Bridging the Gap between Macroscale Drug Delivery Systems and

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Bridging the Gap between Macroscale Drug Delivery Systems and Nanomedicines: A Nanoparticle-assembled Thermosensitive Hydrogel for Peritumoral Chemotherapy Pingsheng Huang, Huijuan Song, Yumin Zhang, Jinjian Liu, Ju Zhang, Weiwei Wang, Jianfeng Liu, Chen Li, and Deling Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10416 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 2016

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ACS Applied Materials & Interfaces

Bridging the Gap between Macroscale Drug Delivery Systems and Nanomedicines: A Nanoparticle-assembled Thermosensitive Hydrogel for Peritumoral Chemotherapy Pingsheng Huang

1a

, Huijuan Song

1a

, Yumin Zhang 2, Jinjian Liu 2, Ju Zhang 1,

Weiwei Wang 1*, Jianfeng Liu 2, Chen Li 1, Deling Kong 1* 1

Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical

Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China 2

Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,

Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China

1

ACS Paragon Plus Environment


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ABSTRACT: The objective of this study was to investigate the spatiotemporal delivery of nanomedicines by an injectable, thermosensitive and nanoparticle self-aggregated hydrogel for peritumoral chemotherapy. Doxorubicin (Dox) was taken as the model medicine, which was encapsulated into poly(ε-caprolactone-co1,4,8-trioxa[4.6]spiro-9-undecanone)-poly(ethylene

glycol)-poly(ε-caprolactone-co

-1,4,8-trioxa[4.6]spiro-9-undecanone) (PECT) nanoparticles

(PECT/Dox

NPs).

Macroscale hydrogel was formed by thermosensitive self-aggregation of PECT/Dox NPs in aqueous solution. Drug release from the hydrogel formulation was dominated by sustained shedding of PECT/Dox NPs and the following drug diffusion from these NPs. The hydrogel retention and release pattern of NPs in vivo was further confirmed by fluorescence resonance energy transfer (FRET) imaging. A single treatment with the hydrogel formulation possessed similar cytotoxicity against HepG2 cells compared to triple administrations of free Dox or PECT/Dox NPs in vitro due to enhanced uptake of PECT/Dox NPs and sustained intracellular drug release. Importantly, single peritumoral injection of drug-encapsulated hydrogel in vivo showed advantages over multiple intravenous administrations of PECT/Dox NPs and free Dox, including preferential and prolonged local drug accumulation and retention in tumors, resulting in superior cancer chemotherapy efficiency. Collectively, such a unique thermosensitive and nanoparticle-shedding hydrogel could effectively combine the advantages of nanomedicines and macroscale drug delivery systems, demonstrating great potential in the local nanodrugs delivery. It will open a new promising path for cancer chemotherapy with enhanced treatment efficacy and minimized side effects.

KEYWORDS: Nanoparticle-assembled hydrogel; Nanomedicines; Doxorubicin; Macroscale drug delivery system; Peritumoral chemotherapy.

2


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1. INTRODUCTION During the past decades, application of the nanoparticulate systems for controlled delivery of anticancer agents has significantly advanced the treatment of solid tumors,1-7 leading to rapid development of nanomedicines with improved therapeutic efficiency and patient compliance. Generally, systemic exposure of nanomedicines including drug-encapsulated or conjugated nanoscale formulations can enhance drug solubility, prolong blood circulation, decrease maximal plasma concentration, increase drug targeting efficiency, improve tumor cell endocytosis while also ameliorate multi-drug resistance.8-17 Despite the advances that had been made, limitations including poor tumor accumulation efficiency, insufficient retention at the tumor tissue and undesired detention by reticuloendothelial systems or mononuclear phagocytic systems still remained to restrict the clinical application of these anticancer nanomedicines.18-24 Multiple injections during a treatment cycle are also required to deliver an optimal dose of anticancer agent to tumor. Besides, after surgical resection of primary malignancies, residual small disseminated tumor nodes in the postoperative lesions may greatly impair the enhanced permeability and retention (EPR) effect of tumor blood vessels, resulting in much lower delivery efficiency. Alternatively, it has been demonstrated that macroscale drug delivery devices such as injectable hydrogels or implantable scaffolds, could provide strategic and precise spatiotemporal control over the presentation of encapsulated bioactive payloads.25-34 In this regard, injectable hydrogels hold the potential to cover the postoperative lesion site and any suspicious site of tumor, sustaining effective therapeutic drug concentrations locally over a prolonged time period, resulting in advanced cancer treatment effect in both animal and clinical trials.35-39 However, depending on the free drug diffusion, such systems also suffered from poor drug penetration through cell membranes or in tumor tissues, and rapid drug clearance in tumor tissues. Therefore, a more efficient treatment strategy that could combine the advantages of nanomedicines and macroscale drug delivery systems is urgently desired to further improve the drug 3



