shell nanoplatform for tumor combination therapy

Accepted Manuscript Full length article An RGD-modified hollow silica@Au core/shell nanoplatform for tumor combination therapy Xin Li, Lingxi Xing, Yong Hu, Zhijuan Xiong, Ruizhi Wang, Xiaoying Xu, Lianfang Du, Mingwu Shen, Xiangyang Shi PII: DOI: Reference:

S1742-7061(17)30523-8 http://dx.doi.org/10.1016/j.actbio.2017.08.024 ACTBIO 5035

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

21 February 2017 24 July 2017 16 August 2017

Please cite this article as: Li, X., Xing, L., Hu, Y., Xiong, Z., Wang, R., Xu, X., Du, L., Shen, M., Shi, X., An RGDmodified hollow silica@Au core/shell nanoplatform for tumor combination therapy, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio.2017.08.024

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An RGD-modified hollow silica@Au core/shell nanoplatform for tumor combination therapy

Xin Li a, 1, Lingxi Xing b, 1, Yong Hu a, Zhijuan Xiong a, Ruizhi Wang c, Xiaoying Xu a, Lianfang Du b,

a

*, Mingwu Shen a, *, Xiangyang Shi a, d, *

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China b

Department of Ultrasound, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong

University, Shanghai 200080, P. R. China c

Shanghai Institute of Medical Imaging, Department of Interventional Radiology, Zhongshan

Hospital, Fudan University, Shanghai 200032, P. R. China d

CQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390

Funchal, Portugal ________________________________________________________ * Corresponding author. Tel.: +86 21 67792656; fax: +86 21 67792306 804 (X. Shi); +86 21 67792750; fax: +86 21 67792306 804 (M. Shen); +86-13386259562; fax: +86 21 37798276 (L. Du). E-mail addresses: [email protected] (L. Du), [email protected] (M. Shen), [email protected] (X. Shi). 1

Authors contributed equally to this work.

1

Abstract The combination of chemotherapy and photothermal therapy (PTT) in multifunctional nanoplatforms to improve cancer therapeutic efficacy is of great significance while it still remains to be a challenging task. Herein, we report Au nanostar (NS)-coated hollow mesoporous silica nanocapsules (HMSs) with surface modified by arginine-glycine-aspartic acid (RGD) peptide as a drug delivery system to encapsulate doxorubicin (DOX) for targeted chemotherapy and PTT of tumors. Au NSs-coated HMSs core/shell nanocapsules (HMSs@Au NSs) synthesized previously were conjugated with RGD peptide via a spacer of polyethylene glycol (PEG). We show that the prepared HMSs@Au-PEG-RGD NSs are non-cytotxic in the given concentration range, and have a DOX encapsulation efficiency of 98.6 ± 0.7%. The designed HMSs@Au-PEG-RGD NSs/DOX system can release

DOX

in

a

pH/NIR

laser

dual-responsive

manner.

Importantly,

the

formed

HMSs@Au-PEG-RGD NSs/DOX nanoplatform can specifically target cancer cells overexpressing αvβ3 intergrin and exert combination chemotherapy and PTT efficacy to the cells in vitro and a xenografted tumor model in vivo. Our results suggest that the designed HMSs@Au-PEG-RGD NSs/DOX nanoplatform may be used for combination chemotherapy and PTT of tumors.

Keywords:

Hollow

mesoporous

silica;

Gold

Chemo-/photothermal therapy

2

nanostars;

RGD

targeting;

tumors;

1. Introduction Various cancer therapy methods have been adopted to kill cancer [1-3]. Chemotherapy is able to efficiently kill metastatic cancer cells and prevent cancer cell proliferation, but it generally lacks specificity and affect normal cells simultaneously [4]. Therefore, design of novel carrier systems for controlled drug release is able to effectively reduce the side effects of chemotherapy [5, 6]. Photothermal therapy (PTT) is an effective and safe treatment strategy for cancer therapy due to its high precision local therapy efficacy and negligible systemic side effects, but it can only ablate the tumor site under laser irradiation and is unable to completely ablate the tumor in the whole body [7-9]. Neither chemotherapy nor PTT is generally sufficient to achieve highly efficient cancer therapy. Therefore, it is necessary to integrate chemotherapy and PTT within one nanoplatform for combination therapy of cancer, which is expected to significantly improve the cancer therapeutic efficacy [10-12]. The inorganic, organic or hybrid nanoparticles (NPs) or nanocapsules (NCs) for the different biomedical applications, such as disease treatment [13-15], drug delivery [16-18], diagnostic imaging [19-22], and gene transfection [23, 24], have been rapidly emerging in the past few years. In particular, development of the high efficiency nanoplatforms for cancer therapy is of great interest [25-27]. Among them, the stimuli-responsive drug delivery system with various triggers, such as pH [28], light [29], temperature [30], ultrasound [31], magnetic field [32] and redox gradients [33] were intensively investigated.

For

instance,

in

a

recent

work

Du

et

al.

