2 nanocomposites (GVPS) were fabricated facilely using the surface-protecting etching method, and exhibited high photothermal conversion efficiency. Taking the advantage of porous shell and hollow interior, the nanocomposites have abundant space for drug loading and successfully improved the drug loading capacity up to ~19.6%. To construct a multifunctional drug delivery system, GVPS was further functionalized with polyethylene glycol (PEG) and cyclic RGD peptides to improve biocompatibility as well as selectivity towards the targeted cancer cells. Besides, to achieve precise regulation and near-infrared laser activation of the drug release, a phase-changing material, 1-tetradecanol (1-TD, Tm: 39 °C), was employed as gatekeepers in this system. After incubation with a αVβ3 integrin receptor overexpressed cell line, the as-prepared GVPSPR-DOX/TD nanocomposites exhibited great performances in combined photothermal therapy and chemotherapy. It is worth to be noting that the combined therapy showed more superior efficiency in cancer cell killing than chemotherapy or photothermal therapy alone.

Keywords: drug delivery, gold nanorods, mesoporous silica, nanorattles, combined therapy, cancer therapy


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1. Introduction Gold nanorods (GNRs) have attracted great attention in various biomedical applications due to their unique and tunable localized surface plasmon resonance (LSPR).1-3 The LSPR peaks of GNRs were located in the near-infrared (NIR) region, where is optically transparent to tissues and blood.4 Also, GNRs possess high efficiency in converting light to heat. In addition, GNRs are easily surface functionalized, and cetyltrimethyl ammonium bromide (CTAB) non-bound GNRs are intrinsic biocompatible.5,6 Due to these extraordinary merits, GNRs have become one of the most popular photothermal therapy agents.7-10 However, it is difficult to eradicate tumors completely through photothermal therapy with GNRs alone.11 Owning to the light absorption and scattering, the intensities of NIR light will gradually decrease as penetrating into deeper tumor tissues, leading to declined phototherapeutic efficiency. Therefore, combination of photothermal therapy with chemotherapy (i.e. chemo-photothermal therapy) offers an alternative strategy.8,10,12 In this strategy, anticancer drugs are co-delivered with photothermal agents. On one hand, heating can destroy cancer cells directly. On the other hand, elevated temperature promotes the release of anticancer drugs, which enhanced the chemotherapy. In this way, chemo-photothermal therapy has been proved to pave a promising and effective way in cancer therapy. It has also been reported that this combined therapy method shows high therapeutic efficiency in multi-drug resistant tumors.13,14 To load with anticancer drugs, GNRs were always conjugated with various



surface coatings, including liposome,15 mesoporous SiO2,16-19 polymers,20-22 DNA,13,23 metal-organic framework,24,25 etc. Among them, core-shell GNR@mesoporous SiO2 nanoparticles are very attractive carriers for combined chemo-photothermal therapy.26 Mesoporous SiO2 features several advantages, including good biocompatibility, high chemical and mechanical stability, and facial surface functionalization.27-29 Moreover, the high surface-volume ratio, tunable pore sizes, and high pore volumes render mesoporous silica great loading capacity, which is in favor of various cargo molecules encapsulation. The high therapeutic efficiency of the drug-loaded core-shell GNR@mesoporous SiO2 nanostructrues has been well demonstrated in a batch of previous reports.16-18,30-32 Furthermore, the conjunction of targeting moieties, such as peptides, organic small molecules, antibodies, aptamers, and so on, can prominently improve the delivery specificity and efficiency. To avoid premature release and realize precise control of the drug release at diseased tissues, biocompatible thermo-sensitive and phase-changing materials, for instance, 1-tetrahedron (1-TD), were utilized as gatekeepers in the drug delivery system.17,19,31 Notably, cytotoxic surfactants (cetyltrimethyl ammonium bromide，CTAB) were commonly utilized as templates in porous SiO2 layer fabrication. It would be more bio-friendly and useful if the porous could be prepared without using CTAB. It was reported that rattle-type nanostructures, a class of special core-shell nanostructures with movable cores, porous shells and hollow interiors, have demonstrated many important potential biomedical applications that are not available for conventional core-shell nanoparticles, including drug delivery,33-35 biosensing,36


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and imaging.37 For instance, rattle-structure upconversion nanoparticle@porous SiO2 nanocomposites were developed to deliver anticancer drugs and radiosensitizers simultaneously for combined chemotherapy and radiotherapy.33 Wang and co-workers constructed a gold nanocage@porous SiO2 nanorattles for surface-enhanced Raman scattering (SERS) imaging, drug delivery, and photothermal therapy.38 Whereas, targeted delivery and precise drug release was not considered in their work. Rattle-shaped GNR@mesoprous SiO2 were also fabricated by etching of GNR@Ag@mesoprous SiO2 using H2O2, however cancer therapy was not conducted.39 Taking the advantage of rattle structures, the hollow interior provide enough space for cargos, the inner GNR cores are excellent photothermal agents. The outer silica surface is easily functionalized with targeting moieties. The whole nanocomposites are biocompatible. Therefore, rattle-type GNR@mesoporous SiO2 nanoparticles are promising nanocarriers for cancer combined therapy. In this work, employing the “surface-protected etching” strategy,35,40,41 GNR@void@porous-SiO2 (GVPS) rattle-type nanostructures were prepared by a facial process of etching dense SiO2 coated GNRs (GNR@SiO2). The as-prepared nanorattles featured a mesoporous silica shell and a movable GNR core. Subsequently, polyethylene glycol (PEG) molecules and cyclic RGD (Arg-Gly-Asp) peptides were covalently conjugated to the surface of GVPS (named as GVPSPR), providing biocompatibility and targeting capabilities for the nanocomposites. Doxorubicin (DOX) and 1-TD were loaded as antitumor drugs and gatekeepers, respectively. In this way, the GVPSPR-DOX/TD nanocomposites were obtained. In



