A Redox-Sensitive and RAGE-Targeting ... - ACS Publications

Article pubs.acs.org/molecularpharmaceutics

A Redox-Sensitive and RAGE-Targeting Nanocarrier for Hepatocellular Carcinoma Therapy Xiao-Bin Fang,† Ying-Qi Xu,† Hon-Fai Chan,‡ Chun-Ming Wang,† Qing Zheng,§ Fei Xiao,⊥ and Mei-Wan Chen*,† †

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China ‡ Department of Biomedical Engineering, Columbia University, New York, New York 10027, United States § College of Pharmacy and ⊥Department of Pharmacology, School of Medicine, Jinan University, Guangzhou 510632, China S Supporting Information *

ABSTRACT: Hepatocellular carcinoma (HCC) is an aggressive malignancy and the second leading cause of cancer death worldwide. Most current therapeutic agents lack the tumor-targeting efficiency and result in a nonselective biodistribution in the body. In our previous study, we identified a peptide Ala-Pro-Asp-Thr-Lys-Thr-Gln (APDTKTQ) that can selectively bind to the receptor of advanced glycation end-products (RAGE), an immunoglobulin superfamily cell surface molecule overexpressed during HCC malignant progression. Here, we report the design of a mixed micelles system modified with this peptide to target HCC cells. Specifically, we modified Pluronic F68 (F68) with APDTKTQ (F68−APDTKTQ), and we conjugated D-α-tocopheryl polyethylene glycol succinate (TPGS) with poly(lactic-co-glycolic acid) (PLGA) by a disulfide linker (TPGS−S−S− PLGA). We mixed TPGS−S−S−PLGA and F68−APDTKTQ (TSP/FP) to form a micelle, followed by the loading of oridonin (ORI). The prepared micelles showed a homogeneously spherical shape without aggregation, triggered an increased cellular uptake, and induced apoptosis in more cells than did the free ORI. Taken together, these results demonstrate the potential of this APDTKTQmodified ORI-loaded TSP/FP mixed micelle system as a promising strategy for HCC-targeting therapy. KEYWORDS: HCC, RAGE, redox-sensitivity, pluronic F68, oridonin

1. INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most lethal malignances worldwide. It remains the third leading cause of cancer-related deaths due to its aggressiveness and poor diagnosis.1 Surgical resection and liver transplantation are the main treatment modalities for HCC at the early stages, while at advanced stages, chemotherapy becomes the final and major intervention.2,3 However, few chemotherapeutic agents could specifically target HCC cells due to removal by the abundant existence of endoplasmic reticulum.4 Moreover, HCC becomes irresponsive to chemotherapeutic agents because HCC cells fail to recognize these drugs, which leads to a weakened therapeutic effect.5 To increase the specificity of chemotherapeutic agents, a large number of ligand-mediated delivery systems have been designed to target HCC-relevant receptors including asialoglycoprotein epidermal growth receptor (ASGPR),4,6,7 epidermal growth factor receptor (EGFR), 8,9 transferrin receptor (TfR),10,11 hyaluronan receptor (HAR),12,13 and folate receptor (FA).14,15 However, biological data detailing the expression level of these receptors are incomplete. Some receptors, such as ASGPR, are expressed not only on the surface of malignahepatic cells, but also on normal hepatocytes.16 Thus, development of © XXXX American Chemical Society

novel peptides as specific ligands for HCC targeting therapy is still in high demand. The receptor of advanced glycation end-products (RAGE), an immunoglobulin superfamily cell surface molecule, is a multiligand receptor for advanced glycation end products (AGEs), amyloid-β peptides, high-mobility group box 1 (HMGB1), and S100/calgranulins.17−19 In our previous study, we identified a novel peptide (APDTKTQ) that binds to RAGE with high affinity using a phase display library.20 Its role in treating amyloid β peptide-mediated neuronal disorder was documented.20 Considering the increased expression of RAGE in HCC during malignant progression, we hypothesized that the RAGEexpressing HCC could effectively recognize the RAGE-binding peptide decorated on drug carriers and promote cellular uptake of these drugs.21 In this study, we designed a Pluronic F68 (F68)-based mixed micelles system decorated with the RAGE-binding peptide Received: February 9, 2016 Revised: April 18, 2016 Accepted: May 9, 2016

