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Enzyme responsive drug delivery system based on mesoporous silica nanoparticles for tumor therapy in vivo

This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 145102 (http://iopscience.iop.org/0957-4484/26/14/145102) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 128.138.73.68 This content was downloaded on 29/04/2017 at 12:47 Please note that terms and conditions apply.

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Nanotechnology Nanotechnology 26 (2015) 145102 (14pp)

doi:10.1088/0957-4484/26/14/145102

Enzyme responsive drug delivery system based on mesoporous silica nanoparticles for tumor therapy in vivo Yun Liu1,2, Xingwei Ding1, Jinghua Li1, Zhong Luo1, Yan Hu1, Junjie Liu1, Liangliang Dai1, Jun Zhou1, Changjun Hou1 and Kaiyong Cai1 1

Key Laboratory of Biorheological Science and Technology, Ministry of Education College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China 2 Research Center for Medicine and Biology, Zunyi Medical University, Zunyi 563003, People’s Republic of China E-mail: [email protected] Received 25 November 2014, revised 25 February 2015 Accepted for publication 26 February 2015 Published 19 March 2015 Abstract

To reduce the toxic side effects of traditional chemotherapeutics in vivo, we designed and constructed a biocompatible, matrix metalloproteinases (MMPs) responsive drug delivery system based on mesoporous silica nanoparticles (MSNs). MMPs substrate peptide containing PLGLAR (sensitive to MMPs) was immobilized onto the surfaces of amino-functionalized MSNs via an amidation reaction, serving as MMPs sensitive intermediate linker. Bovine serum albumin was then covalently coupled to linker as end-cap for sealing the mesopores of MSNs. Lactobionic acid was further conjugated to the system as targeting motif. Doxorubicin hydrochloride was used as the model anticancer drug in this study. A series of characterizations revealed that the system was successfully constructed. The peptide-functionalized MSNs system demonstrated relatively high sensitivity to MMPs for triggering drug delivery, which was potentially important for tumor therapy since the tumor’s microenvironment overexpressed MMPs in nature. The in vivo experiments proved that the system could efficiently inhibit the tumor growth with minimal side effects. This study provides an approach for the development of the next generation of nanotherapeutics toward efficient cancer treatment. S Online supplementary data available from stacks.iop.org/NANO/26/145102/mmedia Keywords: mesoporous silica nanoparticles, matrix metalloprotease, drug delivery system, tumor microenvironment, in vivo (Some figures may appear in colour only in the online journal) 1. Introduction

solutions was to exploit desired nanocarriers for efficient drug delivery [5–7]. Recently, mesoporous silica nanoparticles (MSNs) attracted much attention as one of the promising drug delivery nanocarriers for potential clinical application [7–11]. MSNs have many advantages, such as good biocompatibility, large surface area, meso-structure, tunable pore size, and plenty of active chemical groups for surface functionalization [4, 12, 13]. As for constructing a MSNs-based drug delivery system, a critical issue is how to ‘seal’ the mesopores of MSNs with desirable sealants and then ‘open’ the mesopores in

Malignant tumors (liver, breast and lung tumors etc) are one of the most fatal diseases worldwide [1]. Although a variety of chemotherapeutics have been invented for treating malignant tumor, these drugs are not widely used due to their damaging side effects on normal organs of a host [2, 3]. During the past decade, the rapid development of nanomedicine provided nanotechnology-based potential solutions for tumor patients, by enhancing the bioavailability of the tumortreating drugs and reducing their side effects [4]. One of the 0957-4484/15/145102+14$33.00

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© 2015 IOP Publishing Ltd Printed in the UK

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responding to biological signals or external stimuli for controlled drug release [14–16]. Generally, end-caps are used to seal the mesopores of MSNs for preventing the leakage of anticancer drugs on their way heading for tumor sites from the system. The efficient end-capping of MSNs thus could reduce the toxic side effects of anticancer drugs on other major organs of the host. Previously, inorganic nanoparticles [17– 19], pseudorotaxanes [20], polymers [21–23] and cyclodextrin [15] etc were employed as end-caps to seal the mesopores of MSNs. In our previous studies, we used macromolecular proteins (collagen, bovine serum albumin (BSA) etc) as endcaps to construct MSNs-based drug delivery system [14, 24]. Meanwhile, different stimuli such as redox reaction [24–26], pH [14, 17, 27], photoirradiation [28, 29], temperature [30], and magnetic actuation [18] etc were investigated for triggering the controlled drug release from MSNs systems. However, most of the stimuli are external triggers, which are difficult to be applied in vivo [31]. Thus, to investigate inherent biological signals deriving from tumor of a host for controlling drug release attracted much attention in the field of cancer therapy. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes, which are over-expressed in the microenvironment of certain kinds of tumors, in particular of liver and colon tumors [32–35]. MMPs could degrade the extracellular matrix via cleaving peptide substrate. The majority of the cleavage sites are the peptide bond between glycine and leucine. Considering the distinguishing feature of MMPs, to develop MMPs-responsive drug delivery systems attracted much attention in recent years [31, 36, 37]. For instance, Zhang et al developed MMP-2 responsive and tumor-atrgeting drug delivery system based on envelope-type MSNs with clinic application potential [36]. Mallik et al exploited MMP9 responsive PEG cleavable nanocarrier for drug delivery [38]. Thus, more reated work need to be conducted to exploit the MMPs responsive drug delivery systems. In this study, we report an approach for the fabrication of a MMPs responsive controlled drug release system based on MSNs for antitumor drug of doxorubicin hydrochloride (DOX) delivery within the microenvironment of tumor tissue in vivo, by using BSA as end-cap, peptide substrate of MMPs (PLGLAR) as intermediate linker and lactobionic acid (LA) as targeting moiety (figure 1(A)). The reason for choosing BSA as end-capping agent was that it beneficial for reducing the immunotoxicity of MSNs [39]. After vein injection, the DOX loaded MSNs would accumulate at liver tumor site via targeting or enhanced permeability and retention (EPR) effect [40–42]. We thus hypothesized that the system would efficiently deliver DOX to tumor tissue triggering by MMPs for tumor growth inhibition in vivo.