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vitro studies demonstrated the as-prepared nanocomposites exhibited good biocompatibility and low premature release. The cellular uptake was significantly increased in cell line with αVβ3 integrin receptor over-expressed compared with non-targeted nanocomposites. The intracellular drug release can be activated by NIR light remotely. Finally, we evaluated the combined chemo-photothermal therapeutic efficiencies with the HeLa cells and MCF-7 cells (Scheme 1).

2. Experimental Section 2.1 Materials and reagents Hydrogen tetrachloroaurate trihydrate (HAuCl4•3H2O, ACS reagent) was purchased from Alfa Aesar. L(+)-ascorbic acid (AA, >99%), and hydrochloride solution (HCl, 36%~38%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), doxorubicin (DOX), Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC), poly(vinylpyrrolidone) (PVP, MW: 20,000), silver nitrate (AgNO3, >99%), CTAB (>98%), and sodium borohydride (NaBH4, 98.0%) were purchased from Aladdin. Thiol-NHS-terminated

PEG

(NHS-PEG-SH,

MW:

5000)

and

thiol-methoxyl-terminated PEG (OMe-PEG-SH, MW: 5000) were obtained from J&K Scientific and Technology. cRGD (cRGDyK, >95%) was obtained from ChinaPeptides Biotechnology. All commercial chemicals were used as received without further purification. All glassware throughout the synthesis of nanoparticles were immersed in aqua regia for 4 h, washed with ultrapure water several times and


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oven-dried before use. 2.2 Synthesis of GNRs To prepare gold seeds, HAuCl4 solution (250 μL, 10 mM) was added to the CTAB solution (9.75 mL, 0.1 M) with vigorous stirring. Then, fresh ice-cold NaBH4 solution (0.6 mL, 0.01 M) was injected. The mixture changed from yellow to brown, indicating the creation of gold seeds. After 5 min stirring, the solution was kept for 1 hour before using. To prepare the growth solution, CTAB (7.0 g) and sodium oleate (1.234 g) were dissolved in water (250 mL). Then AgNO3 solution (24 mL, 4 mM) was added. The solution was then kept undisturbed for 15 min. After that, HAuCl4 solution (250 mL, 1 mM) was added and kept quietly for 60 min. Concentrated HCl (2.1 mL) was added and stirred gently for 15 min. Ascorbic acid (1.25 mL, 0.064 M) was then added and stirred for 30 s. At last, seed solution (800 L) was added and further stirred for 30 s. The solution was kept at 30 °C overnight and then centrifuged at 10,000 rpm for 30 min and washed with water once. The obtained GNRs were finally dispersed in 10 mL water. 2.3 Preparation of GNR@SiO2 SiO2-coated GNRs was prepared by a modified Stöber method. Briefly, To avoid the aggregation of GNRs in ethanol solutions, PEG was grafted to the surface of GNRs firstly. Typically, OMe-PEG-SH (5 mg) was added to a solution of GNRs (10 mL) at room temperature. After stirring for 24 h, PEG-capped GNRs were centrifuged and washed with ethanol. Then PEG-capped GNRs were dispersed with 100 mL of



ethanol. At 30 °C, the diluted ammonia solution (20 mL, 1: 19 v/v) was added. After that, TEOS ethanol solution (1.5 mL, 1: 9 v/v) was added five times with the interval of 2 h under stirring. The mixture was allowed to react for another 3 h at 30 °C. After centrifugation and washing with water, GNR@SiO2 nanoparticles were obtained and redispersed in 10 mL water. 2.4 Synthesis of GVPS GVPS were synthesized by a surface-protected etching process.42,43 GNR@SiO2 (3 mL) were mixed with PVP solution (17 mL, 1.2 mg/mL). After stirring for 30 min, the solution was heated to 105 °C and kept for 2.5 h. The as-prepared GVPS were obtained by centrifugation, and washed with ethanol for five times, and redispersed in ethanol (1 mL). 2.5 Synthesis of GVPSPR Firstly, GVPS were amino-functionalized with APTES. GVPS (1 mL) were mixed with ethanol (19 mL). Then APTES (30 μL) in ethanol (840μL) were added. After stirring for 4 h at 35 °C, the products (GVPS-NH2) were obtained by centrifugation, and washed with water and PBS, and redispersed in PBS (8 mL). Next, NHS-PEG-SH were covalently conjugated onto GVPS-NH2 through cross linking the thiol and amino groups with sulfo-SMCC. Typically, GVPS-NH2 (10 mL) were mixed sulfo-SMCC solution (4.6 mg in 800 μL of water) for 30 min at room temperature. After that, excess sulfo-SMCC were removed by centrifugation. The pellets were redispersed in PBS (10 mL). Then NHS-PEG-SH (200 μL, 20 mg/mL) were added slowly. After stirring for 24 h at room temperature, the NHS-PEG