A

DOI: 10.1021/acs.molpharmaceut.6b00116 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). 2.2. Synthesis of TPGS−S−S−PLGA Copolymer and F68−APDTKTQ. The synthesis of TPGS−S−S−PLGA conjugate consisted two main steps (Figure 1). First, 2,2bis(hydroxymethyl)propionic acid (2.0 mmol), DCC (2.0 mmol), and DMAP (0.1 mmol) were dissolved in DMSO to activate the carboxyl of 2,2-bis(hydroxymethyl)propionic acid for 12 h, and the solution was dropwise added into the TPGS solution (1.0 mmol TPGS was dissolved in 10 mL of DMSO) and reacted under nitrogen protection. After 24 h, the reaction product was dialyzed using dialysis tubing with MWCO 500 to remove the residual 2,2-bis(hydroxymethyl)propionic acid, DCC, and DMAP as well as the byproduct 1,3-dicyclohexylurea (DCU). In addition, the targeted product TPGS−S−S−COOH was obtained by cryodesiccation. Second, TPGS−S−S−COOH (1.0 mmol), PLGA (1.0 mmol), DCC (1.1 mmol), and DMAP (0.1 mmol) were dissolved in DMSO, and the TPGS−S−S− COOH was conjugated with PLGA through esterification reaction. The excess of TPGS−S−S−COOH, PLGA, DCC, DCU, and DMAP was removed by dialyzing with molecular weight cutoff at 5000 kDa, and the resultant product TPGS−S− S−PLGA was obtained. Additionally, TPGS−PLGA was synthesized by esterification reaction to compare the redoxsensitive property with TPGS−S−S−PLGA. F68−APDTKTQ conjugate was synthesized by two steps (Figure 1). First, F68 (1 mmol) and 3-maleimidopropionic acid N-hydroxysuccinimide ester (2.2 mmol) were dissolved in deionized water under stirring condition and reacted under the protection of nitrogen for 24 h. The excess of 3-maleimidopropionic acid N-hydroxysuccinimide ester was removed by dialyzing against dialysis bag with MWCO 3500, and the F68− MAL was obtained by lyophilization. Subsequently, F68−MAL (1 mmol) and thiolated peptide APDTKTQ (2 mmol) were dissolved in deionized water accompanied by ultrasound, and the reacting system was incubated at 4 °C for 24 h. Similarly, the residual thiolated peptide APDTKTQ was removed by dialysis, and F68−APDTKTQ was obtained by cryodesiccation. All of the reactants and the products were characterized by 1H NMR spectroscopy (400 MHz, Bruker German), and deuterated chloroform was used as the solvent. To further validate the successful synthesis of TPGS−S−S−PLGA, TPGS−PLGA, and F68−APDTKTQ, the copolymers were mixed with KBr and pressed into small round pieces, and the fourier transform infrared (FT-IR) spectra were detected by NEXUS 670 FT-IR spectrophotometer (Nicolet, American). The samples were scanned from 500−4000 cm−1. 2.3. Preparation of ORI-Loaded Mixed Micelles. ORIloaded mixed micelles were prepared using the dialysis method.22 Briefly, a certain amount of copolymers (TPGS−S−S−PLGA and F68−APDTKTQ with different ratios) and 1 mg of ORI were dissolved in different volumes of acetonitrile in a roundbottom flask, and ultrasound was conducted to promote dissolution. After the copolymers and free ORI were completely dissolved, the solution was dropwise added into a certain volume of deionized water under stirring condition for 30 min at room temperature. Then the solution was dialyzed in a dialysis tube with MWCO 3500 for 24 h to remove the acetonitrile. The unencapsulated ORI was further removed with the 0.45 μm filter membrane, and the ORI-loaded TSP/FP mixed micelles were obtained. Concurrently, ORI-loaded TPGS−PLGA/F68−MAL (TP/FM) and TPGS−S−S−PLGA/F68−MAL (TSP/FM) mixed micelles and both of their blank mixed micelles were

APDTKTQ to achieve a HCC-targeting therapy. TSP/FP-mixed micelles were prepared for encapsulating the model drug oridonin (ORI) (shown in Scheme 1). The 2,2-bisScheme 1. Drug Delivery Mechanism of TSP/FP Mixed Micelles. Mixed Micelles Are Formed via Self-Assembly and Maintain Long Circulation in the Blood Due to the Presence of Pluronic F68 and TPGSa

a

Once the Micelles Reach the Target Site, Endocytosis Took Place via the RAGE-Mediated Process and Release Drug by Utilizing the RedoxSensitivity of the Disulfide Bond.

(hydroxymethyl)propionic acid (S−S) was used as a disulfide bond linker connecting D-α-tocopheryl polyethylene glycol succinate (TPGS) and poly(lactic-co-glycolic acid) (PLGA), followed by the conjugation of the peptide APDTKTQ onto F68. ORI-loaded mixed micelles were prepared by dialysis, and the central composite design/response surface method (CCDRSM) was used to optimize the preparation conditions of the ORI-loaded mixed micelles. Besides, D,L-dithiothreitol (DTT), a common reducing agent, was employed to examine the redoxsensitivity of mixed micelles. Finally, this drug delivery system was assessed for its physicochemical properties, in vitro drug release, cytotoxicity, pro-apoptotic effects, induction of mitochondrial membrane potential change, and cellular uptake.

2. MATERIALS AND METHODS 2.1. Materials. Pluronic F68 was provided by BASF Co. (Germany). N,N′-Dicyclohexylcarbodimide (DCC), 4dimethylaminopyridine (DMAP), TPGS (D-α-tocopheryl polyethylene glycol succinate), and 2,2-bis(hydroxymethyl)propionic acid were supplied by GL Biochem Ltd. (Shanghai, China). Thiolation peptide HS-Ala-Pro-Asp-Thr-Lys-Thr-Gln was purchased from China Peptides Co., Ltd. (Shanghai, China). PLGA (poly(lactic-co-glycolic acid)) was obtained from Daigang Biomaterial (Jinan, China). ORI and Nile red were purchased from Nanjing Zelang Biomedic Technology Co., Ltd. (Nanjing, China). Pyrene was obtained from Baoman Bio (Shanghai, China). 3-Maleimidopropionic acid N-hydroxysuccinimide ester was provided by Tianjin Heowns Biochem LLC (Tianjin, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin (PS), phosphatebuffered saline (PBS), and 0.25% (w/v) trypsin/1 mM EDTA were purchased from Gibco LifeTechnologies (Grand Island, USA). The 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Hoechst 33342 was purchased from Invitrogen. Primary antibodies against RAGE and GAPDH and B


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Figure 1. Synthetic route of TPGS−S−S−PLGA, TPGS−PLGA conjugate, and F68−APDTKTQ.