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concentration and retention in tumor tissues and cells with reduced systemic toxicity. To this end, a hydrogel-based nanomedicine delivery strategy was proposed in this study, and the overall design and action pathway of this unique drug delivery system were shown in Scheme 1. An injectable, thermosensitive hydrogel constructed by the self-aggregation of drug-loaded nanoparticles was prepared to spatiotemporally control the delivery of drug-loaded nanoparticles. After local injection in vivo, drug-loaded nanoparticles dissociated from the parent depot were supposed to be spatially delivered within tumor by EPR effect, free diffusion and tumor cell endocytosis. Drugs would be intracellularly released and exert their toxic effect. In contrast to conventional multiple intravenous injections of nanomedicines, peritumoral injection of this hydrogel formulation serving as a reservoir of nanomedicines held the potential to combine the advantages of nanomedicines, such as improved drug endocytosis and tissue penetration ability, and macroscale drug delivery systems, including high local therapeutic drug concentration and prolonged drug retention in the localized tissue. Thus, this hydrogel depot was anticipated to not only enhance the local drug accumulation and retention at the tumor site, but also minimize drug accumulation in major organs and reduce toxic side effects associated with chemotherapeutics. Detailedly, Doxorubicin (Dox) was used as the model drug and amphiphilic triblock copolymer poly(ε-caprolactone-co-1,4,8-trioxa[4.6] spiro-9-undecanone)poly(ethylene glycol)-poly(ε-caprolactone-co-1,4,8-trioxa[4.6] spiro-9-undecanone) (PECT) was employed as the drug delivery vehicle. Drug encapsulation, nanoparticle shedding and drug release from the hydrogel were investigated in vitro and in vivo. Thereinto, fluorescence resonance energy transfer (FRET) imaging was used to study the drug release kinetics in vivo. Cytotoxicity and endocytosis were tested against HepG2 cells. Subsequent evaluation of local drug accumulation, distribution and corresponding antitumor activity were carried out using mouse models. Furthermore, terminal transferase dUTP nick-end labeling (TUNEL) staining was also exercised to examine tumor cell apoptosis. 4


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Scheme 1. The schematic diagram of design concept of peritumoral chemotherapy: in vivo peritumoral

injection

of

the

thermosensitive

hydrogel

formulation

aggregated

by

drug-encapsulated nanoparticles, the following nanoparticles shedding from the hydrogel depot, nanoparticles retention in tumor and intracellular drug delivery.

5



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2. EXPERIMENTAL SECTION 2.1 Materials Amphiphilic triblock copolymer PECT was prepared according to our previous study.40,

41

The characterization including proton nuclear magnetic resonance (1H

NMR), fourier transform infrared (FT-IR), gel permeation chromatography (GPC), X-Ray diffraction (XRD) and differential scanning calorimetry (DSC) of PECT were supplemented in Fig. S1. Dox was received from Wuhan Hezhong Biochem Co. Ltd (Wuhan,

China).

4',6-diamidino-2-phenylindole

3,3′-dioctadecyloxacarbocyanine

perchlorate

(DiO)