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conjugated GVPS (GVPSP-NHS) were obtained by centrifugation, and washed with water and PBS, and redispersed in PBS (8 mL). The OMe-PEG-SH were also used to conjugate to GVPS-NH2. The obtained PEG modified GVPS (GVPSP) were non-targeting nanocomposites. Finally, cRGD (200 μL, 10 mg/mL) were added to GVPSP-NHS in PBS (8 mL). After stirring for 24 h at room temperature, the cRGD conjugated nanoparticles (GVPSPR) were obtained by centrifugation. After washing with water and ethanol, the cRGD conjugated GVPSPR was redispersed in ethanol (8 mL) and stored at 4 °C for further use. According to our previous work,44 the amounts of conjugated cRGD molecules were obtained by subtracting the unattached cRGD molecules from the total cRGD molecules in the reactions. The amounts of unattached cRGD molecules in the supernatant were quantified by BCA assays. The yield of the conjugation was ~20%. According the GVPS concentration, it was calculated that about 0.2 wt% of cRGD were conjugated for the targeting nanocomposites. 2.6 Characterization Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1011 transmission electron microscope at a voltage of 80 kV. UV-vis-NIR spectra were recorded on a UV1800 spectrometer (Jinghua Instruments). Fourier transform infrared (FTIR) spectra were recorded on a Nicolet iS10 spectrometer (Thermo-Fisher Scientific). Hydrodynamic diameters and zeta potentials were measured by NanoBrook 90Plus Zeta (Brookheaven). Fluorescence intensities were



measured on a RF-5301PC fluorescence spectrometer (Schimadzu). Confocal laser scanning microscopy (CLSM) images were obtained on a Leica TCS SP8 microscope. Au concentrations in living cells were measured by inductively coupled plasma mass spectrometry (ICP-MS, NexION 350, PerkinElmer). 2.7 Photothermal heating experiment In a typical setup, GVPS in PBS with different concentrations were added to eppendorf tubes (0.5 mL), and then placed in a 37 °C water bath. Each tube was irradiated with an 808 nm diode laser (LE-LS-808, LEO-Photoelectric) at different intensities for 10 min. The solution temperatures were detected every 30 s using a digital thermometer. Control experiment (PBS only) were also performed in all conditions. 2.8 Drug and gatekeeper loading The loading of DOX and 1-TD onto GVPSPR were performed according to previous work with minor modification.45 In brief, GVPSPR (8 mg) were added to DOX solution in ethanol (8 mL, 8 mg/mL). The mixture was stirred at 50 °C for 1 h. After elevating the temperature to 60 °C, 1-TD (4 mg) were added and continuously stirred for 15 min. Then hot water (5 mL, 60 °C) was added. Immediately, the mixture was centrifuged to remove excess 1-TD. Then the drug and gatekeeper loaded GVPSPR (GVPSPR-DOX/TD) were washed with cold water for several times to remove the free DOX. To determine the loaded amounts of DOX, GVPSPR-DOX/TD (2 mg) were dispersed into acetone (2 mL). The solution was sonicated for 10 min to extract the 1-TD and DOX into acetone. After centrifugation, the surfactant was


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collected and measured by UV-vis spectroscopy at 495 nm. The amounts of loaded DOX could be calculated according the calibration curve. The drug loading capacity was calculated following equation: Loading capacity = Amounts of Loaded DOX / Amounts of GVPSPR-DOX/TD. The total amounts of loaded DOX and 1-TD were evaluated by thermogravimetric analysis (TGA, PE Pyris 1 DSC, temperature: 20 to 700 °C at 20 °C/min, air atmosphere). The DOX stability in 60 °C solution was evaluated by UV-vis spectra and fluorescence spectra. The DOX solution were incubated at 60 °C water bath for 4 h and 8 h. Then the solution of UV-vis absorption spectra and fluorescence spectra were recorded. As shown in Figure S6, both types of spectra exhibited negligible change, indicating DOX are substantially stable at 60 °C solution during that time. 2.9 Drug release Drug release was performed in PBS with two pHs (7.4 and 5.0) under or no NIR laser irradiation. Typically, GVPSPR-DOX/TD were diluted with PBS buffer (pH 7.4 or 5.0, DOX concentration: 5 μg/mL). All nanoparticle solutions were incubated at 37 ºC. At the setting time points, the solutions were irradiated with or without NIR laser (5 min, 2 W cm-2). After that, the solutions were centrifuged and the supernatants were collected. The released drug amount in the supernatants was measured using fluorescence spectroscopy with excitation wavelength 480 nm. Fluorescence intensities at 595 nm were used to calculate the DOX concentrations. 2.10 Cell culture