carbodiimide-HCl (EDC), N-hydroxysuccinimide (NHS), 3aminopropyltriethoxysilane (APTS) and fluorescein isothiocynate (FITC) were provided by Sigma-Aldrich (Shanghai, China). LA, 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU), DOX and N,N-Diisopropylethylamine (DIEA) were purchased from J&K (Guangzhou, China). Vectashield mounting media with DAPI was supplied by Vector (Burlingame, USA). Cellulose dialysis tubes (MWCO, 7000) were purchased from Solarbio (Beijing, China). MMP-13 and MMP-13 inhibitor were purchased from R&D systems China (Shanghai, China). TRITClabeled tunnel apoptosis assay kit was provided by KeyGEN BioTECH (Nanjing, China). Fmoc-Peptide–COOH (Fmoc-6aminocaproic acid-PLGLARR-6-aminocaproic acid) was synthesized by Zhejiang Hongtuo Biological Technology (Zhejiang, China). All other reagents were provided by Oriental Chemicals (Chongqing, China). 2.2. Synthesis of MSNs and amino-functionalized MSNs (MSNs–NH2)

MCM-41-type MSNs were synthesized according to previous reports with slight change [24, 43]. Briefly, 1.0 g of CTAB and 0.28 g of sodium hydroxide (NaOH) were dissolved into 480 mL deionized water and heated up to 80 °C in 30 min. Then, 5.0 g of TEOS was dropwisely added and stirred vigorously for 2 h until white precipitant was formed. After centrifugation (8000 r min−1,10 min), the crude nanoparticles was rinsed with deionized water and methanol each for six times. Finally, the sample was dried with a vacuum freezer (0.8 mbar) at −50 °C for 24 h. The obtained sample was denoted as MSNs. The as-synthesized MSNs were then amino-functionalized by refluxing 1.0 g of MSNs with 80 mL of anhydrous toluene containing 0.75 mL of APTS at 60 °C for 36 h. The amino-functionalized MSNs (MSNs-NH2) were named as MSNs-NH2. To remove the template surfactant of CTAB, the MSNs-NH2 were refluxed with methanolic solution composed of 7 mL HCl (37.4 wt%) and 120 mL methanol at 80 °C for 48 h. The refluxed MSNs-NH2 were centrifuged and washed with deionized water for six times. Finally, the sample was dried with a vacuum freezer (0.8 mbar) at −50 °C for 24 h. 2.3. Synthesis of functional peptides functionalized MSNs (MSNs-peptide)

The MSNs-peptide was synthesized as follows: firstly, 100 mg of Fmoc-peptide-COOH (Fmoc-NH(CH2)5COPLGLARR-(CH2)5COOH), 100 mg of HATU and 50 μL DIEA were dissolved into 20 mL DCM (containing 10% DMSO v/v) and stirred at room temperature for 30 min. Meanwhile, MSNs-NH2 (400 mg) was dispersed into 40 mL DCM (containing 10% DMSO v/v). Then, these solutions were mixed and reacted at room temperature for 36 h. The resulting solution was subsequently centrifuged (8000 r min−1, 10 min). After centrifugation, the intermediate product of MSNs-Peptide-Fmoc (Fmoc-NH-(CH2)5CO-

2. Experimental section 2.1. Materials

Tetraethylorthosilicate (TEOS), N-cetyltrimethylammonium bromide (CTAB), BSA, 1-ethyl-3-(3-dimethylaminopropyl) 2

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Figure 1. (A) Schematic illustration of MMPs-responsive MSNs-Peptide-BSA-LA@DOX for controlled drug delivery for tumor therapy

in vivo and (B) the synthetic routes of MSNs-Peptide-BSA-LA@DOX.