[34]

prepared

α-cyclodextrin/aniline-comodified hollow mesoporous silica which possess a pH-responsive controlled release function. Our previous work has also shown that inorganic or organic drug delivery system, such as dendrimers modified with polyethylene glycol (PEG) or laponite nanodisks are able to encapsulate anticancer drug doxorubicin (DOX) and release the drug in a pH-responsive 3

manner [35, 36]. For efficient combination of chemotherapy and PTT, nanoplatforms with near infrared (NIR) absorption feature were used to encapsulate anticancer drug [37-40]. Recently, Li et al. [41] reported the use of DOX-loaded Au nanoflowers for combination chemotherapy and PTT of tumor. They showed that DOX could be released quickly from the Au nanoflowers under acidic pH conditions or NIR laser irradiation, resulting in controllable release of DOX. Meng et al. [5] reported NIR-laser-switchable smart nanocapsules with efficient combination chemotherapy/PTT of the tumors. However, the designed carrier systems either lack the surface modification of an active targeting ligand for efficient tumor penetration and retention, or lack the sufficient drug loading capacity. Development of tumor-targeted carrier systems having sufficient drug loading capacity for stimuli-responsive delivery still remains challenging. It is well known that hollow mesoporous silica (HMS) spheres enable encapsulation of high payload of anticancer drugs (e.g., DOX) and exhibit sustained drug release kinetics with pH-responsiveness [42]. In addition, gold nanostars (Au NSs) with the strong absorption in the NIR region [43, 44] have a higher photothermal conversion efficiency than other Au NPs with particular shapes (e.g., Au nanorods [45] and Au nanoshells [46]). Therefore, Au NSs have been widely used for PTT of cancer [44, 47, 48]. Very recently, we have shown that Au NSs can be deposited onto the HMS spheres and used to encapsulate perflurohexane for multi-mode ultrasound (US)/computed tomography (CT)/photoacoustic (PA) imaging and PTT of tumors [49]. Logically, we hypothesize that the merits of HMSs and Au NSs may be combined to build up a targeted and controlled drug delivery platform with a pH and NIR laser dual-stimuli for combination chemotherapy and PTT of tumors. In

this

present

study,

Au

NS-coated 4

HMSs

were

modified

with

PEGylated

arginine-glycine-aspartic acid (RGD) peptide and used to encapsulate DOX to form a pH/NIR laser dual-responsive nanoplatform for synergic chemotherapy and PTT of tumors. Au NS-coated HMSs formed according to our previous work [49] were modified with PEGylated RGD peptide via Au-S bond, followed by physical encapsulation of DOX (Figure 1). The structure, morphology, composition, cytocompatibility, encapsulation efficiency, and stimuli-responsive release kinetics of the formed HMSs@Au-PEG-RGD NSs were well characterized. Then, the use of the nanoplatform for combination chemotherapy and PTT of cancer cells in vitro and a xengrafted tumor model in vivo was explored. To our knowledge, this is the first work descibing the creation of Au NS-coated HMSs as a novel drug delivery system with pH/NIR laser dual-responsive release manner for combination chemotherapy and PTT of tumors.

2. Experimental section 2.1. Synthesis of HMSs@Au-PEG-RGD NSs HMSs@Au NSs were synthesized according to our previous work [49]. To prepare SH-PEG-RGD segment, SH-PEG-COOH (100 mg) dissolved in dimethylsulfoxide (DMSO) solution (8 mL) was added with 5 molar equiv. of EDC (19.2 mg, in 5 mL DMSO) under vigorously stirring for 30 min, then NHS (11.5 mg, in 3 mL DMSO) was added into the above solution under stirring for another 3 h. After that, equal molar equiv. of the activated SH-PEG-COOH was dropwise added to a DMSO solution of RGD peptide (13.8 mg, 4 mL) under vigorous stirring for 3 days. The reaction mixture was then dialyzed against water (9 times, 2L) using a dialysis membrane with an MWCO of 1 000 for 3 days, followed by lyophilization to obtain the product of SH-PEG-RGD. After that, the SH-PEG-RGD was modified onto the surface of the HMSs@Au NSs via Au-S bond. In brief, 10 mL of the SH-PEG-RGD aqueous solution (10 mg·mL-1) was added into the

5

HMSs@Au NSs solution (2.5 mg·mL-1, 20 mL) and the mixture was stirred for 48 h at room temperature. The resulting solution was centrifuged (8500 rpm, 20 min), washed with water for 3 times, and lyophilized to obtain the HMSs@Au-PEG-RGD NSs. 2.2. Characterization techniques Transmission electron microscopy (TEM), UV-vis spectrometry, 1H NMR, thermal gravimetric analysis (TGA), and inductively coupled plasma-optical emission spectroscopy (ICP-OES) were used to characterize the structure, morphology, and composition of the intermediate or final materials according to standard protocols reported in the literature [44, 48, 49]. 2.3. Photothermal property of the HMSs@Au-PEG-RGD NSs The photothermal property and stability of the HMSs@Au-PEG-RGD NSs were tested. Water, HMSs@Au seed and HMSs@Au-PEG-RGD NSs solution with the volume of 100 µL at different Au concentrations (1, 5, 10 and 20 mM) were irradiated under an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co. Ltd., Shanghai, China) at a power intensity of 1.2 W/cm2 for 350 s. The temperature of each sample was recorded every 5 s using a thermocouple probe (Shenzhen Everbest Machinery Industry Co., Ltd., Shenzhen, China). Quantitative

analysis

of

the

photothermal

conversion

efficiency

(ηPTC)

of

the

HMSs@Au-PEG-RGD NSs was carried out according to the literature [44, 49]. Furthermore, the photothermal stability of the HMSs@Au-PEG-RGD NSs was tested by irradiating the HMSs@Au-PEG-RGD NSs suspension (5 mM, in 200 µL water) with an 808 nm laser for 280 s (1.2 W/cm2) and cooling the suspension down to room temperature for 290 s. The irradiation and cooling down process was performed for 4 times. 2.4. Formation and release kinetics of HMSs@Au-PEG-RGD NSs/DOX complexes HMSs@Au-PEG-RGD NSs/DOX were formed according to our previous reports [36]. Briefly, 6

