Hai Sun

pH-Triggered Charge-Reversal Polypeptide Nanoparticles for Cisplatin Delivery: Preparation and In Vitro Evaluation

A series of pH-responsive random copolymer poly(l-glutamic acid-co-l-lysine) [P(Glu-co-Lys)] were synthesized through the ring-opening polymerization (ROP) of γ-benzyl-l-glutamate N-carboxyanhydride (BLG-NCA) and 3-benzyloxycarbonyl-l-lysine N-carboxyanhydride (ZLys-NCA) and the subsequent deprotection. The chemical structure of the P(Glu-co-Lys)s was confirmed by NMR. Critical aggregation concentration and transmission electron microscopy measurements indicated that the P(Glu-co-Lys)s could self-assemble into aggregates in phosphate buffer. The surface charge of P(Glu-co-Lys) aggregates was greatly affected by the solutions pH and l-glutamic acid/l-lysine ratio because the carboxyl and amino groups present on the P(Glu-co-Lys) aggregates could be protonated or deprotonated to become charged. The pH value of the solution at which the surface charge of the P(Glu-co-Lys) aggregates reversed could be manipulated by the feed ratio of BLG-NCA and ZLys-NCA. In vitro methyl thiazolyl tetrazolium assays demonstrated that negatively charged P(Glu-co-Lys)s were nontoxic and biocompatible. Positive charged P(Glu-co-Lys)s showed some cytotoxicity to Hela cells. Cisplatin (CDDP) was used as a model anticancer drug to evaluate the charge-reversal drug delivery system. By the manipulation of CDDP loading content, the surface charge of the CDDP/P(Glu-co-Lys) nanoparticles could be reversed to positive from negative at tumor extracellular pH (pHe 6.5-7.2). An enhanced drug uptake and inhibition of cancer cell proliferation were observed for the tumoral pHe triggered charge-reversal CDDP/P(Glu-co-Lys) drug delivery system. These indicated that the CDDP/P(Glu-co-Lys) nanoparticles could be used as intelligent drug delivery systems for cancer therapy.

Doxorubicin-loaded amphiphilic polypeptide-based nanoparticles as an efficient drug delivery system for cancer therapy.

An amphiphilic anionic copolymer, methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine) (mPEG-b-P(Glu-co-Phe)), with three functionalized domains, was synthesized and used as a nanovehicle for cationic anticancer drug doxorubicin hydrochloride (DOX·HCl) delivery via electrostatic interactions for cancer treatment. The three domains displayed distinct functions: PEG block chain for prolonged circulation; poly(phenylalanine) domain for stabilizing the nanoparticle construct through hydrophobic/aromatic interactions; and the poly(glutamic acid) domain for providing electrostatic interactions with the cationic drug to be loaded. The copolymer could self-assemble into micellar-type nanoparticles, and DOX was successfully loaded into the interior of nanoparticles by simple mixing of DOX·HCl and the copolymer in the aqueous phase. DOX-loaded mPEG-b-P(Glu-co-Phe) nanoparticles (DOX-NP) had a superior drug-loading content (DLC) (21.7%), a high loading efficiency (almost 98%) and a pH-triggered release of DOX. The size of DOX-NP was ∼140 nm, as determined by dynamic light scattering measurements and transmission electron microscopy. In vitro assays showed that DOX-NP exhibited higher cell proliferation inhibition and higher cell uptake in A549 cell lines compared with free DOX·HCl. Maximum tolerated dose (MTD) studies showed that DOX-NP demonstrated an excellent safety profile with a significantly higher MTD (15 mg DOX kg(-1)) than that of free DOX·HCl (5 mg DOX kg(-1)). The in vivo studies on the subcutaneous non-small cell lung cancer (A549) xenograft nude mice model confirmed that DOX-NP showed significant antitumor activity and reduced side effects, and then enhanced tumor accumulation as a result of the prolonged circulation in blood and the enhanced permeation and retention effect, compared with free DOX, indicating its great potential for cancer therapy.

Nanoscaled Poly(l-glutamic acid)/Doxorubicin-Amphiphile Complex as pH-responsive Drug Delivery System for Effective Treatment of Nonsmall Cell Lung Cancer

Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Herein, we develop a polypeptide-based block ionomer complex formed by anionic methoxy poly(ethylene glycol)-b-poly(L-glutamic acid) (mPEG-b-PLG) and cationic anticancer drug doxorubicin hydrochloride (DOX·HCl) for NSCLC treatment. This complex spontaneously self-assembled into spherical nanoparticles (NPs) in aqueous solutions via electrostatic interaction and hydrophobic stack, with a high loading efficiency (almost 100%) and negative surface charge. DOX·HCl release from the drug-loaded micellar nanoparticles (mPEG-b-PLG-DOX·HCl) was slow at physiological pH, but obviously increased at the acidic pH mimicking the endosomal/lysosomal environment. In vitro cytotoxicity and hemolysis assays demonstrated that the block copolypeptide was cytocompatible and hemocompatible, and the presence of copolypeptide carrier could reduce the hemolysis ratio of DOX·HCl significantly. Cellular uptake and cytotoxicity studies suggested that mPEG-b-PLG-DOX·HCl was taken up by A549 cells via endocytosis, with a slightly slower cellular internalization and lower cytotoxicity compared with free DOX·HCl. The pharmacokinetics study in rats showed that DOX·HCl-loaded micellar NPs significantly prolonged the blood circulation time. Moreover, mPEG-b-PLG-DOX·HCl exhibited enhanced therapeutic efficacy, increased apoptosis in tumor tissues, and reduced systemic toxicity in nude mice bearing A549 lung cancer xenograft compared with free DOX·HCl, which were further confirmed by histological and immunohistochemical analyses. The results demonstrated that mPEG-b-PLG was a promising vector to deliver DOX·HCl into tumors and achieve improved pharmacokinetics, biodistribution and efficacy of DOX·HCl with reduced toxicity. These features strongly supported the interest of developing mPEG-b-PLG-DOX·HCl as a valid therapeutic modality in the therapy of human NSCLC and other solid tumors.

pH and reduction dual-responsive nanogel cross-linked by quaternization reaction for enhanced cellular internalization and intracellular drug delivery

A novel pH and reduction dual-responsive nanogel with improved cellular internalization was prepared through atom transfer radical polymerization and subsequent quaternization reaction. Doxorubicin (DOX), a model anticancer drug, was loaded into the nanogel via dispersion. The DOX-loaded nanogel presented a stable core-cross-linked structure under physiological conditions, but quickly released its payload in an acidic (pH 6.8) and reductive (10.0 mM glutathione) environment. Confocal fluorescence microscopy and fluorescence flow cytometry revealed that the DOX-loaded nanogel could deliver DOX into the cytoplasm and nucleus of cells, more efficiently than that of free DOX. The improved cellular internalization was more significant under acidic and reductive conditions, which was analogous to the pH and reductive conditions in endosomes and cytoplasm. In vitro cytotoxicity studies demonstrated that the pH and reduction responsive DOX-loaded nanogel could inhibit cellular proliferation more efficiently than free DOX. This dual-bioresponsive nanogel with quaternary ammonium salt group has appeared to be highly promising in the further development of intracellular drug transporters.

Anti-tumor efficacy of c(RGDfK)-decorated polypeptide-based micelles co-loaded with docetaxel and cisplatin

There are two important obstacles for the currently applied anti-cancer drug delivery systems. One is the conflict between long-circulation and cellular uptake while the other one is the achievement of ideal anti-cancer efficacy. To solve these problems, we designed a polypeptide-based micelle system that combined the advantages of receptor mediated endocytosis and multi-drug delivery. Firstly, an amphiphilic PLG-g-Ve/PEG graft copolymer was prepared by grafting α-tocopherol (Ve) and polyethylene glycol (PEG) to poly(l-glutamic acid) (PLG). Then docetaxel (DTX) and cisplatin (CDDP) were co-loaded into the PLG-g-Ve/PEG micelles via hydrophobic and chelation effect. After that, the surface of the dual-drug-loaded micelles was decorated with an αvβ3 integrin targeting peptide c(RGDfK). The targeted dual-drug-loaded micelles showed synergistic cytotoxicity and enhanced internalization rate in mouse melanoma (B16F1) cells. In vivo tests demonstrated that remarkable long circulation, anti-tumor and anti-metastasis efficacy could be achieved using this drug delivery system. This work revealed a strategy for the design and preparation of anti-cancer drug delivery systems with reduced side effect, enhanced anti-tumor and anti-metastasis efficacy.

Polypeptide-based combination of paclitaxel and cisplatin for enhanced chemotherapy efficacy and reduced side-effects.

A novel methoxy poly(ethylene glycol)-b-poly(l-glutamic acid)-b-poly(l-phenylalanine) (mPEG-b-P(Glu)-b-P(Phe)) triblock copolymer was prepared and explored as a micelle carrier for the co-delivery of paclitaxel (PTX) and cisplatin (cis-diamminedichlo-platinum, CDDP). PTX and CDDP were loaded inside the hydrophobic P(Phe) inner core and chelated to the middle P(Glu) shell, respectively, while mPEG provided the outer corona for prolonged circulation. An in vitro release profile of the PTX+CDDP-loaded micelles showed that the CDDP chelation cross-link prevented an initial burst release of PTX. The PTX+CDDP-loaded micelles exhibited a high synergism effect in the inhibition of A549 human lung cancer cell line proliferation over 72 h incubation. For the in vivo treatment of xenograft human lung tumor, the PTX+CDDP-loaded micelles displayed an obvious tumor inhibiting effect with a 83.1% tumor suppression rate (TSR%), which was significantly higher than that of a free drug combination or micelles with a single drug. In addition, more importantly, the enhanced anti-tumor efficacy of the PTX+CDDP-loaded micelles came with reduced side-effects. No obvious body weight loss occurred during the treatment of A549 tumor-bearing mice with the PTX+CDDP-loaded micelles. Thus, the polypeptide-based combination of PTX and CDDP may provide useful guidance for effective and safe cancer chemotherapy.

