Dacarbazine-Loaded Hollow Mesoporous Silica

Jun 7, 2017 - Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong ... cytotoxicity to melanoma cells compared with DTIC@HMSNs ...
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Dacarbazine-Loaded Hollow Mesoporous Silica Nanoparticles Grafted with Folic Acid for Enhancing Anti-Metastatic Melanoma Response Qianqian Liu, Nan Xu, Liping Liu, Jun Li, Yamin Zhang, Chen Shen, Khurram Shezad, Lianbin Zhang, Jintao Zhu, and Juan Tao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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Dacarbazine-Loaded Hollow Mesoporous Silica Nanoparticles Grafted with Folic Acid for Enhancing Anti-Metastatic Melanoma Response Qianqian Liu,1,

#

Nan Xu,2,

#

Liping Liu,1 Jun Li,2 Yamin Zhang,

2

Chen Shen,2

Khurram Shezad,1 Lianbin Zhang,1 Jintao Zhu1, 3, *, and Juan Tao2, *

1

Key Laboratory of Materials Chemistry for Energy Conversion and Storage (HUST),

Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan 430074, China 2

Department of Dermatology, Union Hospital, Tongji Medical College, HUST,

Wuhan 430022, China 3

Shenzhen Research Institute of HUST, Shenzhen 51800, China

#

These authors contributed equally to this work

*Corresponding authors, E-mail: [email protected] (J. Zhu); [email protected] (J. Tao)

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ABSTRACT: Dacarbazine (DTIC) is one of the most important chemotherapeutic agents for the treatment of melanoma, while its poor solubility, photosensitivity, instability and serious toxicity to normal cells limit its clinical applications. In this paper, we present a rationally designed nanocarrier based on hollow mesoporous silica nanoparticles (HMSNs) for the encapsulation and the targeted release of DTIC for eradicating melanoma. The nanocarrier (DTIC@HMLBFs) is prepared by modifying HMSNs with carboxyl groups to enhance the loading of DTIC, followed by further envelop of folic acid (FA)-grafted liposomes which act as melanoma active target for controlled and targeted drug release. In vitro, DTIC@HMLBFs exhibited the strongest cytotoxicity to melanoma cells compared to DTIC@HMSNs and free DTIC. The in vivo investigations demonstrate that the rationally designed nanocarrier loaded with DTIC achieve significant improvement against lung metastasis of melanoma via targeting melanoma cells and tumor-associated macrophage (TAM). This study provides a promising platform for the design and fabrication of multifunctional nanomedicine, which is potentially useful for the treatment of melanoma.

KEYWORDS: Dacarbazine, Hollow mesoporous silica nanoparticles, Folic acid, Melanoma, Target delivery

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1. INTRODUCTION Malignant melanoma, one of the most devastating types of cancer, is notorious for its rapid relapse, high multidrug resistance, and low survival rate.1 Nearly 76,380 new cases of melanoma were diagnosed and reported in the United States in 2016 with an estimated 10130 deaths.2 The average survival time for metastatic melanoma patients is ~ 8-9 months with a 3-year overall survival rate of less than 15%.3 For malignant melanoma treatment, many approaches have been developed,4-8 among which, dacarbazine (DTIC), the only US Food and Drug Administration (FDA) approved chemotherapeutic agent, is currently used as a first line chemotherapy medication against melanoma.9 DTIC is a member of the class of alkylating agents, which destroy cancer cells by adding an alkyl group to its DNA. However, application of DTIC in melanoma therapy is obviously limited because of its disadvantages as follows.9-11 Firstly, it is normally administered intravenously, which is painful and usually reduces patient compliant. Secondly, the absorption of DTIC is generally erratic, slow, and incomplete due to its poor solubility in water. Thirdly, DTIC is light sensitive and unstable. Fourthly, DTIC is myelosuppressive, and its use in combination therapy has been further limited by its short half-life. Moreover, similar to other chemotherapy drugs, it has nonspecific toxicity to normal cells. One promising strategy to overcome these limitations is targeted delivery of DTIC encapsulated within nanocarriers into tumor cells.9 Selective targeting strategies, the specific interaction between ligands conjugated to the nanocarrier surface and receptors expressed on the cell surface of interest, can 3