prepared in the same way. The particle sizes and zeta potentials of all the mixed micelles were detected by dynamic light scattering (DLS) method at 25 °C using a Zetasizer Nano ZSP (Malvern Instruments, Malvern, UK).23 2.4. Optimization of the Preparation Conditions for ORI-Loaded Mixed Micelles. The central composite design-

response surface methodology (CCD-RSM) was used to optimize the preparation conditions for ORI-loaded mixed micelles.24 The three main factors, including the ratio of TPGS− S−S−PLGA to F68−APDTKTQ, the ratio of copolymer to drug, and the ratio of aqueous volume to organic volume, were chosen to optimize the preparation conditions. A central C


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2.8. In Vitro Drug Release. In vitro release of ORI from TSP/FP mixed micelles was examined using a dialysis method.28 Particularly, 0.5% Tween-80 solution with or without 10 mM DTT was used as the release medium. Briefly, 2 mL of ORIloaded mixed micelles as well as free ORI solution was added into the dialysis tubing with MWCO 3500, respectively, and incubated in 40 mL of release medium with the shaking frequency of 100 rpm at 37 °C. At each certain time point, 2 mL of release medium was collected and replaced with the same volume of fresh release medium. HPLC was used to determine the concentration of ORI in the release medium. By using the following equation, the cumulative percentage of ORI release could be calculated:

composite design was performed based on these three factors (factor X1, ratio of TPGS−S−S−PLGA to F68−APDTKTQ ranging from 1−7; factor X2, ratio of copolymer to drug ranging from 2−8; factor X3, ratio of aqueous volume to organic volume ranging from 5−20) with five experimental levels: −1.682, −1, 0, +1, +1.682. Particle size, drug loading (DL%), and drug entrapment efficiency (EE%) were chosen as the responses y1, y2, and y3, respectively. In addition, the overall desirability (OD) value was calculated according to the following equations:25 dmin = (ymax − yi )/(ymax − ymin )

(1)

dmax = (yi − ymin )/(ymax − ymin )

(2)

OD = (d1 × d 2 × ...dn)1/ n

(3)

Mi = C i × 40 mL +

Cumulative release (%) = (Mi / MA ) × 100

(7)

Mi represents the total amount of ORI release at the ith time point. Ci (mg/mL) represents the concentration of ORI in the release medium at ith time point. ∑j=1j=j‑1Cj × 1 mL represents the total amount of ORI release before jth time point. MA represents the initial amount of ORI in the dialysis bag. 2.9. Cell Lines and Cell Culture. Human hepatoma BEL7402 cells, HepG2 cells, and SMMC-7721 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in plastic culture flasks in DMEM supplemented with 10% (v/v) heated-inactivated FBS and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) at 37 °C in a 5% CO2 humid atmosphere. 2.10. Western Blot Analysis. Expression of RAGE receptors in BEL-7402 cells, HepG2 cells, and SMMC-7721 cells was determined by Western blot analysis. Briefly, these three cells were placed in cell culture dishes at a density of 6 × 105 cell per dish the day before the experiment. Then the cells were lysed using RIPA lysis buffer containing 1% protease inhibitor cocktail and 1% phenylmethanesulfonylfluoride (PMSF) to extract total protein. The protein concentrations in cell lysates were determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Equivalent amounts of cell lysate proteins from each group were resolved in 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to PVDF membranes. The membranes were incubated with primary antibodies against RAGE and GAPDH (1:1000) at 4 °C overnight after being blocked with nonfat dried milk for 1 h, followed by the incubation with antimouse IgG (goat) secondary antibodies (1:1000). The specific protein bands were visualized using Western Blotting Detection kit (GE Healthcare). 2.11. Cells Viability Assay. MTT assay was performed to determine the viability of BEL-7402 cells and HepG2 cells when exposed to a series of concentrations of TP/FM, TSP/FM, and TSP/FP. First, exponentially growing BEL-7402 cells and HepG2 cells were seeded in 96-well plates at a density of 5 × 103 cells/well and cultured with 100 μL of DMEM overnight. Then the cells were treated with TP/FM, TSP/FM, TSP/FP micelles, or free ORI at different concentrations ranging from 1.25−20 μM. After 24 and 48 h of incubation, the culture media were replaced with fresh medium containing 1 mg/mL of MTT. After incubation for 4 h, the MTT solution was carefully aspirated with the addition of 150 μL of DMSO to dissolve the formed formazan crystals. The absorbance in each well was measured with a microplate reader (SpectaMax M5, Molecular

DL% = amount of drug in micelles/amount of feeding (4)

EE% = amount of drug in micelles/amount of feeding drug × 100

(6)

j=1

2.5. Drug Loading and Encapsulation Efficiency in Mixed Micelles. High-performance liquid chromatography (HPLC) was used to determine the drug concentration of ORIloaded mixed micelles with the maximum absorption 238 nm.26 A certain volume of mixed micelles was dissolved in the same volume of methanol and scrolled for 1 min to disrupt the polymeric shells of the micelles and the ORI would be released and dissolved in the methanol. Waters e2695 HPLC with a reverse phase C18 column (250 × 4.6 mm2) was used to determine the concentration of ORI at a flow rate of 1 mL/min. The mobile phase consisted of methanol/water at ratio of 55:45. The DL% and EE% were calculated based on the following equations:

polymer and drug × 100

∑ j = j − 1C j × 1 mL

(5)