Polymeric topology and composition constrained polyether-polyester micelles for directional antitumor drug delivery.

Amphiphilic linear and dumbbell-shaped poly(ethylene glycol)-poly(lactide-co-glycolide) (PEG-PLGA) copolymers were simply synthesized by the ring-opening polymerization of lactide and glycolide using PEG or tetrahydroxyl-functionalized PEG as the macroinitiator and stannous octoate as the catalyst. The copolymers spontaneously self-assembled into spherical micelles in phosphate-buffered saline at pH 7.4. The self-assembly behavior was dependent on both the polymeric topology and composition. Doxorubicin (DOX), an anthracycline antitumor drug, was loaded into micelles through nanoprecipitation. The in vitro release behavior could be adjusted by regulating the topology or composition of the copolymer, or the pH of the release medium. The effective intracellular DOX release from DOX-loaded micelles was confirmed by confocal laser scanning microscopy and flow cytometry in vitro. DOX-loaded micelles displayed great cellular proliferation inhibition efficacies after incubation for 24, 48 or 72 h. The hemolysis ratio of DOX was significantly reduced by the presence of copolymer. These properties indicated that the micelles derived from linear or dumbbell-shaped copolymers were promising candidates as smart antitumor drug carriers for malignancy therapy.

In vitro evaluation of anticancer nanomedicines based on doxorubicin and amphiphilic Y-shaped copolymers

Four monomethoxy poly(ethylene glycol)-poly(L-lactide-co-glycolide)2 (mPEG-P( LA-co-GA)2) copolymers were synthesized by ring-opening polymerization of L-lactide and glycolide with double hydroxyl functionalized mPEG (mPEG-(OH)2) as macroinitiator and stannous octoate as catalyst. The copolymers self-assembled into nanoscale micellar/vesicular aggregations in phosphate buffer at pH 7.4. Doxorubicin (DOX), an anthracycline anticancer drug, was loaded into the micellar/vesicular nanoparticles, yielding micellar/vesicular nanomedicines. The in vitro release behaviors could be adjusted by content of hydrophobic polyester and pH of the release medium. In vitro cell experiments showed that the intracellular DOX release could be adjusted by content of P(LA-co-GA), and the nanomedicines displayed effective proliferation inhibition against Henrietta Lacks’s cells with different culture times. Hemolysis tests indicated that the copolymers were hemocompatible, and the presence of copolymers could reduce the hemolysis ratio of DOX significantly. These results suggested that the novel anticancer nanomedicines based on DOX and amphiphilic Y-shaped copolymers were attractive candidates as tumor tissular and intracellular targeting drug delivery systems in vivo, with enhanced stability during circulation and accelerated drug release at the target sites.

Polypeptide/Doxorubicin Hydrochloride Polymersomes Prepared Through Organic Solvent-free Technique as a Smart Drug Delivery Platform

Rapid and efficient side-chain functionalization of polypeptide with neighboring carboxylgroups is achieved via the combination of ring-opening polymerization and subsequent thiol-yne click chemistry. The spontaneous formation of polymersomes with uniform size is found to occur in aqueous medium via electrostatic interaction between the anionic polypeptide and cationic doxorubicin hydrochloride (DOX·HCl). The polymersomes are taken up by A549 cells via endocytosis, with a slightly lower cytotoxicity compared with free DOX ·HCl. Moreover, the drug-loaded polymersomes exhibit the enhanced therapeutic efficacy, increase apoptosis in tumor tissues, and reduce systemic toxicity in nude mice bearing A549 lung cancer xenograft, in comparison with free DOX ·HCl.

Thermogel-mediated sustained drug delivery for in situ malignancy chemotherapy

In the past few decades, the in situ sustained drug delivery platforms present fascinating potential in sentinel chemotherapy of various solid tumors. In this work, doxorubicin (DOX), a model antitumor drug, was loaded into the thermogel of poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide). The moderate mechanical property of DOX-loaded hydrogel was confirmed by rheological test. In vitro degradation revealed the good biodegradability of thermogel. The DOX-loaded hydrogel exhibited the sustained release profiles up to 30days without and even with elastase. The improved in vivo tumor inhibition and reduced side-effects were observed in the DOX-incorporated hydrogel group compared with those in free DOX group. The excellent in vivo results were further confirmed by the histopathological evaluation or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The thermogel with great prospect may be used as an ideal controlled drug delivery platform for the designated and long-term antitumor chemotherapy.