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promote nanocarrier binding and internalization.12 We have proved that melanoma highly expresses transferrin receptor, which is regulated by hypoxia inducible factor-1 under hypoxic tumor microenvironment. Targeting these interest receptors has been shown a promising way to treat melanoma.13 And those receptors, which were highly expressed on tumor cells, as well as those immunosupressive cells in tumor microenvironment (e.g., tumor associated macrophage (TAM)), like folic acid (FA) receptor, would be a great target to inhibit tumor growth. Recently, FA has been reported successfully conjugated on quantum dots,14 iron oxide nanoparticles (NPs),15 block copolymer micelles,16 and others. It has been widely used in cancer cell targeting, tumor imaging, and drug delivery.14-16 FA conducts efficient cell specific targeting through FA-FA receptor conjugation. With the development of nanotechnology and application of nanocarriers in medicine, different organic nanocarriers have also been used for the delivery of DTIC, including cubosomes,9 nano-emulsions,10 and biodegradable polylactide or methoxy poly (ethylene glycol)-b-poly (lactide) NPs.11, 18 Yet, these organic carriers are usually physically unstable in physiological environment or have low drug loading capacity.19 Hollow mesoporous silica NPs (HMSNs), one of the excellent inorganic nanocarriers, have been widely used for drug delivery due to their many advantages, including excellent mesoporous structure and an adjustable pore size,20-22 large surface area and pore volume,23 versatile surface functionalization,24,

25

in vivo

biosafety evaluations of cytotoxicity,26 biodegradation,27, 28 tunable bio-distribution and excretion.29, 30 Besides, HMSNs can not only perform as an agent carrier but also 4

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be used as immunoadjuvants to treat cancer by enhancing anti-tumor immune responses.31 For DTIC, it is difficult to be loaded into HMSNs because there is no strong interaction between HMSNs and DTIC, and it is very easy to release because of the open pore of HMSNs. Nowadays, it is still a challenge to encapsulate DTIC into nanocarriers with high loading efficiency. Here, we rationally designed and fabricated a nanocarrier based on HMSNs for the improved loading of DTIC, targeted delivery and controlled release. By introducing carboxyl group in HMSNs, the loading efficiency of DTIC was significantly enhanced. The DTIC-loaded HMSNs (DTIC@HMSNs) were further coated with liposomes through electrostatic interaction to increase the stability of HMSNs and to control the release of drug. Furthermore, modification with FA on the liposomes enveloped HMSNs (DTIC@HMLBFs) further enhanced specific interactions between the targeted tumor cells and ligand-modified nanocarriers, achieving the targeted delivery of DTIC. Interestingly, in vitro experiment indicated that DTIC@HMLBFs exhibited the strongest cytotoxicity to melanoma cells compared to DTIC@ HMSNs and free DTIC. The in vivo investigations demonstrated that the rationally designed nanocarrier loaded with DTIC achieve significant improvement against lung metastasis of melanoma via targeting melanoma cells and tumor-associated

macrophage

(TAM).

Our

rationally

demonstrated great potential against melanoma.

2. EXPERIMENTAL SECTION

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designed

nanocarrier

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2.1. Materials: Dacarbazine (DTIC), folic acid (FA), N, N'-dicyclohexylcarbodiimide (DCC), 3-Triethoxysilylpropylsuccinic acid anhydride (TESPSA), (3-Aminopropyl) triethoxysilane

(APTES),

cetyltrimethyl

ammonium

bromide

(CTAB),

fluoresecinisothiocyanate (FITC) were purchased from Sigma-Aldrich. Dulbecco’s modified eagle medium (DMEM) and FA free Roswell Park Memorial Institute (RPMI)-1640

were

purchased

2-distearoyl-sn-glycero-3-phosphocholine

from (DSPC),

Gibco. cholesterol,

2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene

1, 1,

glycol)2000]