were

(DAPI) purchased

and from

Sigma-Aldrich (St. Louis MO, USA). Colorimetric TUNEL apoptosis assay kit was received from Beyotime Institute of Biotechnology (Jiangsu, China). The synthetic route and detailed method of rhodamine-B and fluorescein isothiocyanate (FITC) labeled PECT polymers (termed as PECT-RB and PECT-FITC, respectively) were shown in supporting information file as Fig. S5 and Fig. S6. 2.2 Preparation of PECT/Dox NPs and thermosensitive hydrogels The Dox-encapsulated PECT nanoparticles (PECT/Dox NPs) were prepared by the nanoprecipitation technology. Generally, Dox (1 mg) and PECT copolymer (100 mg) were dissolved in DMSO and then the mixture was sealed in a dialysis bag (MWCO = 3500 Da) and then dialyzed against distilled water for two days. The resulted solution was freeze-dried to obtain the powders of PECT/Dox NPs. The size and morphology of PECT NPs with or without Dox were detected by DLS (Brookhaven BI-200SM) and TEM (JEM100CXII), respectively. UV-Vis spectrophotometry was used to determine the drug loading amount and encapsulation efficiency. For intravenous injection of PECT/Dox NPs, the feeding drug-loading amount was adjusted to 7% (mass ratio, drug/polymer) and the polymer concentration was enhanced to 10 mg/mL, which was prepared by the same method. The thermosensitive behavior of PECT/Dox NPs was investigated by DLS to characterize the particle size change as a function of temperature. At each temperature, the sample was equilibrated for 2 hours. The freeze-dried powders of PECT/Dox NPs were re-dispersed in water at room 6


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temperature with a concentration of 25% (w/w). The viscosity change of the above aqueous dispersion as a function of temperature was measured by a Fluids Rheometer equipment (Stress Tech, Rheological Instruments AB) according to our previous method.41 2.3 In vitro nanoparticle shedding and drug release In vitro Dox release from the hydrogel was performed according to our previous method.40 Drug release from PECT/Dox NPs was also performed using the dialysis method under neutral (in PBS, pH = 7.2, 0.01 M) or acid (in acetate buffer, pH = 6.45 or 5.0, 0.01 M) environment. Briefly, 1 mL of PECT/Dox NPs dispersion containing about 670 µg Dox was placed in the dialysis bag (MWCO = 3500 Da) and immersed in 5 mL of mediums. At each time points, all medium was withdrawn and isometric fresh medium was added. Dox concentration was determined by UV-Vis at 480 nm and calculated according to a calibration curve. All experiments were set up in triplicate to determine the mean values and standard deviations (SDs). 2.4 In vitro cytotoxicity analysis Culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL). HepG2 cells were maintained in DMEM/High Glucose media (Hyclone) at 37 oC. Cytotoxicity of Dox, PECT/Dox NPs and PECT/Dox NPs-Hg formulations were evaluated in vitro using HepG2 cells and a cell counting kit-8 (CCK-8). Briefly, cells were seeded at a density of 5×103 cells/well in 96-well microtiter plates and pre-incubated for 24 h. Cells were then exposed to a series of 50 µL of Dox or PECT/Dox NPs solutions containing 100 µg Dox for 1, 4, or 7 days (treatment buffers were replaced every other day). For PECT/Dox NPs-Hg formulation, 50 µL of corresponding solution was firstly added in the bottom of plate and allowed to gelatinize at 37 oC. 100 µL of cell suspension was seeded onto the hydrogel surface afterwards. The Dox/PECT NPs-Hg was given only once. After incubation, to determine the cytotoxicity, CCK-8 assay was carried out as previously detailed.5, 29, 42, 43 All experiments were set up in quintuplicate to determine 7



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mean values and standard deviations (SDs). 2.5 Cellular endocytosis evaluation To observe cellular uptake of PECT/Dox NPs, 500 µL of HepG2 cell suspensions were seeded on a confocal microscopic dish at a density of 2 × 105 per dish. A certain amount of sample taken from the release medium of the Dox/PECT NPs-Hg formulation was added and the Dox concentration in the dish was adjusted to 5 µg/mL. After further incubation for 0.5 or 4 h, cells were washed with PBS three times and then stained with 500 µL DAPI (1 µg/mL in PBS) for 5 min. After repeated wash with PBS, cellular endocytosis was examined using a confocal laser scanning microscope (TCS SP8, Leica). Cells treated with native Dox were used as the control. In order to verify if PECT NPs could be internalized by HepG2 cells, PECT-FITC/Dox NPs were prepared and subjected to cellular uptake study. 2.6 In vivo hydrogel degradation and drug release monitored by FRET imaging DiO/RB was used as the FRET pair.44 RB was conjugated to PECT by the detailed procedures described in the Supplementary Information. Due to its lipophilicity, DiO was intended to model drug molecules. DiO was encapsulated into PECT-RB nanoparticles via the dialysis method. Briefly, 100 mg PECT-RB and 3.0 mg DiO were simultaneously dissolved in 3 mL dimethyl sulfoxide and then the solution was dialyzed against neutral PBS for three days. After the final volume was adjusted to 50 mL, the obtained solution was centrifuged at 5000 r/min for 10 min to remove the non-encapsulated DiO and then lyophilized to generate DiO encapsulated PECT-RB nanoparticles (PECT-RB/DiO NPs) powders. The FRET of PECT-RB/DiO NPs was determined under the excitation of 465 nm using a fluorescence spectrophotometer (Varian Cary Eclipase). The powders were re-dispersed in water at a mass concentration of 25% without any visible precipitate. 300 µL of the obtained PECT-RB/DiO NPs dispersion was injected subcutaneously into Balb/c nude mice. At designed time points, mice were anesthetized by the intra-peritoneal injection of chloral hydrate solution (4%, 8.25 µL/g animal body weight) and subjected to a 8


