Dual Stimuli-Responsive Hybrid Polymeric Nanoparticles Self

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Dual Stimuli-Responsive Hybrid Polymeric Nanoparticles SelfAssembled from POSS-Based Star-Like Copolymer-Drug Conjugates for Efficient Intracellular Delivery of Hydrophobic Drugs Qingqing Yang, Lian Li, Wei Sun, Zhou Zhou, and Yuan Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02403 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 13, 2016

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ACS Applied Materials & Interfaces

Dual Stimuli-Responsive Hybrid Polymeric Nanoparticles Self-Assembled from POSS-Based Star-Like Copolymer-Drug Conjugates for Efficient Intracellular Delivery of Hydrophobic Drugs

Qingqing Yang, Lian Li, Wei Sun, Zhou Zhou, and Yuan Huang*

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China

ACS Paragon Plus Environment


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ABSTRACT To

further

finetune

drug

release

and

enhance

therapeutic

effects

of

polyhedral

oligomericsilsesquioxane (POSS)-based nanomedicine, a star-like organic-inorganic conjugate was synthesized by grafting semitelechelic N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to a POSS rigid core through reductively degradable disulfide bonds. The hydrophobic docetaxel (DTX) was attached to the grafts by pH-sensitive hydrazone bonds and also encapsulated into the POSS core (SP-DTX). Thus the final amphiphilic star-shaped conjugates could self-assemble into nanoparticles and exhibited conspicuous drug-loading capacity (20.1 wt%) based on the covalently conjugated accompanied with physically encapsulated DTX. The stimuli-responsive DTX release under acidic lysosomal and reducing cytoplasmic environments was verified, leading to enhanced cytotoxicity against PC-3 human prostate carcinoma cells. To evaluate the in vivo therapeutic effects of the DTX-loaded nanovehicles objectively, a stroma-rich, prostate xenograft tumor model was generated. SP-DTX displayed uniform tumor distribution, and suppressed tumor growth to a more pronounced level (tumor inhibition of 78.9%) than non-redox-sensitive SP-DTX-A (67.4%), SP-DTX-C contained DTX only in the core (65.5%) or linear P-DTX (60.7%) through enhanced depletion of cancer-associated fibroblasts and induction of apoptosis. The hybrid POSS-based polymeric nanoparticles offer an efficient approach to transport hydrophobic drugs for cancer therapy. KEYWORDS: hybrid nanoparticles, redox-responsive, pH-responsive, high drug loading, stroma-rich tomor


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INTRODUCTION During the last few decades, cancer treatment and diagnosis have increasingly attracted tremendous attention and a series of nanomedicines have been proposed and developed.1-4 However, the therapeutic efficacy of some nanomedicines is sub-optimal, mainly because they lack sufficient drug loading efficiency and meanwhile could not release drug on demand. Thus, it is extremely important to develop a drug delivery system that has high and stable drug loading during circulation but specific intracellular drug release in tumor environment. In terms of improving drug loading efficiency, combination strategy of physical encapsulation and covalent conjugation could be utilized. Recently, hybrid nanovehicles composed of both inorganic and organic components have emerged as a promising platform.5 Caged molecular structure such as polyhedral oligomeric silsesquioxane (POSS) has attracted considerable interests in several biomedical applications such as drug delivery, cellular imaging, biosensors and tissue engineering systems due to its organic-inorganic structure, biocompatibility and non-toxicity.6-9 Notably, the Si-O core provides hydrophobic environment for efficient entrapment of drugs by hydrophobic interactions.10-11 However, the drug loading content via physical encapsulation is less than 15 wt% and may lead to the instability problem. Thus we hypothesized to develop a POSS based drug delivery system to loading additional drugs by grafting drug-conjugated polymers to POSS, such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-drug conjugates. In order to ensure highly specific release of therapeutic payloads, intracellular stimuli could be exploited.12-13 For example, the pH values in endo/lysosome compartments of tumor cells further decrease to pH 5.0-5.5, when compared with that in blood circulation and normal tissues (pH 7.4). A variety of pH-sensitive delivery systems have been designed to achieve pH-responsive drug release



