Huiyun Wen

Engineered Redox-Responsive PEG Detachment Mechanism in PEGylated Nano-Graphene Oxide for Intracellular Drug Delivery

In biomedical applications, polyethylene glycol (PEG) functionalization has been a major approach to modify nanocarriers such as nano-graphene oxide for particular biological requirements. However, incorporation of a PEG shell poses a significant diffusion barrier that adversely affects the release of the loaded drugs. This study addresses this critical issue by employing a redox-responsive PEG detachment mechanism. A PEGylated nano-graphene oxide (NGO-SS-mPEG) with redox-responsive detachable PEG shell is developed that can rapidly release an encapsulated payload at tumor-relevant glutathione (GSH) levels. The PEG shell grafted onto NGO sheets gives the nanocomposite high physiological solubility and stability in circulation. It can selectively detach from NGO upon intracellular GSH stimulation. The surface-engineered structures are shown to accelerate the release of doxorubicin hydrochloride (DXR) from NGO-SS-mPEG 1.55 times faster than in the absence of GSH. Confocal microscopy shows clear evidence of NGO-SS-mPEG endocytosis in HeLa cells, mainly accumulated in cytoplasm. Furthermore, upon internalization of DXR-loaded NGO with a disulfide-linked PEG shell into HeLa cells, DXR is effectively released in the presence of an elevated GSH reducing environment, as observed in confocal microscopy and flow cytometric experiments. Importantly, inhibition of cell proliferation is directly correlated with increased intracellular GSH concentrations due to rapid DXR release.

Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers.

A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency.

Multifunctional nanocomposite based on graphene oxide for in vitro hepatocarcinoma diagnosis and treatment

Because of its unique chemical and physical properties, graphene oxide (GO) has attracted a large number of researchers to explore its biomedical applications in the past few years. Here, we synthesized a novel multifunctional nanocomposite based on GO and systemically investigated its applications for in vitro hepatocarcinoma diagnosis and treatment. This multifunctional nanocomposite named GO-PEG-FA/Gd/DOX was obtained as the following procedures: gadolinium-diethylenetriamine-pentaacetic acid-poly(diallyl dimethylammonium) chloride (Gd-DTPA-PDDA) as magnetic resonance imaging (MRI) probe was applied to modify GO by simple physical sorption with a loading efficiency of Gd(3+) up to 0.314 mg mg(-1). In order to improve its tumor targeting imaging and treatment efficiency, the obtained intermediate product was further modified with folic acid (FA). Finally, the nanocomposite was allowed to load anticancer drug doxorubicin hydrochloride via π-π stacking and hydrophobic interaction with the loading capacity reaching 1.38 mg mg(-1). MRI test revealed that GO-PEG-FA/Gd/DOX exhibit superior tumor targeting imaging efficiency over free Gd(3+). The in vitro release of DOX from the nanocomposite under tumor relevant condition (pH 5.5) was fast at the initial 10 h and then become relatively slow afterward. Moreover, we experimentally demonstrated that the multifunctional nanocomposite exhibited obviously cytotoxic effect upon cancer cells. Above results are promising for the next in vivo experiment and make it possible to be a potential candidate for malignancy early detection and specific treatment.

Glutathione-mediated shedding of PEG layers based on disulfide-linked catiomers for DNA delivery

Engineered PEG-detachable catiomers were developed as non-viral gene vectors to detach the PEG layers responsive to the intracellular reducing environment. These catiomers were found to exhibit high DNA binding ability and reduced cytotoxicity, as evidenced in agarose gel electrophoresis and MTT assays. The size of the mPEG-SS-PLL/DNA complexes was around 100 nm with a regular spherical shape, as observed under transmission electron microscopy (TEM). The complexes were stably dispersed in an aqueous medium with 10% serum. However, fast aggregation was observed in the presence of 10 mM glutathione (GSH) due to detachment of the PEG segment via disulfide cleavage. These complexes showed high transfection efficiency in 293T and Hela cells under optimized conditions. The experimental results indicated that the mPEG-SS-PLL catiomers may have promising potential as a non-viral gene vector.

Novel vesicles self-assembled from amphiphilic star-armed PEG/polypeptide hybrid copolymers for drug delivery.