HeLa cells and MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and RPMI 1640 medium, respectively. Cell culture medium was supplemented with 10% FBS, 10 U/mL penicillin, and 10 mg/mL streptomycin. Cultures were maintained at 37 °C under humidified conditions with 5% CO2. 2.11 Cellular uptake measured by ICP-MS HeLa cells or MCF-7 cells were seeded in a 12-well plate at a density of 100,000 cells per well and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. After that, the medium in each well were removed and washed with PBS once. GVPSP or GVPSPR in complete medium (1 mL, 50 μg/mL) were added. The cells were incubated at 37 °C for 1, 2, 4, 8, 12, and 24 h. After that, each well was washed with cold PBS three times. Cells in each well were digested with trypsin and counted. Then aqua regia (100 μL) were added to complete digest the cells. After incubation overnight, the cell digested solutions were diluted with 0.5% HNO3 solution and analyzed by ICP-MS. 2.12 Intracellular drug release visualized by CLSM HeLa cells (20,000 cells) were seeded in a 35 mm confocal dish. After incubation for 24 h, the medium was replaced with fresh medium containing free DOX, GVPSP-DOX/TD, or GVPSPR-DOX/TD (DOX concentration: 2.9 μg/mL). After incubation for 2 h or 4 h, the group of GVPSPR-DOX/TD were replaced with fresh medium, and irradiated with 808 nm laser (2 W cm-2) for 5 min. Then the cells were washed with cold PBS three times and Hoechst 33342 (2 μg/mL) was added to stain the cell nuclei. After 30 min of incubation, the cells were washed with PBS three


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times again and analyzed by CLSM. 2.13 Cytotoxicity of blank nanocarriers HeLa cells and MCF-7 cells were seeded in a 96-well plate (5,000 cells/well). After incubation for 24 h, medium containing GVPSP and GVPSPR (1, 5, 10, 50, 100, 250, and 500 μg/mL) were added. Following 24 h of incubation, the media were removed and each well was rinsed with cold PBS once. Afterwards, fresh medium (100 μL) and CKK-8 solution (20 μL) were added to each well. After incubation for 4 h, the absorbance of each well was measured at 450 nm by a microplate reader. Each experiment was performed in triplicates. 2.14 Combined chemo-photothermal therapy Briefly, HeLa cells and MCF-7 cells were seeded in a 96-well plate (5,000 cells/well). After incubation for 24 h, cells were treated with free DOX, GVPSP-DOX/TD and GVPSPR-DOX/TD (DOX concentration: 0.01, 0.1, 0.5, 1, 5, and 10 μg/mL). After incubation for 4 h, the cell culture media were replaced with fresh media. For photothermal therapy, cells were irradiated with 808 nm laser (2.5 W cm-2, 5 min). For chemotherapy only, cells were incubated with fresh medium without laser irradiation. After incubation for another 20 h, the media were removed and each well was rinsed with cold PBS once. Afterwards, fresh medium (100 μL) and CKK-8 solution (20 μL) were added to each well. After incubation for 4 h, the absorbance of each well was measured at 450 nm by a microplate reader. Each experiment was performed in triplicates. 2.15 Live-dead cell assay



HeLa cells were seeded in a 6-well plate (200,000 cells/well). After incubation for 24 h, the cells were treated with free DOX, GVPSPR, and GVPSPR-DOX/TD (DOX: 10 μg/mL). After incubation for 4 h, the culture medium was replaced with fresh medium. For photothermal therapy, cells were irradiated with 808 nm laser (2.5 W cm-2, 5 min). Cells without any treatments were used as control. All cells were stained with Calcein-AM and propidium iodide (PI) solutions for 15 min. After washing with PBS for several times, cell imaging were carried out with fluorescence microscopy.

3. Results and discussion 3.1 Synthesis and characterization of GNR@void@porous-SiO2 The synthesis procedure of rattle-type GNR@void@porous-SiO2 (GVPS) is illustrated in Figure 1A. GNRs with the aspect ratio of ~4.5 (68 × 15 nm) were firstly synthesized by a seed-mediated growth method (Figure 1B).46,47 Secondly, to avoid the aggregation, the GNRs were modified with a layer of PEG by Au-S bonds. Using a modified stöber mthod, a dense SiO2 shell was fabricated and SiO2-coated GNRs (GNR@SiO2) was obtained. As shown in Figure 1C, each GNRs was well encapsulated inside a uniform and dense SiO2 shell with a thickness of ~35 nm. Then, the surface of GNR@SiO2 was protected with PVP. After refluxing at 100 °C for 2.5 h, rattle-type GVPS with mesoporous shell and movable GNR core were successfully prepared by etching the PVP protected GNR@SiO2 using hot water (Figure 1D).42,43 According to TEM images, the size of GVPS were statistically measured, which is