2.6. Redox-Sensitivity of Mixed Micelles. The average particle size of the TSP/FP mixed micelles with or without 10 mM DTT was detected by the DLS instrument at 25 °C (treating with DTT for 24 h). Besides, their morphologies were imaged by transmission electron microscopy (TEM) (HRTEM, Tecnai G20, FEI Company, American). Briefly, 50 μL micellar samples (1 mg/mL) were dropped onto the Formvar/carbon film grid and were negative stained with 2% phosphotungstic acid after partially air-drying. Moreover, the redox-sensitivity drug release behavior of the TSP/FP mixed micelles was investigated by Nile red assay. To examine the redox-sensitivity drug release, Nile redloaded mixed micelles solutions were treated with 10 mM DTT, and the fluorescence of the solution was detected at several certain time points (0, 2, 4, 8, 12, 24, and 48 h). Meanwhile, the Nile red-loaded mixed micelles solution treated without DTT was also detected at 0, 24, and 48 h. 2.7. Critical Micelle Concentration (CMC). The CMC of TSP/FP mixed micelles was determined by fluorescence measurements using a fluorescence spectrophotometer (LUMINA, Thermo Scientific, USA). In this experiment, pyrene was used as a hydrophobic probe, of which the emission spectra were recorded from 360−450 nm at an excitation wavelength of 339 nm.27 The concentrations of copolymer ranged from 0.0002−1 mg/mL with a constant concentration of pyrene (6.0 × 10−7 M). The ratio of fluorescence intensities at 373 nm (I373) to 384 nm (I384) of each sample was calculated. D


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were washed with PBS and fixed in 4% PFA for 10 min. Then the cells were amplified and stained with Hoechst 33342 for better visualization. The cells were detected by merging fluorescent DAPI and FITC images. 2.16. Statistical Analysis. Statistical analysis was based on three separate experiments and performed with GraphPad Prism 5 software. Assessed by one-way ANOVA or t-test, statistical significance was considered with a p-value < 0.05.

Devices) at 570 nm. The viability, expressed as a percentage of the viability of treated cells to unexposed cells, was determined according to triplicate assays. 2.12. Nuclear Morphology of BEL-7402 Cells. Hoechst staining assay was carried out to observe morphology changes and chromosome condensation of BEL-7402 cells. Cells, seeded in 96-well plates the day before experiment, were treated with TP/FM, TSP/FM, TSP/FP micelles, blank TSP/FP micelles, or free ORI solution at an equivalent ORI concentration of 10 μM. After exposure for 24 h, cells were fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, and stained with Hoechst 33342 (10 μg/mL) at room temperature for 15 min. Cells were visualized and imaged by IN Cell Analyzer 2000 (GE Healthcare Life Sciences, USA). 2.13. Annexin V/PI Staining Assay. Apoptotic cells were distinguished using an Annexin V-FITC/PI assay according to manufacturer’s instruction. To investigate the effect of TP/FM, TSP/FM, TSP/FP on apoptosis, BEL-7402 cells were plated in six-well plates at a density of 2 × 105 cells/well and treated with 10 μM free ORI solution or each micelle. After incubation for 24 h, the unattached cells in cell medium were collected by centrifugation, while the attached cells were trypsinized, combined with the cells collected earlier, and washed twice with PBS. All cells were suspended in 100 mL of binding buffer containing 5 mL of Annexin-FITC. After incubation in the dark for 15 min, 10 mL of PI solution was then added before flow cytometry analysis was performed. 2.14. Mitochondrial Membrane Potential (MMP). The MMP was monitored by a flow cytometer using the mitochondrial permeability lipophilic tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) fluorescence dye. BEL7402 cells were placed in 24-well plates and treated with 10 μM free ORI solution or each micelle for 24 h. The cells were then incubated in PBS containing JC-1 (10 μg/mL) at 37 °C in the dark for 20 min to allow JC-1 to enter the mitochondrial membrane. Cells were then rinsed twice with PBS and subsequently measured by FACS flow cytometry (Beckman coulter, California, USA). 2.15. Cellular Uptake Studies. To monitor the cellular uptake of the micelles, the fluorescent TP/FM, TSP/FM, and TSP/FP micelles loading fluorescent probe Nile red instead of ORI were prepared. BEL-7402 cells were placed in 12-well plates at a density of 6 × 104 cells/well. Cells in RAGE group were pretreated with 100 μL of medium containing 1/1000 RAGE antibody. After 1 h of incubation, the media were discarded, and the cells were rinsed twice with PBS. Then free ORI solution, TP/FM, TSP/FM, and TSP/FP micelles at an equivalent concentration of Nile red at 100 ng/mL were added at the specified time point. Untreated cells were served as control in this assay. After the 1, 2, and 4 h incubation period, respectively, cellular uptake was terminated by gently washing cells twice. By using flow cytometry, the PE fluorescence of the collected cells was read in the FL-2 channel. The cellular uptake amount was expressed as the ratio of fluorescence intensity in Nile red-loaded micelles treated cells relative to untreated cells. The cellular uptake of each micelle in BEL-7402 cells was imaged using the In Cell Analyzer (GE Healthcare, USA). After being seeded in 96-well plates at a density of 8000 cells per well overnight, cells were treated with aforementioned micelles at an equivalent concentration of Nile red 100 ng/mL for 4 h at 37 °C. Similarly, cells in RAGE group were pretreated with 100 μL of medium containing 1/1000 RAGE antibody for 1 h before incubation with Nile red-loaded micelles. After incubation, cells

