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A54 Peptide Modified and Redox-Responsive Glucolipid Conjugate Micelles for Intracellular Delivery of Doxorubicin in Hepatocarcinoma Therapy Na Liu,† Yanan Tan,† Yingwen Hu,† Tingting Meng,† Lijuan Wen,† Jingwen Liu,† Bolin Cheng,† Hong Yuan,† Xuan Huang,‡ and Fuqiang Hu*,† †
College of Pharmaceutical Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China Department of Pharmacy, School of Medicine Science, Jiaxing University, Zhejiang 314001, PR China
‡
S Supporting Information *
ABSTRACT: Redox-responsive nanomaterials applied in drug delivery systems (DDS) have attracted an increasing attention in pharmaceutical research as a carrier for antitumor therapy. However, there would be unwanted drug release from a redox-responsive DDS with no selection at nontarget sites, leading to undesirable toxicities in normal tissues and cells. Here, an A54 peptide modified and PEGylated reduction cleavable glucolipid conjugate (A54-PEG-CSO-ss-SA, abbreviated to APCssA) was designed for intracellular delivery of doxorubicin (DOX). The synthesized APCssA could be assembled via micellization selfassembly in aqueous water above the critical micelle concentration (54.9 μg/ mL) and exhibited a high drug encapsulation efficiency (77.92%). The APCssA micelles showed an enhanced redox sensitivity in that the disulfide bond could be degraded quickly and the drug would be released from micelles in 10 mM levels of glutathione (GSH). The cellular uptake studies highlighted the affinity of APCssA micelles toward the hepatoma cells (BEL-7402) compared to that toward HepG2 cells. In contrast with the nonresponsive conjugate, the drug was released from APCssA micelles more quickly in 10 mM level of GSH concentration (tumor cells). Moreover, the DOX-loaded APCssA micelles displayed an increased cytotoxicity which was 1.6- to 2.0-fold that of unmodified and nonresponsive micelles. In vivo, the APCssA micelles had stronger distribution to liver and hepatoma tissue and prolonged the circulation and retention time, while the drug release only occurred in the tumor tissue. The APCssA/DOX showed the tumor inhibition rate equal to that of commercial doxorubicin hydrochloric without negative consequence. This study suggested that the APCssA/DOX showed promising potential to treat the tumor for its special tumor targeting, selective intracellular drug release, enhanced antitumor activity, and reduced toxicity on normal tissues. KEYWORDS: glycolipid-like micelles, active targeting, redox-responsive, triggered release, chemotherapeutics cytotoxicity reduction
1. INTRODUCTION Liver cancer, as the second most common cancer related deaths all around the world, has been playing a threat to human health. Its global incidence has been reported to be on the rise, and the overall survival of liver cancer patients is dismal.1,2 Doxorubicin (DOX) is widely applied for treating liver cancer.3,4 The exact action mechanism of DOX remains unclear, but it is generally accepted that it interacts with DNA through intercalation and inhibits topoisomerase II.5,6 However, DOX is largely limited in the clinical field due to the severe cardiotoxicity and myelosuppression.7−9 During the past years, many efforts have been made to construct stimuli-responsive nano drug delivery systems (NDDS) to achieve the best therapeutic effect of the antitumor drugs at tumor sites and reduce the damage to the healthy ones at a minimum.10−13 Redox-responsive NDDS have received tremendous attention for triggered drug release in response to different redox conditions between the extra- and intracellular environments (100- to 1000-folds) and the higher concen© XXXX American Chemical Society
tration of reductive substances (7- to 10-fold) in tumor cells compared to that in normal cells.14−16 Unfortunately, it should be noted that this redox potential difference exists in both the normal and tumor tissues,17,18 which means a nonselective redox-responsive NDDS may result in unwanted drug release in normal cells before reaching the target sites and subsequent side-effects and toxicity. In our previous study, we designed and prepared a chitosanbased glycolipid-like redox-responsive conjugate (CSO-ss-SA, abbreviated to CssA), which showed excellent cellular uptake and selective drug release in tumor cells.19 Nevertheless, it is far from optimal for its fast elimination and short retention time. With the modification by polyethylene glycol (PEG), NDDS can avoid the phagocytosis against macrophage and extend the circulation time in the blood. However, the cellular uptake may Received: July 31, 2016 Accepted: November 16, 2016 Published: November 16, 2016 A