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~120 nm in length and ~60 in width with narrow size distribution (Figure S1). The Brunauer-Emmett-Teller (BET) surface area and total pore volume of GVPS analyzed by N2 adsorption-desorption isotherm assays were 156 m2 g-1 and 0.29 cm3 g-1, respectively (Figure S2). The pore size distribution was wide, ranging from 2~30 nm, due to the hollow interior and non-uniform pores of silica. The optical properties of nanoparticles were also studied with UV-vis-NIR spectra. As shown in Figure 1E, the LSPR peak of GNRs is around 780 nm. After coating with dense SiO2, the LSPR peak showed an obvious red-shift from ~780 nm to ~830 nm, which was attributed to the increase of the refractive index near GNRs. However, after etching, the LSPR peak showed ~20 nm blue-shift, in which the LSPR peak of GVPS appeared at ~810 nm. It is worth noting that the LSPR absorption spectra did not broaden in all experimental process, indicating no aggregation of GNRs was formed during this entire synthesis procedure. The dependence of the fabrication of rattle-type nanostructures on reaction time was further investigated. As shown in Figure 2, the SiO2 layer showed no obvious decomposition within the first 30 min. Actually, mesoscale pores were gradually formed on the surface of SiO2 shell, so that etchant could diffuse into the particles. Due to the lack of protection, the etching rate of silica near GNRs increased significantly. At 90 min, silica around GNRs was etched away and a hollow interior appeared. As the reaction time increasing, the pores of silica shell became larger. As demonstrated in Figure 2C, after reaction for 2.5 h, the shell become thinner and hollower compared with particles in Figure 2B. The pores on silica shell were



obvious, and interior GNRs were movable. When the reaction time was prolonged to 180 min, the outer silica shell was completely destroyed (Figure 2D). Therefore, the rattle-type nanostructures formed at reaction time 2.5 h were chosen for the following drug delivery experiments. 3.2 Functionalization of GVPS with targeting moiety and drug loading Integrin receptors were over-expressed in several cancer cells,48,49 such as hepatoma, cervical carcinoma, and so on. To targeted binding with integrin receptors in cancer cells, the surface of GVPS was covalently modified with cRGD peptides.50,51 The modification procedures were presented in Figure 3A. The surface of GVPS were firstly amination with APTES. Then HS-PEG-NHS was covalently conjugated onto the surface of amino-functionalized GVPS by the thiol groups using sulfo-SMCC as cross-linking reagents. Finally, cRGD were conjugated through the condensation reaction between -NH2 group and activated -COOH group (NHS group), and the as-prepared nanocomposites were named as GVPSPR. The hydrodynamic diameters of functionalized GVPS were increased from ~150 nm to ~210 nm (Figure 3B). Meanwhile, the zeta potentials were measured (Figure 3C). After amino modification, the zeta potential was positive, and after conjugation with PEG, the zeta potential turned to negative. The corresponding change in zeta potential confirmed that the APTES and PEG molecules were successfully modified on the surface of GVPS. To verify the successfully conjugation of cRGD, FTIR spectra of cRGD, GVPS, and GVPSPR were recorded. As shown in Figure 3D, compared to the FTIR spectrum of GVPS, the emerging peaks at 2877 and 2907 cm-1 could be caused by


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methyl and methylene groups in PEG and cRGD. The absorption at 1653 cm-1 became stronger due to the stretching vibration of carbonyl groups. For GVPSPR, the small peaks at 1460 and 1350 cm-1 verified the presence of aromatic rings in cRGD. The strong absorption peak at 1083 cm-1 corresponding to Si-O-Si bond in the spectrum of GVPS shifted to 1103 cm-1, due to C-O-C bond incorporation in the spectrum of GVPSPR. The results indicated that PEG and cRGD molecules were successfully grafted on the surface of GVPS. DOX and 1-TD molecules were subsequently loaded into GVPSPR (Figure 4A), and the obtained nanocomposites were named as GVPSPR-DOX/TD. Because of the mesoporous shell and hollow interior, DOX was easily loaded into the GVPSPR. The loaded DOX was verified by the increase of UV-vis absorption at ~500 nm (Figure 4B). The loading amount could be calculated by extracting DOX in acetone solution under sonication, which was determined to be ~19.6%. Notably ， the drug loading capacity in this work was almost 2 times higher than the previous work using core-shell GNR@mesoporous SiO2 nanoparticles,17 which could be attributed to mesoporous silica shell and hollow interior in the unique rattle-type nanostructure. TGA analysis revealed that the overall fraction of DOX and 1-TD was ~26% in weight (Figure 4C). Accordingly, the amounts of incorporated 1-TD was estimated to be ~6.4%. 3.3 Photothermal effects The photothermal effects of GVPS was studied by monitoring the increase of solution temperature under the laser irradiation at 808 nm. As shown in Figure 5A, at