stirred at room temperature for 24 h. The drug-loaded product was named as MSNs-peptide@DOX. BSA was used as endcapping agent to seal mesopores of MSNs for preventing dug leaking. Briefly, BSA (44 mg), EDC (22 mg) and NHS (10.7 mg) were added to the above solution and stirred at room temperature for 36 h. The sealed MSNs were named as MSNs-peptide-BSA@DOX. To fabricate cell specific targeting MSNs, 100 mg of LA was added to the solution containing MSNs-peptide-BSA@DOX. The LA would covalently couple with MSNs-peptide-BSA@DOX with assistance of coupling agents of EDC/NHS mixture (EDC: 50 mg, NHS: 25 mg). The solution was then stirred at room temperature for another 24 h. Finally, the solution was

PLGLARR-(CH2)5CO-MSNs) was washed with DCM (containing 10% DMSO v/v) for six times. Finally, the Fmoc of the intermediate product was cut off with 30 mL DMF (containing 20% piperidine v/v) for 20 min to obtain MSNsPeptide (NH2-(CH2)5CO-PLGLARR-(CH2)5CO-MSNs). The MSNs-peptide was centrifuged and washed with deionized water for six times and dried with a vacuum freezer (0.8 mbar) at −50 °C for 24 h.

2.4. Fabrication of MSNs-peptide-BSA-LA@DOX

To load DOX, 100 mg of MSNs-peptide and 20 mg of DOX were dispersed into 100 mL MES buffer solution (pH 6.0) and 3

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and 100 μg mL−1 of streptomycin. The culture media was changed every two days.

centrifuged and washed with deionized water for six times, respectively. All supernatants (containing DOX) were mixed and measured for determining the DOX loading reference to a standard calibration curve of DOX. The loading efficiency of MSNs-peptide-BSA@DOX was calculated using the equation as follows [44]:

2.8. Cytotoxicity assay by CCK-8

HepG2 cells were seeded at 8 × 103 cells per well in 96-well plates and pre-incubated for 12 h before cytotoxicity assay. The culture medium was replaced with fresh medium containing DOX (2 μg mL−1), MSNs@DOX, MSNs-PeptideBSA@DOX, MSNs-Peptide-BSA-LA@DOX (40 μg mL−1). After the cells were cultured at 37 °C for another 6, 12 and 24 h, the CCK-8 solution (10 μL) was added to the wells. And the cells were incubated at 37 °C under 5% CO2 atmosphere for another 2 h. Finally, the absorbance values of the solution per well were determined with a spectrophotometric microplate reader (Multiskan Spectrum, Thermo Fisher, USA) at a wavelength of 450 nm [24, 45].

Drug loading(%) ⎡ Total drug added − Drug in supernatant ⎤ =⎢ ⎥ × 100%. ⎣ ⎦ The weight of nanoparticles

2.5. Characterization of MSNs and MSNs-peptide-BSA-LA

Scanning electron microscopy (FESEM, su-8020, Hitachi Japan) and transmission electron microscopy (TEM, 2100f, Jeol Japan) were used to characterize the morphologies of MSNs and MSNs-peptide-BSA-LA, respectively. To monitor the grafting procedures approaching for MSNs-Peptide-BSALA, Fourier transforms infrared spectroscopy (model 6300, Bio-Rad USA) and Zeta potential measurements (Nano ZS90Zetasizer, Malvern Instruments UK) were utilized in this study. Fluorescamine detection was used to qualitatively analyze the contents of NH2 groups grafted to MSNs at each step [24] (Fluorescence microplate reader, Varioskan Flash, Thermo Fisher USA). Brunauer-Emmett-Teller (BET) and Barett-Joyner-Halenda (BJH) (ASAP2020M, Micromeritics Instrument USA) were employed to characterize the surface areas and the pore size distribution of different MSNs particles, respectively. Powder x-ray diffractometer (XRD, xpert pro, PANalytical Holland) was employed to detect the crystalline characteristic of MSNs. A thermal analyzer (thermal gravitational analysis (TGA)/DSC1, stare, Mettler Switzerland) was employed to perform the TGA at 10 °C min−1 under Ar protection.

2.9. Intracellular distribution of nanoparticles

The distribution of nanoparticles within HepG2 cells was observed by TEM [47, 48]. Briefly, HepG2 cells were incubated with MSNs, MSNs-Peptide-BSA and MSNs-PeptideBSA-LA (70 μg mL−1) for 24 h. Next, the cells were centrifuged and fixed with glutaraldehyde (2.5% w/v) at 4 °C for 24 h. The intracellular distribution of nanoparticles within cells was observed by TEM (Hitachi-7500, Hitachi, Japan). The distribution of nanoparticles within HepG2 cells was also studied using confocal laser scanning microscopy (CLSM) [16, 46]. HepG2 cells growing onto a glass slide were incubated with MSNs, MSNs-Peptide-BSA, MSNsPeptide-BSA-LA (FITC-labeled, 70 μg mL−1) at 37 °C for 12 and 24 h, respectively. Next, trypan blue (200 μg mL−1) was added to the media to quench extracellular fluorescence for 10 min. The treated cells were fixed with 4% paraformaldehyde for 20 min. Then, 0.2% Triton X-100 was used to permeabilize the cells at room temperature for 3 min. After that, the treated cells were stained with rhodamine-phalloidin (5 U mL−1) at 37 °C for 1 h. Finally, the stained cells were mounted with 5 μL Vectshield mounting media with DAPI (Alexis, USA) and observed by CLSM (TCS SP2, Leica, Germany).