3. RESULTS AND DISCUSSION 3.1. Characterization of TPGS−S−S−PLGA Conjugate and F68−APDTKTQ. The synthesis of TPGS−S−S−PLGA and TPGS−PLGA conjugate as well as F68−APDTKTQ were shown in Figure 1 and evidenced by 1H NMR and FT-IR spectra (Figure S1). In the 1H NMR spectra of TPGS−PLGA and TPGS−S−S−PLGA conjugate, the typical signals for TPGS were observed including the peaks for −OOC−CH2−CH2O− groups (δ = 3.62−3.82 ppm), −OOC−CH2−CH2−COO− groups (δ = 2.59−2.82 ppm), and −CH2CH(CH3)2 groups (δ = 0.85−1.80 ppm). Meanwhile, we also observed the typical signals for PLGA including the peaks for −OOC−CHO− groups (δ = 5.12−5.30 ppm), −OOC−CH2O groups (δ = 4.66−4.92 ppm), and −CH3 groups (δ = 1.48−1.63 ppm). Particularly, we observed the conspicuous signals for −(H3C)HC−S−S− CH(CH3)− groups (δ = 3.19−3.26 ppm) in the 1H NMR spectra of TPGS−S−S−PLGA conjugate but not in TPGS− PLGA. Moreover, from the FT-IR spectra of TPGS−PLGA conjugate, the stretching vibrational peak at 1650 cm−1 was detected, indicating the formation of ester bonds between the −OH groups of TPGS and the −COOH groups of PLGA. Similarly, ester bonds were also formed between the −OH groups of TPGS, PLGA, and the −COOH groups of 2,2bis(hydroxymethyl)propionic acid. Taken together, the 1H NMR spectra and FT-IR spectra confirmed the successful synthesis of TPGS−PLGA and TPGS−S−S−PLGA. 1 H NMR spectra and FT-IR spectra of F68, F68−MAL, and F68−APDTKTQ were shown in Figure S2. In the 1H NMR spectra of F68−MAL, the typical signals for maleimide groups (δ = 6.86−6.89 ppm) were observed. However, it was absent in the 1 H NMR spectra of F68−APDTKTQ. Meanwhile, the typical signals for amino groups of APDTKTQ were observed (δ = 7.94−8.31 ppm), indicating that the maleimide groups were replaced by the peptide. In the FT-IR spectra of F68−MAL, the peaks at 1650 cm−1 referred to the CC groups of the maleimide, and the peaks between 1710 and 1750 cm−1 indicated the formation of ester bond between −OH groups of F68 and −COO− groups of 3-maleimidopropionic acid N-hydroxysuccinimide ester. Besides, we could also detect the CO groups in maleimide. In the FT-IR spectra of F68−APDTKTQ, the characteristic peak at 1650 cm−1 attributed to CC groups of maleimide was much weaker than that of F68−MAL. Two new peaks appeared at 3180 and 3350 cm−1 due to the presence of amino groups in APDTKTQ peptide. In addition, the peaks between 1710 and 1750 cm−1 reflected CO groups in APDTKTQ peptide and the ester bond formed by the reaction between F68 and 3-maleimidopropionic acid N-hydroxysuccinimide ester. 3.2. Optimization of the Conditions for Preparing ORILoaded Mixed Micelles. The single-factor test showed that the weight ratios of TSP/FP and polymer/drug as well as the volume ratio of aqueous/organic significantly affected the particle size, drug loading, and encapsulation efficiency of ORI-loaded mixed micelles. Consequently, these three factors on five levels (0, ± 1, E


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Molecular Pharmaceutics Table 1. Experimental Design and Results of the Central Composite Design (CCD) preparation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

factors

response

TPGS−S−S−PLGA/F68−APDTKTQ (mg/mg)

polymer/drug (mg/mg)

aqueous/organic (mL/mL)

particle size (nm)

DL (%)

EE (%)

OD value

2.22 5.78 2.22 5.78 2.22 5.78 2.22 5.78 1 7 4 4 4 4 4 4 4 4 4 4

3.22 3.22 6.78 6.78 3.22 3.22 6.78 6.78 5 5 2 8 5 5 5 5 5 5 5 5

8.04 8.04 8.04 8.04 16.96 16.96 16.96 16.96 12.5 12.5 12.5 12.5 5 20 12.5 12.5 12.5 12.5 12.5 12.5

125.4 109.6 124.2 112.6 120.6 110.8 120.4 111.4 130.4 118.4 105.4 104.8 114.6 105.4 103.8 103.6 104.4 104.2 105.6 104.8

21.24 21.18 10.21 10.24 21.18 21.99 9.99 10.5 12.44 15.04 27.6 9.33 12.5 13.68 15.04 14.96 15.12 14.96 14.76 15.16

68.4 68.2 69.2 69.4 68.2 70.8 67.7 71.2 62.2 75.2 55.2 74.6 62.5 68.4 75.2 74.8 75.6 74.8 73.8 75.8

0.85 0.51 0.22 0.36 0.47 0.44 0.20 0.39 0.47 0.67 0.94 0 0.55 0 0.56 0.53 0 0.52 0.51 0.67

(from 94.8 ± 3.6 nm to 106 ± 5.4 nm) while having no influence on the particles’ zeta potential (−35.8 ± 2.8 mV and −35.2 ± 2.6 mV). This was due to the addition of a mixture of uncharged groups of Ala, Pro, Thr, Gln, the negatively charged groups of Asp, and the same proportion of positively charged groups of Lys onto the micelle surface. Moreover, the DL% and EE% of TSP/ FM were 16.42 ± 0.42% and 73.88 ± 1.88%, respectively, similar to those of TSP/FP (16.16 ± 0.36% and 72.72 ± 1.62%), which suggested the introduction of the peptide had a negligible effect on the DL% and the EE% of the mixed micelles. Compared to the reported ORI-loaded nanoparticles, the prepared ORI-loaded mixed micelles had smaller particle size but higher EE%, indicating the favorable design and preparation of the ORIloaded mixed micelles.30 3.4. Redox-Sensitivity of the Mixed Micelles. It has been reported that the concentration of the glutathione (GSH) in cancer cells is approximately 2−10 mM, which is much higher than that detected in extracellular matrices (about 2−20 μM).31 This results in a higher likelihood of disulfide bond dissociation in cancer cells. To study the fate of mixed micelles with disulfide bonds in different concentrations of GSH, DTT, with a reductive property similar to GSH, was used as a simulate of the microenvironment within the cancer cells as well as in extracellular matrices. As shown in Figure 3, panel A, the particle size of the mixed micelles displayed a unimodal distribution when treated with 20 μM DTT for 24 h. The TEM image showed that the mixed micelles maintained their uniform spherical shape, indicating that the low concentration of DTT had no effect on the mixed micelles. However, when treated with 10 mM DTT for 24 h, the particle size of the mixed micelles transformed to double-humped distribution, and ruptured spheres were observed in the TEM image (Figure 3B). These observations suggested that the disulfide bond of TPGS−S−S−PLGA was disrupted in the presence of excessive DTT. To further confirm the redox-sensitivity of the mixed micelles, Nile red release profiles of the mixed micelles were investigated. It has been reported that the fluorescence intensity of Nile red