an DOX·HCl aqueous solution (1 mg·mL-1, 1.34 mL) was added into an aqueous suspension of the HMSs@Au-PEG-RGD NSs (1 mg · mL-1, 20 mL) under stirring for 36 h. The HMSs@Au-PEG-RGD NSs/DOX complexes were obtained by centrifugation (8000 rpm, 5 min) and rinsing with water for 3 times, and finally stored in the dark at 4 oC before use. The DOX encapsulation efficiency (η DOX) of HMSs@Au-PEG-RGD NSs and the DOX release behavior of HMSs@Au-PEG-RGD NSs/DOX under different conditions was analyzed according to previously published reports [5, 36]. The concentration of free DOX in the collected supernatants of HMSs@Au-PEG-RGD NSs/DOX complexes after 4 times centrifugation was measured by UV-vis spectroscopy at 480 nm using the standard calibration curve. The ηDOX was calculated according to the equation (1): [36]

η DOX = (M 0 − M f ) M 0 × 100%

(1)

Where Mf and M0 are the masses of the free DOX and the initial DOX, respectively. The DOX release kinetics from the HMSs@Au-PEG-RGD NSs/DOX complexes under different pH conditions or under laser irradiation (5 min with an 808 nm laser, 1.2 W/cm2)/no laser exposure were tested. HMSs@Au-PEG-RGD NSs/DOX (1 mg) dissolved in 1 mL PBS (pH = 7.4) or acetate buffer (pH = 5.0) were placed in a dialysis bag with an MWCO of 5000 and suspended in the corresponding buffer medium (9 mL) in the polyethylene (PE) tube. The release systems were placed in a vapor-bathing constant temperature vibrator at 37 oC. At each predetermined time interval, 1 mL of outer phase buffer medium from different systems was taken out and measured by UV-vis spectroscopy at 480 nm, then the same volume of the corresponding buffer medium was replenished. 2.5. Cell culture U87MG cells were regularly cultured and passaged in fresh DMEM with 10% FBS, 100 U/mL 7

penicillin, and 100 U/mL streptomycin at 37 oC in a 5% CO2 incubator. The U87MG cells cultured in RGD-free medium were identified as U87MG-HR, which have high expression of αvβ3 integrin, while U87MG cells cultured in medium containing free RGD ([RGD] = 2 µM) for 3 h were identified as U87MG-LR, which have low expression of αvβ3 integrin. Without specific declaration, U87MG cells always represent U87MG-HR cells. 2.6. In vitro cytotoxicity and cellular uptake assays CCK8 assay was used to evaluate the cytotoxicity of the HMSs@Au-PEG-RGD NSs at different concentrations according to protocols described in the literature [7]. Furthermore, the morphology of cell cytoskeleton and cell nuclei was observed using a Carl Zeiss LSM 700 confocal laser scanning microscopy (CLSM, Jena, Germany) according to the literature [7]. The coverslips were placed in 12-well plate and soaked using DMEM for 24 h. The U87MG cells were seeded into each well with coverslip at a density of 2.0 × 105 cells/well and incubated overnight, then the cells were incubated with 500 µL fresh medium containing saline (50 µL) or HMSs@Au-PEG-RGD NSs (50 µL) with different concentrations (50-500 µg/mL) for another 24 h. The culture medium was removed and each well was washed 3 times with saline, and the cells were fixed with fresh paraformaldehyde (4.0%) for 30 min at room temperature. Then, the cells were permeabilized with 0.1% Triton® X-100 in saline for 10 min and blocked with 1% bovine serum albumin (BSA) in saline for 30 min. The cells were subjected to F-actin staining with FITC-phalloidin for 30 min and DAPI for 5 min before CLSM imaging. After each step of processing, the cells were washed 3 times with saline. Specific cellular uptake of the HMSs@Au-PEG-RGD NSs/DOX within U87MG-HR or U87MG-LR cells was demonstrated by CLSM imaging and quantitative ICP-OES analysis according to the literature [49, 50]. 8

2.7. In vitro chemotherapy and photothermal ablation of U87MG cells U87MG cells were seeded into a 96-well plate at a density of 0.8 × 10 4 cells/well with fresh DMEM. After overnight incubation, the medium was replaced with 100 µL of fresh medium containing 10 µL of saline, HMSs@Au-PEG-RGD NSs or HMSs@Au-PEG-RGD NSs/DOX dispersed in saline with the final Au concentrations ranging from 0.1 to 0.8 mM and the molar ratio of DOX/Au at 1:16 for the HMSs@Au-PEG-RGD NSs/DOX. The DOX concentration of HMSs@Au-PEG-RGD NSs/DOX was from 0.006 to 0.050 mM based on the Au concentration. After cultured for another 12 h, the cells were washed 3 times with saline and irradiated by an 808 nm laser with a power density of 1.2 W/cm2 for 10 min. For comparison, the control groups received no laser irradiation. After another 2 h incubation, the cell viability was evaluated via CCK8 assay as described above. The viability of U87MG cells was also qualitatively evaluated by DAPI staining using a fluorescence microscope (Carl Zeiss, Axio Vert. A1, Jena, Germany). The U87MG cells (0.8 × 10 4 cell/well) were seeded into each well of 96-well plate and incubated overnight. After the cells was incubated with 100 µL fresh medium containing 10 µL of saline, HMSs@Au-PEG-RGD NSs or HMSs@Au-PEG-RGD NSs/DOX with the final Au concentrations of 0.4 mM for another 12 h. The cells were treated according to the above procedure, fixed with glutaraldehyde (2.5%) for 15 min at 4 oC, and counterstained with DAPI for 5 min. For comparison, the control groups received no laser irradiation. To further prove the efficacy of chemo- and photothermal therapy, the morphology of cytoskeleton and cell nuclei of U87MG cells after different treatments was observed by CLSM according to protocols described above. The differences were that the cell seeding density was 0.5 × 105 cells/well and the cells were incubated with 500 µL fresh medium containing saline (50 µL), 9