(ammonium salt) (DSPE-PEG2k-NH2) were obtained from Avanti Polar Lipids Inc. Tetraethyl orthosilicate (TEOS) and dimethyl sulphoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Ultrapure mili-Q water was used in all experiments. All experiments involving DTIC were done in dark. 2.2. Synthesis of Functional HMSNs: HMSNs were synthesized according to the procedure shown in Scheme 1. Typically, silica (sSiO2) NPs were first synthesized through Stöber method.32 Briefly, 100 mL ethanol and 8 mL water were mixed with 4 mL ammonia solution (~37-38%), and then 3 mL TEOS was added. After stirring for 6 h at 30 oC, the sSiO2 NPs were obtained. Subsequently, mesoporous silica shell was formed around sSiO2 (sSiO2@mSiO2 core/shell NPs). The as-prepared sSiO2 NPs suspension was poured into a solution containing 220 mL water, 10 mL ethanol and 1200 mg CTAB, and the mixture was stirred for 30 min. Then, 1.075 mL TEOS was added and the dispersion was stirred overnight. Precipitate was collected by 6

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centrifugation and redispersed in water. The sSiO2@mSiO2 core/shell NPs were thus obtained. Subsequently, we employed a selective etching approach to produce HMSNs.33,

34

The as-prepared sSiO2@mSiO2 core/shell NPs were collected by

centrifugation and redispersed in 150 mL Na2CO3 (0.4 M) aqueous solution, and the mixture was stirred for 2 h at 50 oC to remove the solid silica core.35 HMSNs were obtained after removal of CTAB micelles in the mesoporous shell by repeated washing with concentrated HCl/ethanol (v/v=1:10) and water for 3 times with the help of sonication.36, 37 For modification of carboxyl groups, 0.1 mL TESPSA was added to the HMSN suspension with 50 mL HCl (0.1M), and the mixture was stirred at 50 oC for 5 h. The resulting carboxyl groups modified HMSNs (denoted as HMSNs, used in all experiments except where noted) were collected by centrifugation and washed with deionized water for 3 times. HMSNs were labelled with FITC through the following procedure. Briefly, amine group modified HMSNs were synthesized by adding 107.5 mL APTES together with 967.5 mL TEOS during the sSiO2@mSiO2 core/shell NPs formed and treated with similar procedure as mentioned above. Then, 20 mg amine group modified HMSNs were dispersed into ethanol containing 0.4 mg FITC and reacted at 30 °C for 12 h under dark shaking condition.38 The product was centrifuged and rinsed with deionized water and ethanol each for 3 times to remove free FITC. Surface chemistry and structure of the HMSNs were characterized by Fourier

transform Infrared (FTIR, VERTEX 70, Bruker company, Germany), transmission electron microscope (TEM, Tecnai G220, FEI company, Holland) and N2 adsorption 7

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investigation (TriStar II 3020, Alpha technologies company, USA). 2.3. Synthesis of FA Grafted Lipid: DSPE-PEG2K-FA was synthesized according to the previously reported procedure.39 In brief, 40 mg DSPE-PEG2k-NH2 and 0.2 mL pyridine were added to a DMSO solution containing 10 mg/mL FA, followed by the addition of 13 mg DCC. The reaction was carried out at room temperature for 4 h. The resulting DSPE-PEG2K-FA was characterized by thin-layer chromatography (TLC) (75:36:6 chloroform/methanol/water). Pyridine was removed by vacuum while 5 mL deionized water was added to the reaction mixture, and the solution was filtered to remove residual of any insoluble entities. The supernatant was dialyzed by dialysis tubing (MW cut off: 4,000) against saline (50 mM, 2000 mL) and water (2000 mL) for 3 times. The powder of DSPE-PEG2K-FA was obtained by lyophilization of the dyalizate. 2.4. Generation of Lipid Enveloped HMSNs (HMLBFs): Firstly, liposomes made of DSPC,

cholesterol,

DSPE-PEG2k-NH2

and

DSPE-PEG2k-FA

(molar

ratio:

3:2:0.75:0.75) were prepared by thin-film hydration method.40 The solvent was removed by N2 flow, followed by solvent evaporation for more than 1 h through a rotary evaporator at room temperature, resulting in the formation of lipid film. The thin film was hydrated with PBS and the hydrated liposome solution was formed through probe sonication. Liposomes were mixed with HMSNs at a mass ratio of 1.1:1 in PBS,40-42 followed by incubation at 60 oC for 45 min. The suspension was then centrifuged and washed with PBS to obtain the HMLBFs. The lipid layer on HMSNs was characterized by cryogenic TEM (Cryo-TEM, Tecnai G20 Twin, FEI), 8

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thermo gravimetric analyzer (TGA, Pyrisl, PerkinElmer) and dynamic light scattering (DLS, Malvern Instruments Ltd., Nano-ZS90, UK). The in vitro stability of HMSNs and HMLBFs was further studied in PBS for 5 days at room temperature. 2.5. DTIC Loading and Release in Vitro: DTIC-loaded HMSNs were prepared by a solution-solvent evaporation approach. Typically, 5 mg DTIC was first dissolved in 1.0 mL mixed solution of methanol and water (volume ratio 7:3), and then 0.4 mL of the as-prepared DTIC solution was added into 5.0 mg HMSNs suspension. After sonication for 10 min, the solution was evaporated under vacuum conditions. The precipitant was washed with PBS for 3 times to remove free DTIC. For DTIC-loaded HMLBFs (DTIC@HMLBFs), the as-prepared DTIC@HMSNs were incubated with liposomes and treated with similar procedure as mentioned above. The encapsulation efficiency (EE) of the DTIC was calculated by analyzing the supernatant with high-performance liquid chromatography (HPLC, Agilent Technologies Inc., 1100S, USA) with a UV-vis detector at wavelength of 319 nm. Chromatographic separations were performed on a reversed phase-C18 column (Hypersil ODS2 C18 5 µm, 250 × 4.6 mm). Methanol/water (30/70, v/v) containing 0.1% trifluoroacetic acid was used as the mobile phase at a flow rate of 1 mL/min.18 The in vitro release profile of DTIC was obtained through a dialysis method in pH 7.4 or pH 5.5.43 An amount of 25 mg DTIC@HMSNs or DTIC@HMLBFs was dispersed in PBS in a dialysis tubing (MW cut off: 4000) and then incubated in 40 mL pH 7.4 or pH 5.5 PBS at 37 °C in a water bath shaker at 120 rpm. The concentration of DTIC in the dialysate was monitored. At different time intervals, 1 mL of dialysate 9

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was then taken out from the dialysis solution for the determination of the concentration of the drug by HPLC. Meanwhile, 1 mL fresh PBS was added to the dialysate to keep the dialysate volume constant. 2.6. Cell Lines and Culture Conditions: Human melanoma cell line A875 and mouse melanoma cell line B16/F10 were purchased from American Type Culture Collection (ATCC), and were cultured in Dulbeccos Modified Eagles Medium supplemented with 10% (v/v) fetal bovine serum (Gibco, USA). Human umbilical endothelial cell line HUVEC was purchased from Shanghai Maisha Biotech., China. And HUVEC was cultured for 3-6 passages in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% (v/v) fetal bovine serum and 1% endothelial cell growth supplement (ECGS). All cell lines were cultured at 37 °C in 5% CO2. 2.7. Detection of FA Receptor Expression by Flow Cytometry: A875 melanoma cells were incubated with mouse anti-human FA receptor and isotype IgG2a monoclonal antibody respectively for 1 h, then washed with PBS twice and re-incubated with goat FITC anti-mouse IgG antibody for another 30 min. The whole procedure was performed on ice, and the fluorescence signal was measured by LSR

flow

cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). 2.8. Cellular Uptake Assay: A875 and B16/F10 melanoma cells were seeded in 6-well tissue plate, cultured in FA-containing Dulbeccos modified eagle medium to bring the cell to 70% confluence. A875 and B16/F10 melanoma cells were treated with FITC labelled DTIC@HMSNs or DTIC@HMLBFs with the concentration of 10 µg/mL (counted as HMSNs) in FA-free 1640 for indicated time points. Subsequently, cells 10