the concentration of 70 μg/mL and the NIR laser intensity of 1.5 W cm-2, a rapid temperature elevation was observed. The temperature increased from 37 °C to 55 °C within 10 min. Whereas, the PBS without GVPS only increased less than 1 °C under the same condition. The results demonstrated that GVPS could convert light to heat efficiently. It is clearly observed from Figure 5A that GVPS could achieve moderate hyperthermia temperature (~43 °C) from 37 °C within 2 min. This temperature was confirmed to be high enough to kill cancer cells and have minimal effects on healthy tissues. Considering human body temperature around 37 °C ， heating up to 43 °C could be easily achieved at lower irradiation intensity, shorter irradiation time or lower nanoparticle concentration. Therefore, the influences of laser intensity and particle concentration on photothermal effects were further investigated. As shown in Figure 5A and 5B, the temperature increase showed a dose- and laser intensity-dependent manner. By increasing the laser intensity from 1.0 to 2.0 W cm-2, the temperature of GVPS (70 μg/mL) could increase 11 °C at 1.0 W cm-2, 18 °C at 1.5 W cm-2, and 27 °C at 2.0 W cm-2, respectively. By increasing the concentration of GVPS from 30 to 120 μg/mL, the temperature could increase 5 °C at 30 μg/mL, 11 °C at 70 μg/mL, and 19 °C at 120 μg/mL, respectively, under the laser intensity of 1.0 W cm-2. The excellent light-to-heat conversion efficiency suggested that GVPS hold great potential for cancer photothermal therapy. In addition, photostability of GVPS was also investigated. As shown in Figure S3, the photothermal capability of GVPS was characterized by irradiating every other 10 min over 5 on/off cycles. It was observed that the temperature elevation did not


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show much change during the 5 cycles. Furthermore, the UV-vis-NIR absorption spectrum of GVPS was measured after the 5 on/off cycles of irradiation (Figure S4). The LSPR peak was observed a small widening, due to the reshaping of GNRs. TEM images of GVPS after 5 on/off cycle 808 nm laser irradiation were shown in Figure S5. The morphologies of GVPS didn’t change much after laser irradiation. On the basis of the statistical results, the length of GNRs was a bit shorter than before. 3.4 Dual-stimulus drug release Controllable drug release is an important function for drug carriers to obtain better therapeutic efficiency and avoid the side effects caused by burst release of drug. Herein, we employed 1-TD as gatekeepers to regulate drug release. 1-TD is a biocompatible phase-change material, and its melting temperature (Tm) is 39 °C. Below this temperature, drugs were entrapped in the carriers. When the environmental temperature was elevated by NIR laser, drugs could diffuse out along with the 1-TD fluid. Under acidic environment, DOX were protonated and water solubility was increased, which accelerated the drug release (Figure 6A). To test our design, drug release profiles of GVPSPR-DOX/TD were studied in PBS solution at pH 7.4 or pH 5.0 with and without NIR laser irradiation (Figure 6B and 6C). Due to the presence of 1-TD, without NIR laser irradiation, the drug released very slowat 37 °C in both pH solutions, and the release rates were ~7% and ~18%, respectively. Comparing with our previous work, the DOX release rate in the similar core-shell system but without gatekeepers could reach as high as 40% (pH 5.0, 37 °C).52 These results revealed 1-TD were efficient in trapping loaded drugs. Furthermore, given the reversible



phase-changing property of 1-TD, multiple NIR laser irradiation on/off treatments were performed to achieve “on-demand” drug release behaviors. Typically, the solution of GVPSPR-DOX/TD was irradiated with NIR laser for 5 min, then the laser was switched off for a period of time. Overall 5 cycles were conducted. It was observed that the drug release rate was significantly improved when the laser was on. Once the laser was switched off, the drug release rate decreased sharply. After 5 on/off cycles, the total drug release rates were ~22% and ~51% at pH 7.4 and pH 5.0, respectively. Hence, the drug delivery systems constructed in this work were pH and NIR laser dual-stimulus responsive. The drug release of nanocomposites loaded with 1-TD could be finely controlled by external NIR laser irradiation. 3.5 Cellular uptake and intracellular NIR-activated drug release Herein, cRGD was covalently conjugated on the surface of GVPS to achieve tumor-selective drug delivery. HeLa cell line with over-expressed integrin αVβ3 receptors was used to evaluate the targeting efficiency of GVPSPR. As a control, PEG conjugated GVPS (GVPSP) was also synthesized. The internalized nanocomposites were quantitative analyzed by measuring gold content using ICP-MS.44,53 As shown in Figure 7A, after 24 h incubation, cellular uptake amounts of targeted GVPSPR were 4 times higher than that of non-targeted GVPSP. Whereas, after incubating with MCF-7 cell with negative expression of integrin αVβ3 receptors, the cellular uptake amounts of targeted and non-targeted nanocomposites showed no difference and were both much lower than that in HeLa cells (Figure 7B). Results above confirmed that targeted GVPSPR were mainly internalized through integrin receptor-mediated