HMSs@Au-PEG-RGD NSs (50 µL) or HMSs@Au-PEG-RGD NSs/DOX (50 µL) with the final Au concentration of 0.4 mM for another 12 h. 2.8. In vivo chemo- and photothermal therapy of tumors All animal experiments were performed following the protocols approved by the institutional committee for animal care and the policy of the National Ministry of Health. According to a previous protocol [51], mice-bearing U87MG tumor model was established to have a tumor volume of 0.07-0.12 cm3. The tumor mice were randomly divided into six groups with five mice in each group: Saline, Saline + Laser, HMSs@Au-PEG-RGD NSs, HMSs@Au-PEG-RGD NSs + Laser, HMSs@Au-PEG-RGD NSs/DOX and HMSs@Au-PEG-RGD NSs/DOX + Laser. Saline (100 µL), HMSs@Au-PEG-RGD NSs or HMSs@Au-PEG-RGD NSs/DOX dispersed in saline ([Au] = 30 mM, 100 µL) were intratumorally injected and at 30 min postinjection laser irradiation was performed using an 808 nm NIR laser at a power density of 1.2 W/cm2 for 5 min. After 5 days, the tumor mice were treated again under the same conditions. At each time point postinjection, the tumor volume, body weight and survival rate of all mice were recorded and the pictures of mice were taken by digital camera. The tumor volumes (VT) and the survival rate (ηSR) were evaluated by equations (2) and (3): VT = 0.5 × L A × L2B

(2)

η SR = N 1 N × 100%

(3)

Where LA and LB are the length and width of tumor, N1 and N are the number of surviving mice and total mice in each group, respectively. 2.9. TUNEL staining

Standard TdT-mediated dUTP Nick-End Labeling (TUNEL) staining tests were performed by protocols described in our previous work [48]. U87MG tumor-bearing nude mice were laser 10

irradiated for 10 min after different treatments, and euthanized at 4 h postinjection. Then the tumor in each group was removed, fixed in 10% formalin, sectioned, and stained. The morphology of tumor sections of each group was observed using a Leica DM IL LED inverted phase contrast microscope, and the tumor cell apoptosis rate was quantified. The percentage of TUNEL-positive cells in each sample was determined from five random selected fields. 2.10. Statistical analysis

One-way analysis of variance (ANOVA) statistical method was performed to evaluate the experimental data. A value of 0.05 was selected as the significance level and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.

3. Results and discussion 3.1. Synthesis and characterization of HMSs@Au-PEG-RGD NSs

We followed the protocols reported in our previous work to prepare HMSs@Au NSs [49]. The HMSs@Au NSs were characterized by TEM (Figure 2a and Figure S1, Supporting Information), and Au NSs (~ 60 nm) were successfully coated on the surface of the HMSs-SH. The formation of Au NSs onto the surface of the HMSs was further confirmed by UV-vis spectroscopy, where a strong surface plasmon resonance (SPR) peak at 816 nm emerges (Figure 2b). To modify RGD peptide onto the surface of HMSs@Au NSs. SH-PEG-RGD was first synthesized by reacting SH-PEG-COOH with RGD peptide via EDC chemistry. Through NMR integration, the number of RGD peptide conjugated to each PEG was calculated to be 0.4 (Figure S2, Supporting Information). The SH-PEG-RGD was then reacted with the HMSs@Au NSs via Au-S bond according to the previous report [48]. The amount of SH-PEG-COOH modified onto the HMSs@Au NSs was quantified by TGA (Figure 2c). It is apparent that the weight loss of 11

HMSs@Au-PEG-RGD NSs was calculated to be 63.2% at the temperature up to 700 oC, while the weight loss of pristine HMSs@Au NSs was determined to be 2.4%. According to the results of TGA data, the percentage of SH-PEG-COOH modified onto the HMSs@Au NSs was calculated to be 60.8%. After the whole modification, the loading of Au NSs onto the HMSs@Au-PEG-RGD NSs was quantified by ICP-OES to be 324.96 µg/mg. 3.2. Photothermal properties of the HMSs@Au-PEG-RGD NSs

The excellent NIR absorption feature renders the HMSs@Au-PEG-RGD NSs with photothermal

property.

