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Home > A pH-Responsive Charge-Reversal Drug Delivery System with Tumor-Specific Drug Release and ROS Generation for Cancer Therapy
A pH-Responsive Charge-Reversal Drug Delivery System with Tumor-Specific Drug Release and ROS Generation for Cancer Therapy
Introduction
During the past decades, polymeric prodrug-based drug delivery systems (PPDDSs) have emerged as a promising platform for cancer therapy. Compared with conventional chemotherapeutic drugs, PPDDSs for cancer therapy have the following advantages: improved drug solubility, prolonged circulation time, improved tumor selectivity, and reduced side effects.3,4 Along with their advantages in terms of prodrug delivery and stimuli-responsive drug release capabilities, PPDDSs could obviously improve the drug accumulation in the tumor tissue via the enhanced permeability and retention (EPR) effect, thereby remarkably increasing the therapeutic effects.5–7 However, the poor tumor cells’ internalization and incomplete drug release are the two major drawbacks hindering the clinical translation of PPDDSs.
Poly(ethylene glycol) (PEG) is mostly used as the hydrophilic component of PPDDSs because of its good hydrophilicity, biocompatibility, and sustained blood circulation.2,8 However, PEG surface modification could impede the uptake of PPDDSs by cancer cells.9–11 It has been reported that the positively surface-charged nanoparticles can improve cellular uptake via electrostatic attraction to negatively charged cell membranes.12,13 However, rapid body elimination and tissue toxicity hindering the application of positively surface-charged nanoparticles.14,15 Recently, a new strategy of combining the advantages of PEGylation and positively surface charged, named as surface charge-reversal strategy, has been used for drug delivery.12–16 Such PPDDSs could maintain a negative charged under physiological conditions (pH 7.4) to reduce protein adsorption and avoid clearance by the reticuloendothelial system (RES) and then charge to a positive surface charge under conditions of extracellular acidity (pH 6.0–7.0) to improve cellular uptake.
After internalization in cancer cells, drugs should be released selectively at the tumor site in a tumor-specific manner, using cues such as an acidic environment, increased secretion of enzymes, or high levels of glutathione (GSH) or reactive oxygen species (ROS).17,18 Because both cancer and healthy cells have lysosomes with an acidic pH and high intracellular levels of GSH, pH- and GSH-triggered drug release mechanisms offer limited selectively between healthy and cancer cells.19 However, ROS levels in cancer cells are tens-to-hundreds times higher than those in healthy cells, and therefore ROS-responsive drug delivery systems provide more tumor-specific drug release.20,21 Various ROS- sensitive linkages such as thioketal (TK), boronic ester, and alkylene sulfide have been widely evaluated in ROS- responsive drug delivery systems for cancer treatment.22,23 Unfortunately, variations in endogenous ROS concentrations because of tumor heterogeneity result in incomplete complete drug release from ROS-sensitive drug delivery systems in vivo.5,24,25 Therefore, pH/ROS charge-reversal PPDDSs with ROS production capabilities represent a promising alternative strategy to overcome the drawbacks of PPDDSs.
Menadione, also named VK3, a quinone-type natural molecule, showed an anti-tumor effect against prostate, lung, hepatic, and breast cancer.26,27 Moreover, many studies have shown that VK3 can produce ROS under catalysis by NAD(P)H: quinone oxidoreductase-1 (NQO1).25,27,28 Because NQO1 is specifically overexpressed in cancer cells, VK3 can specifically increase ROS levels in tumor cells rather than in healthy cells.29,30 Therefore, ROS- responsive PPDDSs co-loaded with VK3 could remarkably amplify ROS levels for complete drug release.
To overcome the aforementioned drawbacks, in the current study, we report a self-amplifiable drug release PPDDS with charge reversal capability, created by loading VK3 in a pH/ROS dual-responsive micelle nanoparticle (Scheme 1). In this nanosystem, a ROS-sensitive paclitaxel (PTX) pro-drug (PTX-TK) and 2,3-dimethyl maleic anhydride (DMA) were conjugated to the amino groups of PEG-b-PLL to produce a pH/ROS dual-responsive component: PEG-bP((LL-g-TK-PTX)-(LL-g-DMA)), and then VK3 was encapsulated into the polymer micelles formed by PEG-bP((LL-g-TK-PTX)-(LL-g-DMA)) (denoted as PVD-NPS). After intravenous administration to mice, PVD- NPs could ideally maintain a negative surface charge in blood circulation, which quickly changed to a positive charge when they reached tumor tissue, allowing them to be quickly ingested by cancer cells. Finally, endogenous ROS can trigger PTX and VK3 release, and the released VK3 could induce ROS generation, consequently amplify- ing drug release.
Experimental Section
Materials
PTX was purchased from Beijing Huafeng United Technology Co., Ltd (Beijing, China). Vitamin K3, 2.3-dimethyl maleic anhydride (DMA), succinic anhydride (SA) and 3-mercaptopropionic acid were purchased from Aladdin Reagent Company (Shanghai, China). Poly(ethylene glycol)-b-poly(L-lysine) (PEG-b-PLL) was synthesized as previously reported.1 Dichlorofluorescindiacetate (DCFA-DA), a BCA kit, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) and DAPI were purchased from Beyotime Institute of Biotechnology (Shanghai, China).