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fluorescence imaging equipment (Kodak In-Vivo Imaging System FX Pro, excitation wavelength, 465 nm; emission wavelength, 535 or 600 nm) to determine the fluorescence decay and the FRET efficiency. All animal experiments were performed in accordance with the protocol approved by Chinese Academy of Medical Sciences and Peking Union Medical College. 2.7 In vivo drug accumulation, distribution and anti-tumor activity To measure in vivo drug accumulation and drug distribution in tumor and metabolic organs (heart, liver, spleen, lung and kidney), mice were sacrificed by cervical dislocation at hour 3, day 1 and 3 for intravenous injections of Dox or PECT/Dox NPs, and at day 1, 3, 7, 14 and 21 for peritumoral injection of PECT/Dox NPs-Hg (all after administration of Dox at a dose of 5 mg/kg per animal). Tumor tissues and organs were harvested immediately at the specified times and washed, and the fluorescence intensities of which were observed and recorded using the Kodak fluorescence imaging equipment (excitation wavelength, 555 nm; emission wavelength, 600 nm). The in vivo anti-tumor activity was conducted using xenograft tumor models. Female Balb/c nude mice (6 - 7 weeks) subcutaneously implanted with Bcap-37 tumors were obtained from Cancer Institute and Hospital, Chinese Academy of Medical Sciences. Animals developed breast cancer tumors of approximately 100-150 mm3 were included in subsequent in vivo assessments. Mice were then randomly assigned to one of the following four groups: Group 1 (n = 8), mice were intravenously injected with 0.9% saline; group 2 (n = 8), mice were given triple shots of intravenous injections of Dox at day 1, 4, 7; group 3 (n = 8), mice were received triple intravenous administrations of PECT/Dox NPs at day 1, 4, 7; group 4 (n = 8), mice were peritumorally administrated once with PECT/Dox NPs-Hg dispersion. The total dose of Dox administrated was 20 mg/kg per animal and the injection volume for each intravenous administration of PECT/Dox NPs or peritumoral administration of PECT/Dox NPs-Hg was 200 µL. Tumor volumes were measured using a caliper at designated times and calculated according to the formula, tumor volume = a2×b/2, where a is the shorter diameter and b is the longer one. Body weight of each animal 9



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was also recorded every two days. Apoptotic cells in tumor were identified using a TUNEL assay kit following the manufacturer’s protocol. Briefly, paraffin-embedded tissues were sequentially de-waxed twice in xylene for 5 min, anhydrous ethyl alcohol for 5 min, 90% ethyl alcohol for 2 min, 70% ethyl alcohol for 2 min, and distilled water for 2 min. The tissues were incubated with 20 µg/mL proteinase K without DNase in TE buffer at 37 o

C for 20 min, followed by PBS wash for three times. After incubation with 50 µL

TUNEL test liquid for 60 min at 37 oC in dark, the slides were washed by PBS three times. The slides were mounted and the immune fluorescent images were obtained using an Axio Imager Z1 microscope (Zeiss, Germany). The fluorescence signals were counted and measured by the Image J software (National Institutes of Health, USA). 2.8 Statistical analysis All data are presented as mean ± standard deviations (SDs). The differences among two or three groups were determined using student′s t-test or ordinary one-way ANOVA multiple comparisons (GraphPad Prism 6.0), respectively and p

Bridging the Gap between Macroscale Drug Delivery Systems and

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