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endocytosis. Moreover, confocal laser scanning microscopy (CLSM) was performed to examine the cellular uptake and intracellular NIR-activated drug release (Figure 8). After loading into GVPS, the red fluorescence signal of DOX was quenched by GNR (Figure S7). Few red fluorescence signals could be observed both for GVPSP-DOX/TD and GVPSPR-DOX/TD nanocomposites. This is consistent with our previous results that few DOX were released without NIR laser irradiation. Under NIR laser irradiation, the red fluorescence signals enhanced significantly in both groups of GVPSP-DOX/TD and GVPSPR-DOX/TD nanocomposites, indicating NIR-activated drug delivery systems were feasible in vitro. Meanwhile, red fluorescence signals for targeted GVPSPR-DOX/TD were much stronger than that of GVPSP-DOX/TD, confirming that the cellular uptake efficiency were improved significantly by the conjugated targeted moieties. Prolonging the incubation time from 2 h to 4 h, it was observed that DOX were located in the nucleus (Figure S8), indicating DOX could bind with DNA and caused the cytotoxicity. 3.6 Combined chemo-photothermal therapy Cell counting kit-8 (CCK-8) assays were employed to evaluate the cytotoxicity of as-prepared nanocomposites. In both HeLa cell lines and MCF-7 cell lines, the cell viabilities were higher than 80% after incubation with different doses of GVPSPR or GVPSP (ranging from 1 to 500 μg/mL) for 24 h (Figure 9A and S9). The results revealed the nanocomposites have good biocompatibility. CCK-8 assays were also used to evaluate the therapeutic effects of combined



Page 22 of 40

chemo-photothermal therapy. HeLa cells were treated with free DOX, GVPSPR with NIR,

non-targeted

GVPSP-DOX/TD

with

or

without

NIR,

and

targeted

GVPSPR-DOX/TD with or without NIR. After 4 h of incubation, fresh media were replaced and NIR laser irradiation were performed for 5 min. Then cells were further incubated for 20 h and cell viabilities were measured. As shown in Figure S10A and 9B, the dose-dependent anti-cell proliferation effects were displayed in HeLa cells for various treatments. At the DOX concentration of 10 μM, targeted GVPSPR-DOX/TD showed higher cytotoxicity than that of non-targeted GVPSP-DOX/TD, indicating targeted moieties improved cellular uptake ability and enhanced the cell killing effects. More importantly, after NIR laser irradiation, the therapeutic efficiency of GVPSPR-DOX/TD and GVPSP-DOX/TD improved significantly. This could be explained in three expects: (1) Heat generated by GNRs resulting from NIR laser irradiation could damage tumor cells directly;50,54 (2) intracellular DOX concentration were increased due to local heat melting of the gatekeepers; (3) It is possible cytotoxicity of DOX was enhanced by heating.25,55 Likewise, due to the targeted capability, after NIR laser irradiation, the cytotoxicity of GVPSPR-DOX/TD was much higher than that of GVPSP-DOX/TD. However, in MCF-7 cells with negative expression of integrin αVβ3 receptors, the therapeutic efficiencies of targeted GVPSPR-DOX/TD did not exhibited significant difference to non-targeted GVPSP-DOX/TD (Figure S10B and 9C). In addition, live-dead cell assays were performed to evaluate the therapeutic effects of combined chemo-photothermal therapy. The live cells stained by calcium


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AM can emit green fluorescence, while the dead cells stained by PI showed red fluorescence. As shown in Figure 9E and 9F, almost no cell died by treating with GVPSPR

under

NIR

laser

irradiation

(photothermal

therapy

only)

or

GVPSPR-DOX/TD (chemotherapy only). For combined therapy group, cells were treated with GVPSPR-DOX/TD for 4 h and further treated with NIR laser for 5 min. We can observe that almost 100% cells were killed (Figure 9G). The results demonstrated once again GVPSPR-DOX/TD could carry out chemotherapy and photothermal therapy simultaneously, which resulted in obviously higher antitumor effects than any single therapy method alone.

4. Conclusions In summary, the targeted DOX-loaded GNR@void@porous-SiO2 nanorattles (GVPSPR-DOX/TD) were prepared as NIR-activated drug delivery systems for combined cancer chemo-photothermal therapy. Due to the hollow interior, the as-prepared drug delivery systems exhibited higher drug loading capacity than conventional core-shell nanostructures. This novel system possesses excellent phototherml conversion capability and photostability for efficient photothermal therapy. “On-demand” drug release behaviors were realized by using phase-changing molecules, 1-TD, as gatekeepers. The overall in vitro investigations were performed using HeLa cells, which overexpressed integrin αVβ3 receptors. The cellular uptake was significantly promoted by the targeting moiety. The NIR/pH dual-stimuli responsive drug release profile was confirmed in vitro. In addition, the



Page 24 of 40

GVPSPR-DOX/TD exhibited combined cancer chemo-photothermal therapy, which was much more efficient in inhibiting cancer cell proliferation than chemotherapy or photothermal

therapy

alone.

These

results

render

the

rattle-type

GVPS

nanocomposites a great promise in building biocompatible and multifunctional nanoformulations for effective tumor eradication. As reported, GNRs were capable in photoacoustic imaging and computed tomography imaging.56,57 These unique features may enable the rattle-type GVPS nanocomposites to be developed as theranostic agents to realize cancer therapy and diagnosis simultaneously.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21705082) and Natural Science Foundation of Jiangsu Province in China (BK20170444).