DOI: 10.1021/acsami.6b09333 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
peptide was activated by EDC/NHS (3:3, mol/mol) for 1.5 h. Thereafter, 24 mg of NH2−PEG−NH2 in DMSO was added dropwise and stirred for another 12 h followed by the addition of 1.6 mg of DSC and then stirring for additional 12 h. Subsequently, the reaction mixture was gradually injected to CssA aqueous solution, following stirring for 24 h. Finally, 2 M HCl solution was used to get rid of the Boc-producting group. After dialyzing, the mixture was obtained by lyophilizing, and the APCA was synthesized as the control group (Figure S1). The FITC-labeled APCssA was obtained as follows. In brief, the FITC ethanol solution was injected to APCssA aqueous solution, with a molar ratio 1:1. After stirring for 12 h in dark, the reaction solution was dialyzed by water for 24 h. The chemical structures of CssA and APCssA conjugates were determined with 1H nuclear magnetic resonance (NMR) spectroscopy, and these chemicals were dispersed in DMSO-d6 or D2O. The degree of amino substitution (SD%) of CssA was detected with 2,4,6trinitrobenzenesulfonic acid (TNBS) test, and the critical micelle concentration (CMC) of CssA and APCssA were evaluated with pyrene as the fluorescent probe. Dynamic light scattering (DLS) assays were completed with the Zetasizer. Transmission electron microscopy was applied for the observation of micellar morphology. 2.3. Preparation and Characterization of Drug-Loaded Micelles. Doxorubicin base (DOX) was used as the model drug which was prepared as previous study.34 Briefly, 10 mg of polymers was dispersed in 5 mL of deionized water, and the DOX was dissolved in DMSO of 2 mg/mL. Then, the DOX/DMSO was gradually injected to polymers solution with a weight ratio 1:10. After stirring for 4 h in dark, the mixture was dialyzed. Finally, we obtained the product by centrifuging at 4000 rpm for 10 min to get rid of unencapsulated DOX. The sizes and zeta potentials of drug-loaded micelles were determined with DLS, and the morphological examinations were carried out by TEM. The amount of encapsulated DOX in the micelles was measured by fluorescence spectrophotometer. Excitation wavelength was 505 nm, while emission wavelength was 565 nm. The drug loading content (DL%) and drug encapsulation efficiency (EE%) were counted according to the formulas as follows:
be weakened and result in the compromising antitumor activity.20−22 In order to balance the relationship between the plasma elimination rate and the cellular uptake, NDDS were functionalized with various ligands, such as antibodies,23 homing peptides,24,25 aptamers,26,27 growth factors,28,29 and so on for active targeting, which could selectively recognize and internalize into tumor cells excellently and subsequently induce the cell death. Meanwhile, it could reduce the unwanted risk for normal tissues remarkably on account of the faster and stronger distribution to the tumor site. The homing peptides with low molecular weight and noncytotoxicity, were extensively studied as the ligands for active targeting.30−32 The peptide A54 (sequence: AGKGTPSLETTP) has the highest affinity with the human hepatoma cell line BEL-7402, which was obtained from a phase-display random peptide library.33 In the present work, A54 peptide modified and PEGylated chitosan-based reduction cleavable glucolipid nanocarrier (APCssA) was developed for intracellular DOX delivery in human hepatoma. The reduction sensitivity and drug release behaviors in different reduction environment simulating the intra- and extra-cellular thiol concentration were investigated. The cellular uptake, intracellular drug release, and cytotoxicity were carried out against human hepatoma cells BEL-7402 and HepG2. And then a series of studies in vivo were conducted to evaluate the efficiency of this novel NDDS in tumor therapy.