The

temperature

changes

of

water,

HMSs@Au

seed

and

HMSs@Au-PEG-RGD NSs solution at different Au concentrations (1.0-20.0 mM) under an 808 nm laser irradiation (1.2 W/cm2) were monitored for 350 s (Figure 3a). Clearly, the temperature of HMSs@Au-PEG-RGD NSs solution significantly increases as a function of time, and the increasing tendency is enhanced with the increase of Au concentration. At an Au concentration of 20 mM, the temperature of HMSs@Au-PEG-RGD NSs solution can dramatically increase from 22 oC to 62.4 oC. The temperature change (∆T) of HMSs@Au-PEG-RGD NSs solution at different Au concentrations from 1.0 to 20.0 mM was quantified to be 20.1, 27.3, 31.5, 34.6 and 40.4 oC, respectively (Figure S3, Supporting Information). In comparison, the temperature of the water and HMSs@Au seed solution just slightly increases under the same experimental conditions, and their maximum ∆Ts is less than 6 o

C. Furthermore, under 808 nm laser irradiation for 5 min, the HMSs@Au-PEG-RGD NSs solution

([Au] = 1 mM, in 1 mL water) can be thermally imaged using an infrared camera (Figure S4, Supporting Information), suggesting their potential for in vivo thermal imaging applications. The photothermal conversion efficiency (ηPTC) of the HMSs@Au-PEG-RGD NSs solution was further measured. The aqueous solution of HMSs@Au-PEG-RGD NSs ([Au] = 1 mM) was first irradiated for 290 s, then the laser was turned off (Figure 3b). The rapid cooling of the 12

HMSs@Au-PEG-RGD NSs solution suggests the excellent thermal conversion performance of the HMSs@Au-PEG-RGD NSs, which can be linearly fitted in the typical photothermal profile (Figure 3c). The ηPTC of HMSs@Au-PEG-RGD NSs was calculated to be 70.8%, which is higher than that of HAPP (67.1%) reported

in our previous work [49]. The increase in ηPTC of

HMSs@Au-PEG-RGD NSs may be due to the higher loading amount of Au NSs on the surface of HMSs. In addition, the photothermal stability of the HMSs@Au-PEG-RGD NSs in aqueous suspension was evaluated by several cycles of laser irradiation/cooling down. The temperature of the HMSs@Au-PEG-RGD NSs solution is able to reach 52.8 oC from the initial 22.7 oC for at least 4 cycles

of

laser

irradiation/cooling

down

(Figure

S5,

Supporting

Information).

These results indicate that the HMSs@Au-PEG-RGD NSs possess an excellent photothermal stability, which is of significance for their PTT applications. 3.3. In vitro cytotoxicity of the HMSs@Au-PEG-RGD NSs

It is necessary to test the cytocompatibility of the HMSs@Au-PEG-RGD NSs by CCK8 cell proliferation assay before their biomedical applications (Figure 3d). The results show that the viability of U87MG cells treated with HMSs@Au-PEG-RGD NSs do not have any significant changes in the concentration ranging from 50 to 500 µg/mL ([Au] = 0.08-0.8 mM), similar to the control of saline. This implies that the developed HMSs@Au-PEG-RGD NSs are quite cytocompatible in the studied concentration range. The integrity of cytoskeleton and cell nuclei of U87MG cells treated with the HMSs@Au-PEG-RGD NSs at different concentrations (from 50 to 500 µg/mL) was further observed to qualitatively confirm the cytocompatibility of the particles (Figure 3e). It is clear that the U87MG cells treated with the HMSs@Au-PEG-RGD NSs at different concentrations do not show any 13

appreciable morphological changes in terms of the cytoskeleton (green) and cell nuclei (blue) when compared to those control cells treated with saline. 3.4. Formation and release property of the HMSs@Au-PEG-RGD NSs/DOX complexes

Then, the formed HMSs@Au-PEG-RGD NSs were used to encapsulate DOX, which can be confirmed

by

UV-vis

spectroscopy

(Figure

4a).

Clearly,

in

comparison

with

the

HMSs@Au-PEG-RGD NSs before DOX encapsulation, the HMSs@Au-PEG-RGD NSs/DOX complexes exhibit a 480 nm absorption peak, which is associated to the encapsulated DOX. The encapsulation efficiency of DOX (ηDOX) was calculated to be 98.6±0.7% according to a standard calibration curve (Figure S6, Supporting Information). The DOX release kinetics from HMSs@Au-PEG-RGD NSs/DOX were explored under different pH conditions or laser irradiation (Figure 4b). It can be found that DOX is slowly released and the cumulative release is only 13.3% after 8 h in the PBS (pH = 7.4). In contrast, the DOX is quickly released and the cumulative release reaches 43.4% for 8 h in acetate buffer (pH = 5.0). The pH-responsive release of DOX from the HMSs@Au-PEG-RGD NSs/DOX complexes could be due to the fact that DOX·HCl has a better water solubility under acidic conditions than under basic pH conditions, thus having increased potential to be released from the platform, in agreement with the literature [52, 53]. Thus, HMSs@Au-PEG-RGD NSs/DOX may be a promising drug delivery system, which is beneficial for the inhibition of cancer cells due to the different pH environments between normal tissues (pH = 7.4) and tumor tissue (pH = 5-6). Furthermore, the DOX release under NIR laser irradiation at pH 5.0 was also tested (Figure 4b). Clearly, once the HMSs@Au-PEG-RGD NSs/DOX complexes in aqueous suspension were irradiated under an 808 nm NIR laser for the 5 min intervals, the amount of DOX release was dramatically increased. After 8 h, the cumulative DOX release reached 64.7%. The phenomenon of 14

enhanced DOX release is likely attributed to the NIR-induced photothermal conversion property that is able to heat the drug carrier to increase the diffusion rate of DOX due to the heat transfer, in accordance with the literature [41]. In the context, it is not necessary to make the mesoporous silica-based nanoplatform decompose, since the carrier itself has pores that allow the drug to diffuse out. Our data suggest that the developed HMSs@Au-PEG-RGD NSs/DOX complexes possess a pH/NIR laser dual responsive drug release behavior, which is beneficial for combination of chemotherapy and PTT of tumors. 3.5. In vitro RGD-targeted cellular uptake