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in tumor cell.14-16 Nevertheless, the drug release induced only by pH difference is often deficient.17-18 Furthermore, as another applicable physiological stimulus, the redox potential in tumor cytosol has drawn great attention recently, which is more reducing in comparison with the extracellular components.19-21 In this study, we aimed to develop a pH and reduction dual-bioresponsive POSS-based drug delivery system, which can release drug under acidic endosomal and lysosomal compartments, then further elevate drug release in the cytoplasm of tumor cells in a glutathione-induced manner. Herein, a bioresponsive star-like organic-inorganic conjugate was synthesized. In the conjugates, the rigid core formed by POSS was grafted with semitelechelic HPMA copolymers through reductively degradable disulfide bonds. Then the resultant amphiphilic star-like conjugates could self-assemble into nanoparticles. The hydrophobic docetaxel (DTX) was incorporated into the designed system by both conjugating onto HPMA copolymer through pH-sensitive hydrazone bonds and physically encapsulating into POSS core. We hypothesized that EPR-mediated targeting of the nanoparticles could direct drugs into the tumor tissue; after endocytosis into cancer cells, a first stage of DTX release would occur due to the degradation of hydrazone linkage in acidic lysosome; upon the arrival of cytoplasma, glutathione-triggered breakage of the disulfide bonds could evoke disassembly of the nanoparticle, thus further facilitating the second stage of DTX release after nanoparticles collapse (Figure 1). The potency of this drug delivery system was evaluated on PC-3 human prostate carcinoma cells and in mice bearing stroma-rich prostate tumor xenografts.


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Figure 1. (A) Synthesis procedure of pyridyldisulfanyl-functionalized POSS (POSS-PDS). (B) Fabrication of SP-DTX nanoparticles which self-assembled from amphiphilic star-shaped POSS-based conjugates. (C) Illustration of tumor accumulation and intracellular trafficking pathway of SP-DTX nanoparticles.

EXPERIMENTAL SECTION Materials OctaAmmonium polyhedral oligomeric silsesquioxanes (POSS-NH2) was purchased from Hybrid



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Plastics (Hattiesburg, USA). Docetaxel (DTX) was bought from Dalian Meilun Biotech Co., Ltd. (Shandong, China). Polyclonal rabbit anti-β-tubulin antibody, Cy3-conjugated mouse anti-rabbit IgG antibody, monoclonal mouse anti-alpha smooth muscle actin (α-SMA) antibody and Cy3-conjugated goat anti-mouse IgG antibody were purchased from ABclonal (Cambridge, USA). Cy5.5-NHS ester was bought from Lumiprobe (Hallandale beach, USA).Annexin V-FITC Kit was obtained from Dojindo Laboratory (Kumamoto, Japan). Matrigel was bought from BD Biosciences (Bedford, USA). Nile red (NR), 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), reduced glutathione (GSH), 4′,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA), RNase A and propidium iodide (PI) were bought from Sigma-Aldrich (St. Louis, USA). All other chemicals were bought from Aladdin Reagent Co., Ltd. (Shanghai, China). The human prostate carcinoma cells PC-3 and the mouse embryonic fibroblast cells NIH/3T3 were bought from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China), and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco) with 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Hyclone) in an atmosphere with 5% CO2 at 37 °C. All experiments were carried out on cells in the logarithmic growth phase. Synthesis of Star Copolymer-Docetaxel Conjugates POSS-based star copolymers were synthesized by the reaction of thiol groups in semitelechelic HPMA

copolymers

(P-SH,

Supporting

Information)

with

PDS

groups

of

pyridyldisulfanyl-functionalized POSS (POSS-PDS, Figure 1A and S1) as follows: P-SH (10 µmol SH groups) was dissolved in dimethylsulfoxide (DMSO) and added to a stirring solution of POSS-PDS (8.13 µmol PDS groups) in DMSO under argon atmosphere. After 4 h of agitation, the mixture was diluted with methanol and the products were purified by gel filtration (Sephadex LH-20,