2. MATERIALS AND METHODS 2.1. Materials. Stearylamine (SA) was provided by Fluka (Milwaukee, WI, USA). Chitosan (deacetylation degree 95%, 450.0 kDa,) was purchased from Yuhuan Marine Biochemistry Co., Ltd. (Zhejiang, China). Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) supplied 2-carboxyethyl disulfide (DTPA). N,N′-Dicyclohexylcarbodiimide (DCC), 4-dimethylaminepyridine (DMAP), and di-t-butyl pyrocarbonate ((Boc)2O) were provided by Shanghai Medped (Shanghai, China). ChinaPeptides Co., Ltd. (Shanghai, China) synthesized AGKGTPSLETTP peptide (A54). NH2−PEG−NH2 was supplied by Sigma-Aldrich (St. Louis, Missouri, USA). N-Hydroxysuccinimide (NHS), 1-(3-dDimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC·HCl), and pyrene were provided by Aladdin Reagent Co., Ltd. (Shanghai, China). N-Succinimidyl carbonate (DSC) was supplied by BIO BASIC Inc., USA. Methylthiazoletetrazolium (MTT), 2,4,6-trinitrobenzenesulfonic acid (TNBS), and 5-fluorescein isothiocyanate (FITC) were provided by Sigma Chemical Co. Hangzhou Simbos Pharm Co., Ltd. (Hangzhou, China) supplied doxorubicin hydrochlorate (DOX·HCl). 1,1′Dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (DiR) was supplied by Life Technologies (Carlsbad, CA, USA). SigmaAldrich (Diegem, Belgium) offered Nile red (NR) and L-glutathione (GSH). Dulbecco’s minimum essential medium (DMEM) and porcine trypsin were provided by Gibco (Merelbeke, Belgium). 2.2. Synthesis and Characteristics of A54-Modified and PEGylated Chitosan-ss-stearylamine (APCssA). The chitosan-ssstearylamine conjugate (CssA) was prepared following a two-step process. Briefly, the reaction of SA (208 mg) and DTPA (162 mg) dissolved in 37 mL of anhydrous DMSO was performed in the presence of DCC and DMAP (10:1, mol/mol) for 24 h at 60 °C under N2. After filtration, the filtrate was activated by corresponding amount EDC/NHS for 0.5 h and then transferred into the preheated chitosan aqueous solution with addition of 2 mL of DMSO to avoid precipitation. After stirring for 8 h, the reaction solution was dialyzed against pure water for 48 h. Subsequently, the product CssA was obtained after lyophilizing and washing with hot ethanol three times. For the synthesis of APCssA conjugate, all of the reactions were conducted at room temperature. Briefly, 10 mg of A54 peptide was dispersed in 0.5 mL of anhydrous DMSO, then 5.2 μL of (Boc)2O was added in dark. After stirring for 12 h, the carboxyl group of A54
DL (%) =
weight of encapsulated DOX × 100% weight of DOX − DOX loaded micelles
EE (%) =
weight of encapsulated DOX × 100% weight of DOX in feed
2.4. GSH-Triggered Destabilization of APCssA Micelles. The redox-sensitivity of APCssA micelles was assessed by the fluorescence spectrophotometry with the probe pyrene. Briefly, APCssA micelles were dispersed in PBS containing 2 μM or 10 mM GSH. The size changes of blank micelles were detected by DLS in response to different GSH concentrations (0 mM, 2 μM, and 10 mM). The morphology changes of the micelles after incubated with 10 mM GSH for different times (0, 4, 10, and 24 h) were observed by TEM. 2.5. In Vitro GSH-Triggered Drug Release. In vitro drug release profiles were conducted by dialysis diffusion method. Specifically, 1 mL of DOX-loaded micelles solution corresponding to 40 μg of DOX was placed in dialysis tube and suspended in 25 mL of PBS (pH 6.8, 0.1M) with different GSH levels (0 and 10 mM) at 37 °C under shaking (75 rpm) using an incubator shaker (HZ-8812SB, Hualida Co., China). All of the solution in the dialysis tube were taken out for fluorescence spectrophotometer, then changed to fresh buffer medium at predesigned point in time. 2.6. Cell Culture. HepG2 and BEL-7402 cells were cultured at 37 °C in DMEM medium supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2. 2.7. In Vitro Cellular Uptake. For the qualitative cellular uptake of drug-loaded micelles, 3 × 104 cells/well of HepG2 or BEL-7402 cells were cultured in a 24-well plate with glass coverslips and kept in the incubator at 37 °C for 24 h, respectively. Subsequently, 3 μg/mL drug-loaded micelles were injected and the cells further incubated for B