± 1.68) were tested. Twenty experiments were conducted by the response surface methodology depending on the central composite design. The experimental design and results were shown in Table 1. By applying to the response surface methodology,29 a suitable polynomial equation model and a quadratic model were generated from the Design-Expert 8.0 software. The best particle size, DL%, and EE% were selected from several statistical parameters based on the following polynomial equation model. In addition, the quadratic model was responsible for the overall evaluation of particle size, DL%, and EE% statistically: OD value = 0.57 + 0.014X1 − 0.12X 2 + 0.063X3 + 0.069X1X 2 + 0.090X1X3 − 0.15X 2X3 − 0.11X12 − 0.081X 2 2 + 5.39 × 10−3X32

Additionally, the 3D models that fit to this equation were shown in Figure 2. According to this generated polynomial equation and the OD values, the optimized values of the three factors could be calculated. In this study, the optimized ratios of TSP/FP (X1), polymer/drug (X2), and aqueous/organic (X3) were 4.47 mg/mg, 4.55 mg/mg, and 9.84 mL/mL, respectively. The three factors were practically modified in number to 4.5 mg/ mg, 4.5 mg/mg, and 10 mL/mL, respectively. As shown in Table 2, the predicted errors of particle size, drug loading, and encapsulation efficiency were 1.68%, 6.23%, and 2.85%, respectively. These results revealed that the central composite design model could be applied to predict the effectiveness of preparing the ORI-loaded mixed micelles. 3.3. Characterization of ORI-Loaded Mixed Micelles. Prior to the adoption of optimal design, the optimized prescription of these three crucial factors was used to prepare the ORI-loaded mixed micelles. As shown in Table 3, the particle size, zeta potential, DL%, and EE% were explored for comparison. Little difference existed between the mixed micelles of TP/FM and TSP/FM. However, decoration with the APDTKTQ peptide increased the particle size of TSP/FP F


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Figure 2. Response surface results of overall desirability (OD) values of particle size, drug loading percentage (DL%), and drug encapsulation efficiency (EE%) by changing weight ratio of TSP/FP, weight ratio of polymer/drug, and the volume ratio of aqueous/organic.

Table 2. Optimized Conditions for Preparing ORI-Loaded Mixed Micelle with the Predicted and Observed Responses factors polymer/drug aqueous/organic TPGS−S−S−PLGA/F68−APDTKTQ

predicted responses 4.5 10 4.5

particle size drug loading entrapment efficiency

observed responses 105.60 17.05 74.30

was extremely low in a hydrophilic environment. In contrast, a strong fluorescence signal could be detected when Nile red was

particle size drug loading entrapment efficiency

predicted error (%) 107.40 16.05 72.24

1.68 6.23 2.85

located in a hydrophobic environment such as in the core of the micelles.32 Taking advantage of this property, we used the G


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group, indicating that the TSP/FP micelles should be considered biocompatible in drug delivery application. On the other hand, TSP/FP micelles tested at all concentrations exhibited enhanced cytotoxicity over free ORI of the micelles without conjugating the peptide. Specifically, 42.8% of BEL-7402 cells survived after the treatment with 10 μM TSP/FP micelles for 24 h, whereas 52.3% of BEL-7402 cells in the TSP/FM groups and more than 60% of BEL-7402 cells in both the free ORI and TP/FM groups were alive. However, in HepG2 cells with low RAGE expression, 78.9% of HepG2 cells survived after the treatment with 10 μM free ORI for 24 h, and similar cell viability was observed when treated with 10 μM TP/FM (63.9%), TSP/FM (61.5%), and TSP/FP micelles (60.1%), respectively (Figure 5B). Such results suggested that the increased cytotoxicity of TSP/FP micelles might be due to the existence of the APDTKTQ peptide, which had an affinity to the RAGE protein on the surface of BEL-7402 cells and promoted the endocytosis of the F68 mixed micelles, leading to an enhanced cytotoxic effect against BEL-7402 cells. 3.9. Apoptotic Effect on BEL-7402 Cells. ORI shows apoptosis-inducing activities to multiple cell lines through extrinsic apoptotic pathways or partly intrinsic pathways,34 for example, ORI-induced apoptosis of human breast cancer MDAMB-231 cell line through regulation of apoptosis-associated tarnscriptonal factors such as NF-κB, caspase-8, and Bcl-2/Bax.35 Herein, the pro-apoptotic activities of ORI loaded TSP/FP micelles were observed by cell nuclear morphology and assessed by AnnexinV-FITC/PI double staining. As shown in Figure 6, Hoechst 33342 staining revealed significant nuclear morphological changes in BEL-7402 cells after exposure to 10 μM TP/ FM, TSP/FM, TSP/FP micelles for 24 h. Untreated cells, which served as control, as well as the cells treated with 15 μg/mL of blank TSP/FP micelles, had regular-shaped nucleic that emitted weak and homogeneous blue signal. However, in the groups of free ORI or any micelle treatment, bright chromatin condensation was observed at higher frequency. Particularly, in the group of TSP/FP micelles treatment, cell number remarkably decreased and bright chromatin condensation and nuclear fragmentation were visualized, indicating that TSP/FP micelles exerted a stronger apoptotic effect than free ORI, TP/FM, and TSP/FM micelles. To further confirm the results, AnnexinV-FITC/PI double staining was performed to quantitatively monitor apoptosis. As shown in Figure 7, few apoptotic BEL-7402 cells were identified in control group and blank TSP/FP micelle group. Treatment with 20 μM ORI loaded TP/FM, TSP/FM, and TSP/FP micelles for 24 h significantly increased the population of apoptotic cells, with an apoptotic rate of approximately 78.2% for TSP/FP group compared with 51.2% for free ORI-treated groups. 3.10. Mitochondrial Membrane Potential (MMP). Since mitochondria play a vital role in cell’s fate and loss of MMP indicates cell apoptosis,36 efforts were made to measure the changes of the MMP in BEL-7402 cells. JC-1 probe, a fluorescent cationic dye, changes from red to green when cell apoptosis occurs and subsequently MMP decreases.37 As shown in Figure 8, homogeneous red fluorescence was observed in untreated cells and cells treated with blank TSP/FP micelles, while cells treated with free ORI or ORI loaded micelles emitted green fluorescence. Moreover, the green fluorescence was more pronounced in the TSP/FP group compared with that in the free ORI group, suggesting an enhanced early apoptotic effect induced by ORI-loaded TSP/FP micelles. The result was further confirmed using flow cytometry. Cells cultured on TSP/FP