The RGD-mediated specific targeting of the HMSs@Au-PEG-RGD NSs/DOX complexes to αvβ3 integrin-expressing cells was next explored by CLSM imaging by virtue of the red fluorescence of DOX (Figure 5). It is apparent that with the increase of HMSs@Au-PEG-RGD NSs/DOX concentration, the signal of red fluorescence gradually enhances in the cytoplasma and cell nuclei. Importantly, the red fluorescence intensity of DOX in cytoplasma and cell nuclei of U87MG-HR cells is higher than that of U87MG-LR cells at the same concentrations, demonstrating the RGD-mediated targeting specificity. In order to further analyze the endocytosis of DOX, large scale fluorescence microscopic images showing more cells in different fields are also given (Figure S7, Supporting Information). Clearly, HMSs@Au-PEG-RGD NSs/DOX complexes are able to be endocytosed in the cytoplasma and cell nuclei of both U87MG-HR and U87MG-LR cells regardless of the surface αvβ3 integrin expression level. However, the red fluorescence intensity of each U87MG-HR cell is significantly higher than that of each U87MG-HR cell, implying that the amount of DOX uptaken by the U87MG-HR cells is significantly higher than that by the U87MG-LR cells. The targeting specificity of the HMSs@Au-PEG-RGD NSs/DOX complexes was further 15

evaluated by quantitative ICP-OES analysis of the Au uptake in U87MG-HR or U87MG-LR cells (Figure 6a). The Au uptake in the U87MG-HR cells is 1.3, 1.5 and 1.6 times higher than that in U87MG-LR cells (p < 0.01) at the particle concentration of 100, 50 and 25 µg/mL, respectively. Taken together, the modification of RGD peptide renders the HMSs@Au-PEG-RGD NSs/DOX complexes with targeting specificity to αvβ3 intergrin-overexpressing U87MG cells. 3.6. Combination chemotherapy and PTT of cancer cells in vitro

To explore the effectiveness of combination chemotherapy and PTT of U87MG cells in vitro, the proliferation of cells treated with saline, HMSs@Au-PEG-RGD NSs or HMSs@Au-PEG-RGD NSs/DOX at different Au concentrations (0.1-0.8 mM) with/without 808 nm laser (1.2 W/cm2, 10 min) was evaluated by CCK8 assay (Figure 6b). It is noted that the cellular Au uptake was performed after 6 h incubation of the nanoplatform, while the therapeutic efficacy of the particles were tested after 12 h incubation. This is because internationalization of particles in cells can occur at 6 h and the encapsulated DOX can exert its therapeutic efficacy after 12 h according to the literature [54]. Compared to the single mode of PTT treatment using the HMSs@Au-PEG-RGD NSs with laser irradiation and single mode of chemotherapy treatment using the HMSs@Au-PEG-RGD NSs/DOX complexes without laser, the treatment of HMSs@Au-PEG-RGD NSs/DOX with laser irradiation is able to significantly enhance the therapeutic efficacy (p < 0.05, for Au concentrations of 0.2, 0.4, and 0.8 mM). The cancer cell inhibition trend is enhanced with the Au concentration for each single mode or bimode treatment. In contrast, the U87MG cells treated with saline, saline + laser, and HMSs@Au-PEG-RGD NSs without laser do not seem to have decreased viability. At the Au concentration of 0.8 mM (equivalent to DOX concentration of 0.05 mM), the viability of U87MG cells treated with the HMSs@Au-PEG-RGD NSs/DOX complexes with laser irradiation decreases to 6.2%, which is significantly lower than that treated with the HMSs@Au-PEG-RGD 16

NSs with laser irradiation (56.2%, p < 0.001) or HMSs@Au-PEG-RGD NSs/DOX without laser (12.8%, p < 0.05). It can be found that the HMSs@Au-PEG-RGD NSs/DOX with laser irradiation show a higher potency to inhibit the proliferation of cancer cells when compared to the chemotherapy or PTT treatment alone. It is worth noting that due to the fact that the cells were incubated with the HMSs@Au-PEG-RGD NSs for 12 h, laser irradiated for 10 min, then incubated for 2 h before cell viability analysis, the efficacy of single mode PTT in vitro was worse than that of single mode chemotherapy, where the particle/DOX complexes used to treat the cells lasted for a longer time period. Hence the therapeutic efficacy of single mode PTT and chemotherapy in vitro is different than that in vivo (see below). The enhanced efficiacy of combination chemotherapy and PTT of cancer cells using the HMSs@Au-PEG-RGD NSs/DOX complexes with laser irradiation was further assessed by fluorescence microscopic observation of the morphology of U87MG cells (Figure 6c). Clearly, cells treated with saline, saline + laser, and HMSs@Au-PEG-RGD NSs without laser display regular cell shapes with cell nuclei being stained with DAPI and the living cells stained with a bright blue fluorescence can be seen in the entire region of the cell well. In contrast, cells treated with MSs@Au-PEG-RGD NSs + Laser and HMSs@Au-PEG-RGD NSs/DOX display dark regions, which is associated to the dead cells exfoliated from the bottom of cell wells. Significantly, the synergetic chemotherapy and PTT occurred in the HMSs@Au-PEG-RGD NSs/DOX + Laser group and only the smallest amount of cells are alive. To further illustrate the mechanism of chemotherapy and PTT of cancer cells, the cytoskeleton and cell nuclei of U87MG cells after different treatments were visualized via CLSM (Figure 6d). The cell functions are reflected by the cytoskeleton and cell nuclei [55, 56], and the chemotherapy and PTT of cancer cells may result in the cytoskeleton disruption and oxidative DNA damage due to 17