2.6. MMPs triggered drug release

Cellulose dialysis tubes (MWCO, 7000) were employed to investigate the MMP-13 triggered drug delivery of MSNsPeptide-BSA-LA@DOX. Briefly, MSNs-Peptide-BSALA@DOX (5 mg) was suspended into 5 mL Tris buffer (pH 6.8) containing MMP-13 (5 μg mL−1) with or without presence of MMP-13 inhibitor. Subsequently, dialysis tubes were immersed into 15 mL Tris solution and incubated at 37 °C. At the desired time of interval, 0.5 mL incubation solution was taken out and measured the fluorescence intensity of released DOX from the system at an excitation wavelength of 497 nm and emission wavelength of 595 nm with a fluorescence microplate reader (Varioskan Flash, Thermo Fisher, USA). The same volume of fresh Tris buffer was then added for further experiments.

2.10. Cellular uptake study

Flow cytometry was used to quantitatively characterize the cell uptake of FITC-labeled nanoparticles [40, 46]. In brief, both HepG2 cells and HUVEC cells were cultured into sixwell plates at an initial seeding density of 2 × 104 cells/cm−2, respectively. The cells were then treated with different nanoparticles (30 μg mL−1) for 2 h and 4 h, respectively. After that, trypan blue (200 μg mL−1) was employed to quench the extracellular fluorescence at 37 °C for 10 min. Finally, flow cytometry was employed to determine the cell specific uptake of nanoparticles.

2.7. Cell culture

HepG2 cells and human umbilical vascular endothelial cells (HUVEC) were cultured at 37 °C under 5% CO2 atmosphere in DMEM medium and RPMI1640 medium containing 10% fetal bovine serum (FBS, Gibco), 100 U mL−1 of penicillin 4

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were embedded in paraffin and then sliced. The slices were finally stained with hematoxylin-eosins tainting (H and E) for histological examination.

2.11. Intracellular DOX release

HepG2 cells were seeded in 24-well plates and co-cultured with DOX (1 μg mL−1), MSNs, MSNs@DOX, and MSNsPeptide-BSA-LA@DOX (20 μg mL−1) as the procedures mentioned above. After incubation at 37 °C for 24 h, cells were fixed with 4% paraformaldehyde at 4 °C for 30 min. Subsequently, cells were washed with PBS at room temperature for three times. Finally, cells were mounted with 5 μL Vectshield mounting media with DAPI (Alexis, USA). Cell nuclei were observed by CLSM.

2.16. Statistical analysis

All data were expressed as mean ±standard deviation (SD). The statistical analysis was performed using the Student’s ttest and one-way analysis of variance (ANOVA) (OriginPro, version 8.6) at confidence levels of 95% and 99%.

2.12. Establishment of tumor model

3. Results and discussion

Animal experiments were carried out according to the Animal Management Rules of the Ministry of Health of the People's Republic of China (Document No. 55, 2001) and the Guidelines for the Care and Use of Laboratory Animals of Zunyi Medical University. Normal nude mice (6–8 weeks old, male, average weight 19.2 ± 0.3 g) were purchased from Beijing Huahengkang Biological Technology (Beijing, China) for in vivo studies. HepG2 cells (0.1 mL, 2 × 106 cells in saline) were injected into subcutaneous tissue of the nude mice [49, 50].