Table 3. Characterization of ORI-Loaded Mixed Micelles ORI-loaded mixed micelles

particle size (nm)

zeta potential (mV)

drug loading (DL%)

encapsulation efficiency (EE %)

TP/FM TSP/FM TSP/FP

95.6 ± 4.2 94.8 ± 3.6 106 ± 5.4

−34.6 ± 2.2 −35.8 ± 2.8 −35.2 ± 2.6

16.68 ± 0.38 16.42 ± 0.42 16.16 ± 0.36

75.06 ± 1.72 73.88 ± 1.88 72.72 ± 1.62

fluorescent signal as an index to determine the Nile red release behavior and evaluate the state of the micelles. In Figure 3, panel C, after incubation with 10 mM DTT, the fluorescence intensity of Nile red loaded mixed micelles gradually declined; however, no change was observed after the micelles were treated with 20 μM DTT for 48 h (Figure 3D). These results indicated that the mixed micelles could be induced to rupture and release the drug inside the cancer cells containing a high concentration of GSH but remained stable in extracellular matrices. 3.5. CMC of the Mixed Micelles. The CMC value of TSP/ FP reflects the ability of the mixed micelles to self-assemble in aqueous solution and the capacity of dilution resistance. Pyrene was used as a fluorescent probe based on the fact that its fluorescent spectrum will vary when the polarity of its environment is changed. In Figure 3, panel E, the ratio of I373/ I384 increased sharply, confirming the successful formation of the micelles. The CMC value was calculated to be about 0.0138 mg/ mL, which indicated that the mixed micelles could self-assemble in aqueous solution and were capable of resisting dilution. 3.6. In Vitro Drug Release of the ORI-Loaded Mixed Micelles. The in vitro drug release of the free ORI as well as the ORI-loaded TP/FM, TSP/FM, and TPS/FP was investigated using the release medium, 0.5% Tween 80 supplemented with 20 μM, or 10 mM DTT (Figure 3F). The results showed that more than 80% of the free ORI was released, while only about 35% of ORI was released from the micelles in the release medium with 20 μM DTT after 4 h. This showed that the release profile of ORI loaded in the mixed micelles was slow but sustained. Moreover, the release of the ORI from TSP/FM and TSP/FP in 10 mM DTT was similar to that of the free ORI with a burst release (about 75%) within the initial 8 h. These results further confirmed the destabilization of the mixed micelles triggered by its redox-sensitivity. 3.7. RAGE Expression. As reported, RAGE is a receptor for diverse ligands, and the activation of RAGE is believed to mediate multiple cell functions such as cell migration and proliferation.33 Previous study has demonstrated that APDTKTQ peptide can selectively bind to RAGE. To determine the expression level of RAGE protein, total proteins extracted from BEL-7402, HepG2, and SMMC-7721 cells were detected by Western blot analysis. By using GAPDH as control, the level of RAGE protein was detected to be remarkably low in either HepG2 or SMMC-7721 cells, while BEL-7402 cells exhibited a relatively high level of RAGE expression (Figure 4). Therefore, BEL-7402 cells were considered as a high RAGE expression cell line and selected to conduct the subsequent experiments. 3.8. In Vitro Cytotoxicity of TSP/FP Micelles. MTT assay was conducted to assess the cytotoxicity of the ORI loaded TSP/ FP micelles compared with free ORI solution or micelles without the peptide. A dose-dependent cytotoxicity was noted in this assay. In BEL-7402 cells, the order of increasing cytotoxicity was TP/FM micelles ≈ free ORI < TSP/FM micelles < TSP/FP micelles. As shown in Figure 5, panel A, more than 90% BEL7402 cells incubated with blank TSP/FP at the concentration of 1.5 μg/mL for 24 and 48 h were viable compared with the control H