ASSOCIATED CONTENT Supporting Information Size distribution of GVPS, N2 adsorption-desorption isotherm curves of GVPS, Photothermal stability curve of GVPS, UV-vis-NIR absorption spectra, Fluorescence spectra, Confocal fluorescence images of HeLa cells after treatment for 4 h, and Cell viabilities of MCF-7 cells and HeLa cells after incubation with different treatments, were included in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Authors [email protected] (Gaoxing Su); [email protected] (Hongyan Zhu) Notes #Yanyan

Yu and Min Zhou contributed equally to this work. The authors declare no

competing financial interest.



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Cancer Cells PTT GVPS

GVPSPRDOX/TD

APTES

O

S PEG RGD

cRGD PEG

NH2

1-TD

Chemotherapy

DOX

HS-PEG-NHS

SMCC

Scheme 1. Schematic illustration of cRGD modified drug- and gatekeeper-loaded GVPS (GVPSPR-DOX/TD) as pH/NIR dual-responsive nanocarriers for synergistic cancer chemo-photothermal therapy. H N O

O S PEG NHS

O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 40


Page 31 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Synthesis of GVPS. (A) Schematic representation of the synthesis route of GVPS. (B) TEM images of GNRs. (C) TEM images of GNR@SiO2. (D) TEM images of GVPS. (E) UV-vis-NIR spectra of GNRs, GNR@SiO2, and GVPS.



A)

C)

B)

D)

Figure 2. TEM images of GVPS collected at different etching time. (A) 30 min, (B) 90 min, (C) 150 min, (D) 180 min.


Page 32 of 40

A)

NH2

APTES

cRGD

GVPS-NH2

GVPSP-NHS

100 100 50

O2 Si @ R GN

2 S S R M NH SP NH M GV SPV S M G M GV GV

D)

20 20

00

GVPSP-NHS

150

GVPS

200 200

GNR@SiO2

40 40

250

0 0

GVPSPR

C)

300 300

ZetaPotentials Potentials(mV) (mV) Zeta

B) Hydrodyamic Diameters (nm) Hydrodyamic Size (nm)

1) SMCC 2) HS-PEG-NHS

GVPS-NH2

-20 -20

-40 -40 2D Graph 4

RGD GVPS

Transmission

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GVPSPR

Page 33 of 40

GVPSPR

4000

3000

2000

1000

400

Wavenumber (cm-1)

Figure 3. Functionalized GVPS with targeting moieties and characterization. (A) Schematic illustration of the synthesis route of GVPSPR. (B) Hydrodyamic diameters and (C) zeta potentials of GNR@SiO2, GVPS, GVPS-NH2, GVPS-NHS, and GVPSPR. Data were presented as mean ± SD (n=3). (D) FT-IR spectra of cRGD, GVPS, and GVPSPR.



A)

DOX

1-TD

GVPSPR-DOX/TD

GVPSPR-DOX

C) 100

DOX GVPSPR GVPSPR-DOX/TD

Weight (%)

B)1.5 Absorption

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

1.0

0.5

80 60 40

GVPSPR GVPSPR-DOX/TD

20

0 400

600 800 Wavelength (nm)

1000

0 100

200

300 400 500 600 Temperature (ºC)

700

Figure 4. Drug and gatekeeper loading. (A) Schematic shows the loading process of DOX and 1-TD. (B) UV-vis-NIR spectra of DOX, GVPSPR, and GVPSPR-DOX/TD. (C) TGA traces of the GVPSPR and GVPSPR-DOX/TD.


GVPS 2.0 W cm-2 PBS 1.0 W cm-2 PBS 1.5 W cm-2 PBS 2.0 W cm-2

A) 70 Temperature (ºC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GVPS 1.0 W cm-2 GVPS 1.5 W cm-2

GVPS 1.0 W cm-2 GVPS 1.5 W cm-2 GVPS 2.0 W cm-2 PBS 1.0 W cm-2

60

GVPS 1.0 W cm-2 GVPS 1.5 W cm-2 GVPS 2.0 W cm-2 PBS 1.0 W cm-2 PBS 1.5 W cm-2 PBS 2.0 W cm-2

PBS 1.5 W cm-2 PBS 2.0 W cm-2

B)60 60

0 g/mL 30 g/mL 70 g/mL 120 g/mL

55 55

Temperature (ºC) Y Data

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50 50

45 45

50

40 40

40 0

2

4

6

8

Time (min)

10

35 35

0 0

2 2

44

6 6

8

8

10 10

X Data

Time (min)

Figure 5. Photothermal effects of GVPS. (A) Temperature rise of GVPS solutions (70 μg/mL) upon irradiation at different 808 nm laser intensity (1.0, 1.5, 2.0 W cm-2). (B) Temperature rise of GVPS solutions of different concentrations (30, 70, 120 μg/mL) after 808 nm laser irradiation (1.0 W cm-2). Data were presented as mean ± SD (n=3).



A) pH

porous-SiO2 Nanocomposites as Near

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