DOI: 10.1021/acsami.6b09333 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Synthetic route of APCssA conjugate. 1, 3, and 6 h. To study the competitive inhibition, cells were incubated with the free A54 peptide (100 μg/mL) for 0.5 h before adding APCSSA/DOX nanoparticles. Subsequently, cells were stained with Hoechst 33342 solution (0.1 mg/mL) for 15 min, washed with PBS three times, and fixed; CLSM was used to observe the cells with flow cytometry for the quantitative analysis of cellular uptake. 2.8. Intracellular Drug Release. NR was selected as the model drug, and NR-loaded micelles were obtained according to the preparation method of DOX-loaded micelles. First, 3 × 104 cells/ well of HepG2 and BEL-7402 cells were cultured in a 24-well plate containing coverslips, then incubated for 24 h, respectively. Thereafter, the culture medium was changed to fresh medium containing NRmicelles and kept in an incubator for 1, 4, and 12 h. Following the nuclei labeling and cells fixation, all the cell samples were viewed by the CLSM. Further quantify the intracellular drug release was determined with flow cytometry. 2.9. Cytotoxicity Study in Vitro. The cytotoxicity of drug-loaded micelles against HepG2 and BEL-7402 cells was evaluated with MTT assay. In summary, 5 × 104 cells/well were seeded in a 96-well plate and maintained for 24 h at 37 °C; then, a series of concentrations of micelles were added and cultured for additional 48 h. Afterward, 20 μL of MTT at a concentration of 5 mg/mL was added into the cells which were kept for additional 4 h. Then, the medium in each well was replaced with 200 μL of DMSO to dissolve the formazan crystals. Finally, the absorbance of samples at 570 nm was determined using a microplate reader. 2.10. In Vivo Imaging. All the studies were conducted according to the guidelines issued by the Ethical Committee of Zhejiang University. For qualitative evaluation of APCssA micelles biodistribution, the xenografted tumor mice models were established by inoculating 1 × 107 BEL-7402 cells in the nude mice (18−20 g) subcutaneously. Nearinfrared dye DiR was encapsulated by the method of DOX-loaded micelles. When tumor size met the requirement, the DiR-labeled micelles were administered via tail vein injection. At predesigned time points, the mice were imaged using the Maestro in vivo Imaging System. 2.11. In Vivo Drug Release. For the observation of drug release in vivo, the NR-loaded micelles prepared according to the preceding
method were injected (intravenously, i.v.). The mice were sacrificed; then, the main tissues, including heart, liver, and tumor, were collected and embedded in OCT. After quick-freezing in liquid N2, the tissues were sectioned and observed by CLSM. 2.12. Antitumor Activity. The xenografted tumor bearing mice were separated into five groups (n = 3) randomly when the tumor volume reached approximately 200 mm3. Each group was subjected to a vein injections of DOX·HCl, CssA/DOX, APCA/DOX, and APCssA/ DOX at the same DOX concentration of 2 mg/kg every day for first 7 days, and the last group was injected with saline as control group. The tumor sizes and mouse weights were surveyed every other day after the first injection as the evaluation index of the antitumor activity. 2.13. Statistical Analysis. All the data were shown as means ± standard deviation (SD) of three separate experiments. Discrepancies between groups were performed by application of one-way ANOVA followed by Student’s t test, and a p-value