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were incubated with Hoescht 333342 (dyes for nuclear staining) for 5 min and Dil (dyes for cell membrane staining) for 5 min before investigation under confocal laser scanning microscope (CLSM, Olympus FV500, Japan). The intensity of FITC was determined with a LSR

flow cytometer.

2.9. In Vitro Cytotoxicity Assay: 5×103 A875 or HUVEC or B16/F10 cells were seeded in a 96-well plate. After 24 h incubation, the cells were treated with different concentrations

of

free

DTIC,

HMSNs,

HMLBFs,

DTIC@HMSNs,

and

DTIC@HMLBFs. The concentration of DTIC equals to those in DTIC@HMSNs and DTIC@HMLBFs while the concentration of HMSNs equals to HMSNs in all NPs containing groups. PBS was taken as the control group. After 48 h, the cell viability was analyzed using CCK-8 assay (Cell Counting Kit-8, Dojindo, Japan) according to manufacture instructions and absorbance were measured by plate reader (Rayto RT-6100, Ba Jiu, Shanghai, China) at 480 nm. All experiments were performed in triplicate. 2.10. Animals Experiment: 5-week-old C57bl/6 mice were purchased from Institute of Laboratory Animal Science, Beijing. All mice were kept under specific pathogen-free (SPF) conditions in the Animal Center of Huazhong University of Science and Technology. During the experiments, mice were sustained at 25 ± 1 °C and 60 ± 10% humidity and exposed to alternate 12 h cycle of light and darkness. During all animal experiments, Chinese law, and local Ethical Committee Quantita Protocol was followed. 2.11. Biodistribution of HMLBFs in Vivo: 1×106 B16/F10 cells were implanted 11

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subcutaneously into the right leg of C57bl/6 mice (6 week old). After 1 week tumor inoculation, biodistribution and imaging studies were performed. At 2 h or 6 h post intravenous (i. v.) injection of Cy5 labelled HMSNs and Cy5 labelled HMLBFs, the fluorescence images of tumor, lung, heart, kidney, spleen, brain, intestine, and liver were collected by live small animal optical imaging system (IVIS Lumina XR, Caliper, USA). All main organs were imaged at 650 nm excitation and 700 nm emission. 2.12. Lung Metastasis Experiments: B16/F10 cell line was used to establish lung metastasis model of melanoma. 5-week-old C57bl/6 mice were intravenously injected with 1.5×105 B16/F10 cells. After injection for 3 days, C57bl/6 mice were intravenously injected with PBS, DTIC, HMSNs, HMLBFs, DTIC@HMSNs, and DTIC@HMLBFs, respectively (6 mg/kg DTIC, 6 mice for each group) every other day for three weeks. Body weight was measured every other day, and overall condition of the mouse was monitored closely. Finally, all mice were sacrificed to harvest major organs, including lung, heart, brain, liver, kidney, and spleen. All these organs were fixed in 4% paraformaldehyde at 4 °C for 3 days once dissected, and hematoxylin-eosin staining (H&E) slices were taken. Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded lungs to examine the expression of CD163 and CD204. The integrated optical density (IOD) was measured as previously described.44 Three images were taken in each section and the average IOD was calculated. The metastasis of melanoma in the lung was counted and lung weight was measured. 12

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2.13. Statistical analysis: All quantification data is presented as mean ± standard deviation. Data with two groups were evaluated by two-tailed student’s t-tests by using GraphPad Prism6. p