3.1. Preparation of MSNs-based drug delivery system

Our approach involved four steps as follows (figure 1(B)): firstly, MSNs were synthesized and surface decorated with APTS to obtain MSNs-NH2 [14, 24, 35]; Secondly, a peptide substrate of MMPs (Fmoc-Peptide-COOH) containing PLGLAR (sensitive to MMPs) was covalently bound to MSNs-NH2 to produce MSNs-Peptide-Fmoc. The two ends of peptide substrate of MMPs (Fmoc-Peptide-COOH) were decorated with 6-aminocaproic acid, and the N-terminal amine was protected by Fmoc. The 6-aminocaproic acid is an isomeride of isoleucine (Ile) with fairly long active arms which would be helpful for the efficient coupling of peptide substrate of MMPs to MSNs-NH2. Moreover, once the Nterminal amine was protected with Fmoc group, cyclization would not occur in peptide substrate; Thirdly, the Fmoc group was cut off by DMF (containing 20% piperidine v/v) to get peptide functionalized MSNs (MSNs-peptide) with free NH2 groups for further reactions; Fourthly, after loading of antitumor drug of DOX by means of simple diffusion equilibrium [15], BSA and LA were subsequently coupled to MSNspeptide for encapsulating the system. Thus, the system of MSNs-Peptide-BSA-LA@DOX was constructed step by step. SEM and TEM were employed to characterize the morphologies of different MSNs. The unfunctionalised MSNs displayed round sphere features with well-defined mesostructures and relatively good dispersion (figures 2(A) and (B)), which was consistent with a previous study [36]. Their average diameters were around 100 nm. XRD analysis showed that the as-synthesized MSNs had highly ordered lattice array (figure 2(C)). After surface modification step by step, although the treated MSNs were not substantially different from the unfunctionalised ones in terms of their morphologies (figure 2(D)), a small faint borderline along the treated MSNs was observed. The result indirectly suggests that Peptide-BSA-LA was immobilized onto MSNs. Similar phenomenon was also observed in our previous studies [14, 24]. To reveal the coupling process, Fourier transform infrared spectroscopy (FTIR) was employed to characterize different MSNs samples. Figure S1 shows the FTIR spectra of MSNs before and after modification at each grafting step. MSNs demonstrated strong absorption signals at 1092 cm−1 and 960 cm−1, mainly due to the asymmetric stretching of Si-

2.13. Treatment of nude mice bearing tumor

When the tumor size of the mice grew to an average volume of around 100 mm3, 20 mice bearing hepatocellular tumor were randomly divided into four groups. The four groups of mice were treated differently by injecting saline, DOX, MSN@DOX, or MSNs-Peptide-BSA-LA@DOX (DOX equivalent, 2 mg kg−1 DOX) via tail vein, three times each week [51, 52]. The tumor volumes and body weight were measured every two days. The tumor volume was calculated according to the following equation [53]: Tumor volume(V) = Length × Width × Width/2.

After treatment for 18 days, all mice were sacrificed to collect tumor tissues and main organs for further study. 2.14. Evaluation of cell apoptosis in tumor tissues

To evaluate cell apoptosis in tumor tissues, the tumors were frozen-sliced and immobilized onto glass slides [21, 54]. The slides were immersed into 4% paraformaldehyde solution at 4 °C for 30 min, and then washed with PBS for three times. After that, the slides were stained according to the protocol of TRITC-labeled tunnel apoptosis assay kit. Subsequently, 5 μL mounting media with DAPI was dropped onto the tissues of each glass slide. Finally, the apoptotic cells in the tumor tissues were observed by CLSM at excitation wavelength of 543 nm and emission wavelength of 571 nm. 2.15. Histological examination of major organs

The major organs (heart, liver, spleen, lung and kidney) of the mice were collected after the sacrifice of the mice. The organs were washed by PBS and immersed into 4% paraformaldehyde solution at 4 °C for 24 h. Subsequently, these organs 5

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Figure 2. Physical property characterization: (A) SEM image of MSNs; (B) TEM image of MSNs; (C) low-angle XRD pattern of MSNs; and

(D) TEM image of MSNs-Peptide-BSA-LA, respectively.

O-Si bridges and skeletal vibration of the C-O bond stretching, respectively (figure S1(a)). Comparing with MSNs, MSNs-NH2 displayed additional absorption peak at 1558 cm−1 (figure S1(b)), which was attributed to the stretching vibration of amide and -NH2 bending [55, 56]. It suggests that NH2 groups were coupled to MSNs. After modification with Fmoc-peptide-COOH, two distinctive absorption peaks at 1659 cm−1 and 1546 cm−1 were observed (figure S1(c)), which were contributed from the stretching vibration of peptide linkage [51]. After removal of the Fmoc protecting groups, the absorption peaks at 1659 cm−1 and 1546 cm−1 for peptide linkage were slightly shifted to 1655 cm−1 and 1542 cm−1, respectively (figure S1(d)). After further immobilization of BSA to MSNs-Peptide, a new peak at 2960 cm−1 was observed (figure S1(e)), which was assigned to skeletal vibration of C-H bonds in BSA molecules. Furthermore, the intensity of peaks at 1658 cm−1 and 1542 cm−1 increased obviously (figure S1(e)). It was contributed to the stretching vibration of abundant peptide linkage of BSA, which was consistent with a previous report [57]. Meanwhile, the intensity of peaks at 3300 cm−1 (OH) increased with the introduction of LA molecules (figure S1(f))