the intracellular hyperthermia [7] and the inhibition effect of DOX in the nucleus [57], respectively. Clearly, the cells exhibit a significant distruption of actin stress fibers in both the HMSs@Au-PEG-RGD NSs + Laser and HMSs@Au-PEG-RGD NSs/DOX + Laser groups, and the cells nuclei become dimmed in both HMSs@Au-PEG-RGD NSs/DOX and HMSs@Au-PEG-RGD NSs/DOX + Laser group (Figure S8, Supporting Information). For comparison, the cells in control groups display intact cytoskeleton and nuclei. Our data suggest that the PTT treatment is to kill the cancer cells via the destruction of the cytoskeleton, while the chemotherapy is to inhibit the cancer cells via the damage of the nuclei even if the cytoskeletion remains relatively intact. Therefore, the treatment of HMSs@Au-PEG-RGD NSs/DOX under laser irradiation is able to exert combination therapeutic efficiacy of cancer cells in vitro by combination chemotherapy and PTT. 3.7. Combination chemotherapy and PTT of a xenograft tumor model in vivo

We next explored the use of HMSs@Au-PEG-RGD NSs/DOX for combination chemotherapy and PTT of tumors in vivo (Figure 7a-c). It can be seen that the tumors in the Saline, Saline + Laser, and HMSs@Au-PEG-RGD NSs groups gradually grow with the time postinjection (Figure 7a and Figure S9, Supporting Information). In contrast, the tumors treated with the HMSs@Au-PEG-RGD NSs + Laser group and HMSs@Au-PEG-RGD NSs/DOX group are able to be inhibited to some extent. Notably, the tumors in HMSs@Au-PEG-RGD NSs/DOX + Laser group are able to be completely ablated at day 12 post-treatment and the tumor did not recur during the experimental time period. Apprarently, the tumor inhibition efficacy follows the order of HMSs@Au-PEG-RGD NSs/DOX + Laser > HMSs@Au-PEG-RGD NSs + Laser > HMSs@Au-PEG-RGD NSs/DOX. The single PTT treatment using the HMSs@Au-PEG-RGD NSs is better than the single chemotherapy treatment using HMSs@Au-PEG-RGD NSs/DOX, which is different from the in vitro results. This difference should be due to the difference in the treatments, where laser irradiation in vivo was 18

performed for two times on Day 0 and Day 5, and the evaluation of tumor inhibition was performed in a much longer time period. It should be noted that in our work, we just used a xenografted brain tumor model to test our hypothesis that combined chemotherapy/PTT is more effective than single mode of chemotherapy and PTT. The work related to combined chemotherapy/PTT of an orthotopic brain tumor model is under way in our laboratories. In addition, the body weight of the tumor mice in the HMSs@Au-PEG-RGD NSs/DOX + Laser group well maintained, indicating that the HMSs@Au-PEG-RGD NSs/DOX + Laser treatment negligiblely exerts side-effects (Figure 7b), similar to other treatments. In order to further study the combination chemotherapy and PTT efficacy of the HMSs@Au-PEG-RGD NSs/DOX with laser irradiation, the mouse survival rate of the six groups was measured. It is clear that the mice treated with the HMSs@Au-PEG-RGD NSs/DOX + Laser group display a 100% survival rate after 80 days, while mice in the Saline, Saline + Laser, or HMSs@Au-PEG-RGD NSs groups are all dead on day 70, day 76, and day 73. Quantatitive data show that the survival rate of mice in the HMSs@Au-PEG-RGD NSs + Laser group and HMSs@Au-PEG-RGD NSs/DOX group are only 40% and 60% at 80 days post-treatment, respectively (Figure 7c). These results demonstrate that the combined chemotherapy and PTT exerts a higher potency in treating tumors than each single mode of chemotherapy and PTT. 3.8. Histological examinations

The combination chemotherapy and PTT of tumors using the HMSs@Au-PEG-RGD NSs/DOX under laser irradiation was further confirmed by TUNEL staining of the tumor sections (Figure 7d). The tumors treated with Saline, Saline + Laser or HMSs@Au-PEG-RGD NSs without laser display rare apoptotic cells with positive staining. However, a larger area of apoptotic cells with positive staining can be seen in the tumors treated with the HMSs@Au-PEG-RGD NSs/DOX + Laser, 19

HMSs@Au-PEG-RGD NSs + Laser, or HMSs@Au-PEG-RGD NSs/DOX. Quantitative analysis of the cell apoptosis rate shows that the apoptosis rate follows the order of HMSs@Au-PEG-RGD NSs/DOX + Laser (85.7%) >

HMSs@Au-PEG-RGD NSs + Laser group (76.1%) >

HMSs@Au-PEG-RGD NSs/DOX (63.3%) (Figure S10, Supporting Information). These results suggest that the developed HMSs@Au-PEG-RGD NSs/DOX under laser irradiation are able to be used as a highly efficient nanoplatform for combination chemotherapy and PTT of tumors in vivo.