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methanol). Another POSS-based star copolymer (SP-A) with non-degradable amide bonds between polymer grafts and POSS core was synthesized by aminolysis of thiazolidine-2-thione groups of semitelechelic copolymers (P-TT, Supporting Information) with amino groups of POSS-NH2. Star copolymer-DTX conjugates were synthesized after separating the Boc groups from hydrazides of star copolymers using trifluoroacetic acid.22 Then star copolymers were dissolved in anhydrous methanol and the derivative of docetaxel with levulinic acid (DTX-LEV, Figure S2) was added. The reaction was carried out in the dark overnight after addition of acetic acid. The product was purified by gel filtration (Sephadex LH-20, methanol). Cy5.5 labeled conjugates were synthesized by the reaction of Cy5.5-NHS ester with hydrazide groups in star copolymers as previously described.23 Linear HPMA copolymer-DTX conjugates (P-DTX) were prepared by radical copolymerization in accordance with previous reports.24 Self-Assembly of Conjugates and Physically Encapsulating of DTX to Nanoparticles The star copolymer-DTX conjugates (25 mg) and DTX (5 mg) were dissolved in 3.3 mL of DMSO and stirred for 2 h. The mixture was added dropwise into deionized water under sonication and stirred for 4 h, and then dialyzed against deionized water using a dialysis bag (MWCO 14000) for 24 h at 4 °C in the dark. The solution was filtered through a 0.45 µm microporous membrane to remove unloaded DTX and lyophilized to obtain the DTX-loaded nanoparticles (SP-DTX, Figure 1B). DTX-loaded nanoparticles without conjugation of DTX-LEV (SP-DTX-C) were prepared using the similar methods.

In Vitro Disassembly of Nanoparticles and Drug Release



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The disassembly of SP-DTX-A and SP-DTX in response to GSH (10 µM and 10 mM) in 0.1 M phosphate buffer (pH 7.4) was examined by dynamic light scattering (DLS). At predetermined time periods, the mean diameter of the nanoparticles was monitored. The release profiles of covalently conjugated DTX were investigated by a dialysis method. The nanovehicles were dissolved in 0.1 M phosphate buffer (pH 5.0 or 7.4, 0.05 M NaCl) and placed in a dialysis tube with a molecular cutoff of 3500. The tube was immersed in phosphate buffer (pH 5.0 or 7.4) containing Tween 80 (0.1 wt%) at 37 °C. At predetermined time points, 100 µL of solution was collected and an equal volume of fresh media was added. The concentration of DTX was determined by HPLC system. The release profiles of physically encapsulated DTX were studied by incubating the nanoparticles in phosphate buffer (PBS, pH 7.4) containing Tween 80 (0.1 wt%) and different concentrations of GSH (10 µM and 10 mM) at 37 °C for 48 h using a similar dialysis method. Cell Uptake Study For quantitative analysis of cell uptake, PC-3 cells were seeded in 12-well plates and treated with P-DTX-FITC, SP-DTX-A-FITC, SP-DTX-C-FITC or SP-DTX-FITC (equivalent to 10 or 20 µg/mL of FITC) for 2 h. After incubation, cells were collected by trypsinization, washed with cold PBS, and then analyzed the mean fluorescence intensity by flow cytometry (Cytomics FC 500, Beckman Coulter Ltd.). For the microscopic analyses by confocal laser scanning microscope (CLSM), PC-3 cells were grown on sterile glass coverslips placed in 6-well plates. After 2 h or 4 h of incubation with medium containing FITC-labeled samples (equivalent to 20 µg/mL of FITC), cells were fixed with 4% paraformaldehyde, and then DAPI was added to stain the cell nuclei. Coverslips were mounted on glass slides and imaged using CLSM (LSM 510 DUE, Carl Zeiss, Jena, Germany). To observe the intracellular release of encapsulated drug from nanoparticles which containing


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disulfide bonds (SP) or amide bonds (SP-A), fluorescent nile red (NR) was loaded into nanoparticles. After incubation with medium containing SP-NR and SP-A-NR (equivalent to 0.2 µg/mL of NR) for 4 h or 24 h, cells were fixed with 4% paraformaldehyde, stained with DAPI and imaged using CLSM.

In Vitro Cytotoxicity PC-3 cells seeded in 96-well plates (8000 cells/well) were incubated with DTX, DTX-LEV, SP-DTX-A, SP-DTX-C and SP-DTX at a series of concentrations for 24 h. Afterward, the media was discarded, cells were incubated with fresh media for another 24 h. After incubation, MTT reagent was added to each well. The medium was then removed and the formed formazan was dissolved in DMSO (150 µL). The absorbance at 570 nm was recorded using a microplate reader (Varioskan Flash, Thermo, Waltham, MA). IC50 values (µM) were calculated by Graphpad Prism 5 software (Graphpad software Inc.). Similar procedures were conducted to assess the cytotoxicity of blank star copolymers after incubating cells with copolymers for 48 h. Microtubule Polymerization Analysis PC-3 cells cultured in 6-well plates were treated with DTX, DTX-LEV, P-DTX, SP-DTX-A, SP-DTX-C and SP-DTX (equivalent to 0.35 µM of DTX) for 24 h, fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100, rinsed with 3% BSA in PBS and incubated overnight with anti-β-tubulin antibody (1:100) at 4 °C. Cells were then incubated with Cy3-conjugated antibody (1:100) for 1 h, stained with DAPI and imaged through CLSM. Cell Cycle and Apoptosis Assays PC-3 cells were treated with various drug formulations (equivalent to 0.6 µM of DTX) for 24 h. For cell cycle study, treated cells were fixed with 70% ethanol at -20 °C overnight, incubated with