[14]. All results indicate that MSNs-peptide-BSA-LA was successfully fabricated step by step. Furthermore, the process of surface modification of MSNs was characterized by TGA. The TGA curves displayed a progressive decrease tendency in weight after each reaction, indicating the immobilizations of peptide, BSA and LA molecules, as well as DOX loading, respectively. The results indicate that around 5.0 wt% of DOX was loaded into MSNsPeptide-BSA-LA, and the conjugation efficiency of peptide was around 13.1 wt% (figure S2), which might be derived from its relatively high molecular weight (∼1009 Da). Moreover, the drug loading efficiency of MSNs-peptideBSA@DOX was estimated to be around 5.8 wt%, by using a UV-vis measurement method according to a previous study [44]. The result was slightly higher than that of TGA measurement. The successful fabrication of the system was also confirmed by a variety of methods including BET, BJH, zeta potential measurement, and fluorescamine detection, respectively. The sealing of the mesopores of the MSNs with BSA molecules was confirmed by using BET and BJH analysis [14]. After the end-capping process, the BET surface area of 6

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Figure 3. Cumulative release profiles of DOX from MSNs-Peptide-BSA-LA@DOX with the presence MMP-13 or with the presence MMP13 and MMP-13 inhibitor for 120 h (A), and the corresponding enlarged figure for the initial 24 h (B).

the MSNs drastically reduced from 959.47 m2 g−1 to 80.02 m2 g−1 (table S1) [58]. Moreover, the BET pore volumes and BJH pores diameters also decreased along with the treatment processes (figure S3, table S1). In addition, zeta potential results indicated that NH2, Fmoc and BSA molecules were successfully introduced onto MSNs surfaces (table S2). Fluorescamine detection also revealed the related reactions (table S3). 3.2. MMPs enzyme-responsive drug release

To investigate the drug release profiles of MSNs-PeptideBSA-LA@DOX, MMP-13 was used as an external stimulus to trigger the MMPs-responsive release of DOX. The rationale was that MMP-13 was overexpressed in the microenvironment of liver tumor [35, 52]. As shown in figure 3, around 57.9% of DOX released from MSNs-Peptide-BSALA@DOX when the system was exposed to MMP-13 solution for 8 h. In contrast, only around 17.9% of DOX was leaked from the system when the trigger of MMP-13 was inhibited even after incubation for 120 h. Detailed investigations revealed that the relatively good sealing efficiency of the system dominantly contributed to the end-capping with BSA, rather than the conjugation of APTS or peptide (figure S4). The distinct difference suggest that MMP-13 could efficiently break down the intermediate linker (-PLGLAR-), leading to the removal of BSA end-capping agent for quick drug release.

Figure 4. Cytotoxicity assay of MSNs, MSNs-peptide, MSNs-

Peptide-BSA, MSNs-Peptide-BSA-LA, DOX, MSNs@DOX, MSNs-Peptide-BSA@DOX and MSNs-Peptide-BSA-LA@DOX with HepG2 cells.

Next, we observed the cell morphology after various treatments with CLSM [46, 59]. HepG2 cells were treated with DOX and DOX-loaded nanoparticles for 24 h respectively. We found that the nuclei of the HepG2 cells cultured onto tissue culture polystyrene (TCPS, control) and co-cultured with MSNs were in oval or round shape with discernible boundaries (figures S5(a) and (b)). However, the nuclei of the HepG2 cells deformed and ruptured after the cells were treated with DOX, MSNs@DOX, and MSNs-Peptide-BSALA@DOX for 24 h, respectively (figures S5 (c)–(e)). Interestingly, the cell nuclei showed red-blue mixed color after the cells were incubated with DOX or DOX-loaded nanoparticles (figures S5 (c1)–(e1)). The phenomenon could be interpreted that pure DOX or released DOX from MSNs and MSNsPeptide-BSA-LA led to the breakage of the double-stranded DNA of cells [36]. The result suggests that MSNs-PeptideBSA-LA@DOX system had discernable inhibition effects on the growth of HepG2 cells, implying its potential for in vivo tumor therapy.

3.3. Cytotoxicity evaluation

To explore the potential of MSNs-Peptide-BSA-LA@DOX for in vivo tumor therapy, we comparatively investigated the cytotoxicity and inhibition effect of the system on the growth of HepG2 cells [40]. MSNs, MSNs-peptide, MSNs-peptideBSA and MSNs-Peptide-BSA-LA nanoparticles displayed relatively good cytocompatibility. MSNs-Peptide-BSALA@DOX system showed obvious inhibitory effect on the proliferation of HepG2 after culture for 48 h. Its inhibition effect was only slightly lower than that of free DOX with the same concentration (figure 4). 7

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Figure 5. (A) Flow cytometry analysis for specific endocytosis of FITC-labeled MSNs-Peptide-BSA-LA and FITC-labeled MSNs-PeptideBSA by HepG2 cells (n = 6). (B) Flow cytometry analysis for specific endocytosis of FITC-labeled MSNs-Peptide-BSA-LA by HepG2 and HUVEC cells (n = 6).