Animal and Cell Lines
The human prostate cancer cell line PC-3 and mouse embryonic fibroblast NIH-3T3 cells were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). PC-3 cells were cultured in RPMI 1640 culture medium, containing 10% (v/v) fetal bovine serum, 100 IU/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. The NIH-3T3 cells were cultured in DMEM culture medium, containing 10% (v/v) bovine calf serum, 1% glutamate, 1% non-essential amino acids, 1% sodium pyruvate (100 mM), 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere.
BALB/c nude mice (male, 4–6 w, 20 ± 2 g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and all procedures were approved by The First Affiliated Hospital of Nanjing Medical University Care and Use Committee.
Characterization
Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AVANCE III spectrometer at 300 MHz with deuterated dimethyl sulfoxide (DMSO-d6) or D2 O as the solvent. The size, size distribution, and the zeta potential of particles in aqueous solution were determined through dynamic light scattering (DLS, Zs90, Malvern Instruments, Malvern, UK). The morphology of the particles was investigated using transmission electron microscopy (TEM, Hitachi Ltd, Tokyo, Japan).
Synthesis of Thioketal (TK)
The TK linker was prepared as previously reported.24 Briefly, anhydrous 3-mercapto propionic (6.0 g, 56.6 mmol) and anhydrous acetone (6.8 g, 115.6 mmol) were mixed and stirred at room temperature for 6 h under dry hydrogen chloride. At the end of the reaction, the flask was placed in an ice-salt bath until the crystallization was completed. Then, the mixture was filtered, washed with abundant hexane and ice-cold water. The TK product was obtained after drying under vacuum.
Synthesis of TK-PTX
TK-PTX was prepared as previously reported.31 Briefly, TK (201.6 mg, 0.8 mmol), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, 230.4 mg, 1.2 mmol), and N-hydroxysuccinimide (NHS, 138.0 mg, 1.2 mmol) were dissolved in 40 mL of dry N, N-dimethylformamide (DMF) and the reaction mixture was maintained under stirring for 2 h under nitrogen atmosphere at room temperature. Then, PTX (427.0 mg, 0.5 mmol) and 4-dimethylamine- pyridine (73.2 mg, 0.6 mmol) were added to the solution and incubated for a further 24 h under nitrogen atmosphere at room temperature. Finally, the solution was precipitated with 400 mL of 0.1 M pre-chilled diluted hydrochloric acid. TK-PTX was obtained through vacuum drying.
Synthesis of PEG-b-P(LL-g-TK-PTX)
In brief, TK-PTX (2.176 g, 2.0 mmol), EDC (480 mg,
2.5 mmol) and NSH (287.5 mg, 2.5 mmol) were dissolved in 80 mL of dry DMF and stirred for 4 h at room temperature under the nitrogen atmosphere. Then, PEG-b-PLL (2.93 g, 0.4 mmol) was dissolved in 40 mL of dry DMF and added to the mixture, which underwent further stirring at room temperature for 48 h in a nitrogen atmosphere. Finally, the mixture was dialyzed (MWCO, 3500 Da) against DMF to remove unreacted small molecules and then, dialyzed against distilled water to remove DMF. The product PEG-b-P(LL-g-TK-PTX) was obtained through lyophilization.
Synthesis of PEG-bP(LL-g-TK-PTX)-(LL-g-DMA)
Briefly, PEG-bP(LL-g-TK-PTX) (1.7 g, 0.1 mmol) and DMA (252.0 mg, 2.0 mmol) were dissolved in 50 mL of DMF, and then TEA (150 µL) was added under nitrogen atmosphere. The mixture was stirred at room temperature for 12 h. At end of the reaction, the mixture was dialyzed (MWCO 3500 Da) against distilled water at pH 8–9 for 24 h and the product, PEG-bP(LL-g-TK-PTX)-(LL-g-DMA), was obtained through lyophilization.
As a control, SA was reacted with PEG-bP(LL-g-TK- PTX) using the same method mentioned above to obtain a no-charge-conversion polymer prodrug: PEG-bP((LL- g-TK-PTX)-(LL-g-SA)). PTX content in the PEG-bP((LL-g-TK-PTX)-(LL-g-DMA)) and PEG-bP((LL-g-TK-PTX)-(LL-g-SA)) was determined using a 1H NMR and UV spectrophotometer at a wavelength of 254 nm. The PTX content was calculated using the following formula: weight of PTX PTX% ¼ weight of polymer ×100%
Nanoparticles Preparation
VK3 and PTX co-loaded nanoparticles were prepared using the coprecipitation method. Typically, PEG-bP((LL-g-TK- PTX)-(LL-g-DMA)) (15 mg) and VK3 (3 mg) were dissolved in 1 mL of DMF and stirred for 1 h at room temperature. Then, the mixture was added dropwise into 10 mL of distilled water under vigorous stirring and then dialyzed (Mw: 3500 Da) against water for 12 h. The PVD-NPs were obtained after filtration using a Millipore filter (pore size: 0.45 µm) to remove unencapsulated VK3. The control groups used the following treatments: 1) PTX-loaded PPDDSs named PD- NPs; 2) PTX and VK3 co-loaded PPDDSs with no surface charge-reversal capability named PVS-NPs. Moreover, coumarin-6 loaded PVD-NPs and PVS-NPs were also prepared using the same method.
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