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RNase A (0.1 mg/mL), followed by PI (0.1 mg/mL) staining. Cell cycle distribution was analyzed using flow cytometry. The proportions of cells in Sun G1, G0/G1, S, or G2/M phases were depicted as a DNA histogram. For apoptosis analysis, treated cells were suspended in binding buffer, stained for 15 min in the dark with annexin V-FITC and PI, and then analyzed by flow cytometry. Animal Xenograft Model Male BALB/c nude mice (5-8 weeks old, 20-23 g; Animal Centre of the Institute of West China Medical Center) received care in accordance with guidelines of the animal welfare committee at Sichuan University. To establish stroma-rich subcutaneous human prostate tumor xenografts, PBS (100 µL) containing PC-3 cells (3 × 106), NIH/3T3 cells (1.2 × 106) and Matrigel (33 µL) was injected subcutaneously into the right axilla of nude mice. For the common subcutaneous human prostate tumor model, only PC-3 cells (3 × 106) were injected. Tumor volume was measured every three days starting on day 5 after inoculation. The formula V = L×W2/2 was applied to calculate tumor volume, where W and L refer to the shortest and longest diameters, respectively. On day 13, mice were sacrificed and tumors were excised. A portion of tumor was fixed in 10% formalin and embedded in paraffin. For immunofluorescence on alpha smooth muscle actin (α-SMA), paraffin-embedded tumor sections were deparaffinized, antigen recovered, blocked and then incubated with anti-α-SMA antibody (1:100) at 4 °C overnight. Immunocomplexes were visualized followed by incubation with Cy3-conjugated antibody (1:100). Slides were then washed with PBS, stained with DAPI, and observed using CLSM. Biodistribution Study in Stroma-Rich PC-3/NIH 3T3 Xenograft When PC-3/3T3 xenograft tumor volumes reached 300-400 mm3, mice were randomly divided into four groups (n=3), and intravenously administrated with Cy5.5-labeled P-DTX, SP-DTX-A,


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SP-DTX-C or SP-DTX (1.5 mg/kg of Cy5.5). Images were obtained at 1, 2, 3 and 4 d after injection via a Bio-Real Quick View 3000 in vivo imaging system (Geneway International, Austria). Meanwhile, after 48 h post-injection of above mentioned samples, mice were sacrificed. Xenograft tumor, heart, lung, liver, kidneys, brain and spleen were dissected, and then imaged immediately for semiquantitative analysis. The tumors were fixed overnight with 4% paraformaldehyde before being sectioned with 6 µm thick. Tumor sections were stained with DAPI and visualized on a CLSM.

In Vivo Antitumor Efficacy in Stroma-Rich PC-3/NIH 3T3 Xenograft When PC-3/3T3 xenograft tumor volumes reached 80-100 mm3, mice were randomly divided into 7 groups (n=5) to receive one of the following treatments: saline, DTX, DTX-LEV, P-DTX, SP-DTX-C, SP-DTX-A or SP-DTX (equivalent to 5 mg/kg of DTX) by tail injection on days 1, 6, 11 and 16. Tumor volumes were measured and calculated every 3 days until day 21. Animal body weight was also monitored throughout the experiment. After the mice were sacrificed, major organs and tumors were excised. The paraffin-embedded tumors were sectioned for α-SMA immunofluorescence (Cy3 label) staining as described above. Apoptosis

of

paraffin-embedded

tumors

was

analyzed

by

terminal

deoxynucleotidyl

transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay using apoptosis detection kit (KeyGEN BioTECH, Nanjing, China). Images were acquired by an inverted fluorescence microscopy (CFM 500, Carl Zeiss, Jena, Germany). The paraffin-embedded tissues were stained with hematoxylin and eosin (H&E) for histopathological analysis. Statistical Analysis Results are presented as mean ± SD. Differences between groups were performed with SPSS program 16.0 by using two tail Student’s t test. Differences were considered statistically significant if



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the associated p was

Dual Stimuli-Responsive Hybrid Polymeric Nanoparticles Self

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