3.4. Cell targeting assay

3.6. In vivo evaluations

To investigate the cell targeting potential of the system, flow cytometry was employed to quantify the endocytosis efficiency of FITC-labeled MSNs-Peptide-BSA and FITClabeled MSNs-Peptide-BSA-LA by HepG2 cells or HUVEC cells. As shown in figure 5, the percentage of HepG2 cells that endocytosed FITC-labeled MSNs-Peptide-BSA-LA (fluorescence) was about 1.67 and 1.71 folds as that of FITClabeled MSNs-Peptide-BSA after culture for 2 and 4 h, respectively. Moreover, the percentage of HepG2 cells that endocytosed FITC-labeled MSNs-Peptide-BSA-LA (fluorescence) was about 2.17 and 2.02 folds as that of HUVEC cells after culture for 2 and 4 h, respectively. All results suggest that the conjugated with LA molecules played an important role in mediating cell specific uptake via cell targeting [60].

To investigate the curative effects of MSNs-Peptide-BSALA@DOX on tumor growth in vivo, we first established tumor model by subcutaneously injecting HepG2 cells into nude mice [46, 49]. After injection for 4 days, 20 nude mice with similar tumor sizes were randomly divided into four groups (n = 5), which were then treated with saline (control), DOX, MSNs@DOX and MSNs-Peptide-BSA-LA@DOX via tail vein injection, respectively. After treatment for 18 days, tumor tissues were removed from the treated nude mice. In terms of tumor sizes and weights (figures 7(A) and (B)), obvious tumor inhibition could be observed. The mice treated only with saline displayed the highest weight of tumor with average value of 1.49 ± 0.49 g; whereas the mice treated with MSNs-PeptideBSA-LA@DOX showed the lowest weight of tumor with average value of 0.22 ± 0.09 g among all groups, following by average weight of 0.32 ± 0.05 g for MSNs@DOX and average weight of 0.57 ± 0.16 g for DOX group. The tumor weight of the MSNs-Peptide-BSA-LA@DOX group was statistically lower (p < 0.05 or p < 0.01) than those of other groups. The results directly suggest that MSNs-Peptide-BSA-LA@DOX could efficiently inhibit the tumor growth. To investigate the tumor growth process after injection with DOX and DOX-loaded nanoparticles, tumor volumes of the treated mice were also periodically recorded. The tumor growth tendency for all treated mice was visualized with an optical camera (figure S6). The tumor sizes for all treated mice in each group gradually increased along with the feeding time-courses. The tumor volumes of nude mice treated with DOX, MSNs@DOX or MSNs-Peptide-BSA-LA@DOX was significantly smaller than those of the saline-treated group (control). After treatment for 18 days, the tumor volume of the MSNs-Peptide-BSA-LA@DOX group was statistically smaller (p < 0.05 or p < 0.01) than those of other groups (figure 7(C)). The result suggests that MSNs-Peptide-BSALA@DOX had great inhibition effect on tumor growth.

3.5. Cellular uptake assay

TEM was utilized to observe the distributions of endocytosed nanoparticles within HepG2 cells [46, 47]. After co-culture with MSNs, MSNs-Peptide-BSA, MSNs-Peptide-BSA-LA for 24 h, the endocytosed nanoparticles (dash circles) were located at cytoplasm, while not penetrating into the cell nuclei (figure 6(A)). The result was consistent with previous studies [40, 47, 60]. Besides, higher amount of MSNs-Peptide-BSALA was uptaken by HepG2 cells when comparing with those of MSNs and MSNs-Peptide-BSA (figures 6(A)–(d) versus (b), (c)). CLSM was further utilized for observing the distributions of different FITC-labeled MSNs endocytosed by HepG2 cells (figure 6(B)). The efficiency of FITC-labeling of different nanoparticles was around 77% (table S4). The cell internalization efficiency of FITC-labeled MSNs-Peptide-BSALA was higher than that of MSNs and MSNs-Peptide-BSA. The phenomenon could be interpreted that targeting moiety of LA in MSNs-Peptide-BSA-LA contributed to the cell specific endocytosis. In details, the galactose groups of LA molecules are specific ligands for the asialoglycoprotein receptor on the membrane of hepatocytes [61]. 8

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Figure 6. (A) Representative TEM images of HepG2 cells cultured with TCPS (a, control group), MSNs (b), MSNs-Peptide-BSA (c) and

MSNs-Peptide-BSA-LA (d) for 24 h. (B) Representative CLSM images of HepG2 cells incubated with free FITC (a,a1,a2), MSNs (b,b1,b2), MSNs-Peptide-BSA (c,c1,c2), and MSNs-Peptide-BSA-LA (d,d1,d2) for 12 h (a–d), 24 h (a1–d1) and 48 h (a2–d2), respectively. Red: cytoskeleton, blue: cell nuclei, green: FITC-labeled nanoparticles.

tumor sites, since it has only short blood circulation time in vivo [20, 49]. After injection, DOX would lead to a transient high plasma drug concentration and thus led to severe side effect on normal cells and tissues in vivo. Meanwhile, pure DOX would lost its bioactivity during circulation and finally be excreted out through metabolism. On the other hand, the inevitable leakage of DOX from MSNs@DOX would lead to severe toxic side effect on normal organs of a host. In contrast, by employing BSA as end-capping agent, MSNs-Peptide-BSA-LA@DOX could efficiently prevent the leakage of DOX before it reaches the tumor sites, in turn improving its curative effect. More importantly, the LA moiety in MSNs-Peptide-BSA-LA@DOX would improve the accumulation of the system at tumor sites via a targeting