4. Conclusion To conclude, we demonstrate a convenient approach to preparing a novel drug delivery system of HMSs@Au-PEG-RGD NSs/DOX for combination chemotherapy and PTT of tumors. The developed Au NS-coated HMS capsules have both merits of HMS capsules that can be used for high payload drug loading and Au NSs that have NIR laser-induced photothermal conversion efficiency (70.8%) and can be used for PTT of tumors. With the RGD-mediated targeting specificity and the pH/NIR laser dual-responsive drug delivery performance, the developed HMSs@Au-PEG-RGD NSs/DOX complexes are able to specifically target αvβ3 intergrin-overexpressing cells, and exert combination chemotherapy and PTT of cancer cells in vitro and a subcutaneous tumor model in vivo. Overall, our study implies that the developed HMSs@Au-PEG-RGD NSs/DOX may be used as a multifunctional nanoplatform for combination chemotherapy and PTT of different types of tumors.

Acknowledgements

This research is financially supported by the Science and Technology Commission of Shanghai Municipality (15520711400 and 17540712000), the support from the Fundamental Research Funds for the Central Universities (X. Li, X. Shi and M. Shen), the National Natural Science Foundation of China (81571679 and 81271596), and the Program for Professor of Special Appointment (Eastern 20

Scholar) at Shanghai Institutions of Higher Learning. X. Shi acknowledges the support by FCT-Foundation for Science and Technology (project PEst-OE/QUI/UI0674/2013, CQM, Portuguese Government funds) and funding through the project M1420-01-0145-FEDER-000005 Madeira Chemistry Center - CQM + (Madeira 14-20).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2017.00.000.

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Figure captions Figure 1. Schematic illustration of the synthesis of HS-PEG-RGD, HMSs@Au-PEG-RGD NSs, and

HMSs@Au-PEG-RGD NSs/DOX complexes. Figure 2. (a) High-resolution TEM image of HMSs@Au NSs. (b) UV-vis spectrum of HMSs@Au

NSs. Inset shows the picture of the water solution of the HMSs@Au NSs. (c) TGA curves of the HMSs@Au NSs and HMSs@Au-PEG-RGD NSs. Figure 3. (a) Temperature elevation of water and the aqueous solution of the HMSs@Au seed and

HMSs@Au-PEG-RGD NSs at different Au concentrations (1, 5, 10 and 20 mM, respectively) under the irradiation of an 808 nm laser (1.2 W/cm2) as a function of irradiation time. (b) Photothermal effect of an aqueous solution of HMSs@Au-PEG-RGD NSs irradiated by an 808 nm laser (1.2 W/cm2). The laser was turned off after irradiation for 290 s. (c) Plot of cooling time (after 290 s) versus negative natural logarithm of the driving force temperature obtained from cooling stage. (d) CCK8 proliferation assay of U87MG cells treated with the HMSs@Au-PEG-RGD NSs at different concentrations for 24 h. The cells treated with saline were used as control and the data were expressed as mean ± SD (n=3). (e) CLSM images of cells with cytoskeleton stained by FITC-phalloidin (green) and nuclei stained by DAPI (blue) after the cells were incubated with the HMSs@Au-PEG-RGD NSs at different concentrations for 24 h. Figure 4. (a) UV-vis spectra of HMSs@Au-PEG-RGD NSs and HMSs@Au-PEG-RGD NSs/DOX

complexes. (b) Time-dependent release of DOX from the HMSs@Au-PEG-RGD NSs/DOX complexes dispersed in PBS (pH = 7.4), and acetate buffer (pH = 5.0) without or with an 808 nm NIR laser irradiation for 5 min (1.2 W/cm-2). Figure 5. CLSM images of the U87MG-HR (a, c, e) and U87MG-LR (b, d, f) cells incubated with

HMSs@Au-PEG-RGD NSs/DOX under different concentrations: 100 µg/mL (a, b), 50 µg/mL(c, d), and 25 µg/mL (e, f) for 6 h. Figure

6.

(a)

Au

uptake

in

U87MG-HR

and

U87MG-LR

cells

treated

with

the

HMSs@Au-PEG-RGD NSs/DOX at different concentrations for 6 h. (b) CCK8 proliferation assay 27

of the U87MG cells after different treatments for 12 h, followed by irradiation with an NIR laser for 10 min (only for laser groups). (c) Fluorescence microscopic images of cells with nuclei stained by DAPI (blue) after the cells received different treatments for 12 h, followed by irradiation with an NIR laser for 10 min (only for laser groups). (d) CLSM images of cells with cytoskeleton stained by FITC-phalloidin (green) and nuclei stained by DAPI (blue) after the cells were differently treated for 12 h, followed by laser irradiation for 10 min (only for laser groups). Figure 7. (a) The relative tumor volume, (b) body weight, and (c) survival rate of U87MG

tumor-bearing mice as a function of time post treatment (red arrow in a and b represents the point of the sample injection). (d) Representative TUNEL images of the xenografted U87MG tumor sections after different treatments.

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Figure 7 Li et al.

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Statement of Significance We demonstrate a convenient approach to preparing a novel RGD-targeted drug delivery system of HMSs@Au-PEG-RGD NSs/DOX that possesses pH/NIR laser dual-responsive drug delivery performance for combinational chemotherapy and PTT of tumors. The developed Au NS-coated HMS capsules have both merits of HMS capsules that can be used for high payload drug loading and Au NSs that have NIR laser-induced photothermal conversion efficiency (70.8%) and can be used for PTT of tumors.

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