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Figure 3. (A) Particle size and the TEM image of TSP/FP mixed micelles treated with 20 μM DTT for 24 h. (B) Particle size and the TEM image of TSP/FP mixed micelles treated with 10 mM DTT for 24 h. Nile red release profiles with (C) 10 mM or (D) 20 μM DTT at different time points. (E) Intensity ratio of I373/I384 as a function of log concentration of TSP/FP for CMC value determination. (F) Drug release in vitro of ORI-loaded mixed micelles in the media with 20 μM or 10 mM DTT.

loaded ORI micelles (10 μM) had a lower red/green fluorescence intensity ratio (53.9%) than TP/FM micelles (68.2%), TSP/FM micelles (63.3%), loaded ORI, and free

ORI-treated group (78.3%). This result quantitatively demonstrated the ability of ORI loaded TSP/FP to dissipate MMP and trigger early apoptosis in target cells. I


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Figure 6. Nuclear morphology of BEL-7402 cells treated with (C) free ORI, (D) TP/FM, (E) TSP/FM, or (F) TSP/FP at the concentration of 10 μM for 24 h. (A) Untreated cells or (B) cells treated with blank micelles were served as control. Cells were observed after Hochest 33342 staining (Scale bar: 20 μm). Apoptotic cells were recognized by condensed nucleic and indicated with arrows.

intracellular fluorescence emitted by Nile red. As shown in Figure 9, panel A, the intracellular accumulation of Nile red was presented in a time-dependent manner. Uptake of each Nile redloaded micelles increased as the incubation time was prolonged from 1 to 4 h. Notably, when the incubation time lasted for 4 h, the fluorescence value of cells treated with TSP/FP micelles was twice compared to that of TP/FM or TSP/FM micelles, indicating that cellular internalization of Nile red-loaded TSP/FP micelles was significant higher than that of TP/FM or TSP/FM

Figure 4. Expression of RAGE in BEL-7402 cells, HepG2 cells, and SMMC-7721 cells.

3.11. Cellular Uptake. The enhanced cytotoxicity of ORI loaded TSP/FP micelles might be contributed by a higher degree of cellular penetration. We then measured the cellular uptake of TP/FM, TSP/FM, and TSP/FP. The cellular internalization of Nile red-loaded micelles can be measured by detecting

Figure 5. Effects of blank micelles, free ORI, TP/FM, TSP/FM, or TSP/FP on BEL-7402 and HepG2 cell viability after treatment for 24 and 48 h, respectively. J


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Figure 7. Quantitative apoptotic measurement of BEL-T402 cells after incubation with blank micelles, free ORI, TP/FM, TSP/FM, or TSP/FP (20 μM) for 24 h. Data were expressed as mean ± SE of three independent experiments. ∗, p < 0.05.

cytometry result, stronger red fluorescence from Nile red could be observed in cells treated with TSP/FP micelles than TP/FM and TSP/FM micelles after 4 h incubation (Figure 9B), implying

micelles. This result was associated with the specific binding of APDTKTQ peptide with RAGE receptor. This result was confirmed by the fluorescent images. In accordance with the flow K


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that TSP/FP micelles were uptaken by BEL-7402 more efficiently via binding to RAGE receptor. Cells pretreated with RAGE antibody showed little cellular accumulation of Nile red, further confirming the specific binding between APDTKTQ peptide and RAGE.

4. CONCLUSION In the present study, we developed an ORI-loaded TSP/FP mixed micelle system with redox-sensitivity for targeted elimination of HCC cells. TPGS−S−S−PLGA and F68− APDTKTQ conjugates were successfully synthesized by esterification reactions, which were confirmed by 1H NMR and FT-IR spectra. The synthesis conditions of ORI-loaded mixed micelles were optimized by the CCD/RSM. Prepared by the dialysis method, the ORI-loaded mixed micelles exhibited small particle size, negative zeta potential, and high DL% and EE%. On top of these properties, the mixed micelles remained stable in the medium with 20 μM DTT but ruptured when the concentration of DTT increased to 10 mM, evidenced by the changes of particle size, TEM images, and Nile red release pattern. Furthermore, the ORI-loaded mixed micelles posed higher cytotoxicity and induced more apoptosis in BEL-7402 cells, in comparison with free ORI. The micelles also enhanced the cellular uptake via the binding of APDTKTQ peptide to RAGE. Our findings suggest that ORI-loaded TSP/FP mixed micelles system can be a prospective therapeutic strategy for the treatment of hepatocellular carcinoma.

Figure 8. MMP changes in BEL-7402 cells after treatment of free ORI solution, TP/FM, TSP/FM, or TSP/FP for 24 h were stained with JC-1 and analyzed by flow cytometry. Results were expressed as ratio of red fluorescence to green fluorescence.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00116. Characterization of TPGS−S−S−PLGA conjugate; characterization of F68-ADPTKTQ (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Macao Science and Technology Development Fund (062/2013/A2), the Research Fund of the University of Macau (MYRG2014-00033-ICMS-QRCM, MYRG2014-00051-ICMS-QRCM, MYRG2015-00171-ICMSQRCM, MYRG2016-00130-ICMS-QRCM), and the National Natural Science Foundation of China (81403120).

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REFERENCES

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Figure 9. (A) Quantification of cellular uptake of BEL-7402 cells incubated with Nile red-loaded TP/FM, TSP/FM, TSP/FP without or with RAGE pretreatment for 1, 2, and 4 h. Untreated cells were used as control. Data were presented as mean ± SE of three independent experiments. ∗, p < 0.05. (B) Fluorescence microscopy images of BEL7402 cells after 2 h incubation with Nile red-loaded TP/FM (Scale bar: 20 μm) (a) TSP/FM or (b) TSP/FP (c) without or (d) with RAGE pretreatment.

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