To investigate the potential mechanism of tumor inhibition, relevant tumor tissues were stained with a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) apoptosis assay kit. The results showed that large amount of apoptotic DNA (red color) were found in the tumor tissues of the mice which were treated by MSNsPeptide-BSA-LA@DOX when comparing with other groups (figures 7(D), (a)–(d)). It indicates that the good curative effect of MSNs-Peptide-BSA-LA@DOX system was related to its high capacity for inducing cells apoptosis within tumor tissues, in turn inhibiting tumor growth in vivo [21, 54]. Moreover, higher apoptotic DNA was found in the tumor tissues of MSNs@DOX group than that of pure DOX group. It was related to the fact that pure DOX could not easily reach 9

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Figure 7. (A) Photographs of tumors after different treatments for 18 days; (B) final weights of tumor tissues after different treatments; (C) time-course observations of tumor sizes by digital vernier with different treatments. Error bars represent means ±SD (n = 5), *p < 0.05, **p < 0.01; and (D) histological observation of tumor tissues apoptosis with a TUNEL method after treatments with (a) saline, (b) DOX, (c) MSNs@DOX, and (d) MSNs-Peptide-BSA-LA@DOX, respectively. Red: apoptosis DNA; blue: cell nuclei. Scar bar: 50 μm.

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Figure 8. (A) Time-course weight analysis of nude mice at different time intervals (n = 5), **p < 0.01 and (B) histological examinations of major organs of nude mice. Scar bar: 200 μm.

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Acknowledgments

pathway [14, 24, 60]. The MMPs in tumor microenvironment then triggered the release of DOX from MSNs-Peptide-BSALA@DOX within tumor tissues in situ, resulting in efficient tumor growth inhibition. To evaluate toxic side effect of the system, the weight of all the treated mice were periodically recorded, and the major organs were stained with H and E. After treatment with DOX for 18 days, the nude mice displayed average weights around 20 g, approximately equaling to their initial weights. However, for saline, MSNs@DOX and MSNs-Peptide-BSALA@DOX groups, the average weights of the treated nude mice increased to 23.1, 22.3, and 22.5 g, respectively (figure 8(A)). Significant difference regarding weight of mice between DOX group and other groups was observed (p < 0.01). The change of the mice weights indirectly suggests that MSNs-Peptide-BSA-LA system (carrier) was beneficial for reducing the side effect of DOX against a host [21, 50, 53]. H and E observation was further conducted on the major organs (heart, liver, lung, kidney, and spleen) of mice. Only pure DOX induced typical myocardial injury to mice heart (figure 8(B)). In details, the heart suffered prominent cardiotoxicity associating with acute inflammatory cells (arrows). It was consistent with previous studies [50, 62, 63]. Besides, no obvious damage on other organs (liver, spleen, lung, and kidney) was observed, suggesting that MSNs-Peptide-BSALA@DOX had limited toxic side effects on normal organs. It was related to the facts as follows: firstly, normal tissues had linear blood vessels with smooth and intact structure maintaining by pericytes [2], thus highly diminished the EPR effect that commonly occurred at tumor tissues; secondly, the MMPs concentration in normal tissues was too low to trigger the DOX release from MSNs-Peptide-BSA-LA@DOX, and thus considerably reduced the side effect of the system. Taken together, we confirmed our hypothesis that MSNs-PeptideBSA-LA@DOX system could deliver DOX to tumor triggering by MMPs for inhibition of tumor growth in vivo.

This work was financially supported by Natural Science Foundation of Chongqing Municipal Government (CSTC2013kjrc-ljrcpy0004), Natural Science Foundation of China (21274169 and 31170923), Fundamental Research Funds for the Central Universities (Project No. CQDXWL2013-Z002) and the ‘111’ project (B06023).

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4. Conclusion In this study, we designed and constructed a biocompatible, MMPs responsive drug delivery system based on MSNs (MSNs-Peptide-BSA-LA@DOX) for targeted tumor therapy in vivo, by using BSA as end-capping agent, functional polypeptide as intermediate linker, while LA as targeting motif. A series of characterizations by combined techniques (e.g. SEM, TEM, BET, BJH, FTIR, TGA, XRD, zeta potential measurement etc) confirmed the successful fabrication of the system. The fabricated MSNs-Peptide-BSALA@DOX system could be triggered by MMP-13 for efficient delivery of anticancer drug DOX, which demonstrated good performance for the inhibition of tumor growth in vivo, while with limited toxic side effect. The internal MMPs triggers for controlled drug release were over-expressed by tumor microenvironment in situ, which paves the way for the system to be applied in clinic. 12

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