A novel amphiphilic four-armed [poly(ε-benzyloxycarbonyl-L-lysine)]₂-block-poly(ethylene glycol)-block-[poly(ε-benzyloxycarbonyl-L-lysine)]₂ hybrid copolymer has been prepared. The cytotoxicity study shows that the copolymer has good biocompatibility with no obvious inhibition effect on cell growth. The amphiphilic copolymers could self-assemble to form vesicles in aqueous solution. DOX · HCl, as a hydrophilic drug, can be loaded into the vesicles, and then successfully internalized by human breast cancer MCF-7 cells. Importantly, the DOX-loaded vesicles show a greatly improved drug release behavior with a zero-order release at the initial stage, suggesting a great potential as the carrier of hydrophilic drugs for controlled drug delivery.

Highly Efficient Drug Delivery Nanosystem via L‐Phenylalanine Triggering Based on Supramolecular Polymer Micelles

An intelligent drug delivery nanosystem has been developed based on biodegradable supramolecular polymer micelles (SMPMs). The drug release can be triggered from SMPMs responsively by a bioactive agent, L-phenylalanine in a controlled fashion. The SMPMs are constructed from ethylcellulose-graft-poly(ε-caprolactone) (EC-g-PCL) and α-cyclodextrin (α-CD) derivate via host-guest and hydrophobic interactions. It has been found that these SMPMs have disassembled rapidly in response to an additional L-phenylalanine, due to great affinity discrepancy to α-CD between L-phenylalanine and PCL. Experiments have been carried out on trigger-controlled in vitro drug release of the SMPMs loaded with a model porphyrin based photosensitizer THPP. The result shows that the SMPMs released over 85% THPP in 6 h, which is two orders magnitudes faster than that of control. Also investigated is the photodynamic therapy (PDT) of THPP-loaded SMPMs with and without L-phenylalanine on MCF-7 carcinoma cell line. An effective trigger-concentration dependent lethal effect has been found showing promise in clinical photodynamic therapy.

Folate conjugated PEG-chitosan/graphene oxide nanocomplexes as potential carriers for pH-triggered drug release.

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Redox-mediated dissociation of PEG–polypeptide-based micelles for on-demand release of anticancer drugs

Intelligent nanoparticles are capable of prolonged blood circulation without leakage of the payload and fast drug release upon exposure to environmental stimuli, such as redox stimuli, and therefore are highly desirable for cancer therapy. In this study, polymeric micelles were designed and developed with a hydrophilic poly(ethylene glycol) (PEG) shell and a hydrophobic poly-l-phenylalanine (PPhe) core, linked by a redox cleavable bond, i.e. mPEG-SS-PPhe. The mPEG-SS-PPhe micelles were loaded with the anticancer drug doxorubicin (DOX) and shown an on-demand release profile in the presence of redox agents such as glutathione (GSH). Remarkably, the GSH-triggered micellar dissociation accelerated in vitro release of DOX 4.87 fold faster at 10 mM GSH than that without GSH at 12 h. An enhanced inhibitory effect of DOX-loaded mPEG-SS-PPhe micelles was achieved by improving the intracellular GSH levels. Confocal laser scanning microscopy and flow cytometric analyses of HeLa cells further confirmed that DOX accumulation was accelerated by elevating the extracellular GSH concentrations. In addition, mPEG-SS-PPhe micelles showed excellent biocompatibility on L929 and HeLa cell lines. These redox-sensitive polymeric micelles may provide more possibilities as promising carriers for on-demand drug release in a controlled manner.

AMF responsive DOX-loaded magnetic microspheres: transmembrane drug release mechanism and multimodality postsurgical treatment of breast cancer

DOX-loaded magnetic alginate-chitosan microspheres (DM-ACMSs) were developed as a model system to evaluate alternating magnetic field (AMF)-responsive, chemo-thermal synergistic therapy for multimodality postsurgical treatment of breast cancer. This multimodality function can be achieved by the combination of DOX for chemotherapy, with superparamagnetic iron oxide nanoparticles (SPIONs) as magnetic hyperthermia agents and drug release trigger. Both moieties are encapsulated in ACMSs which also allow on-demand drug release. It is demonstrated that the optimized SPION content in DM-ACMSs is about 0.29 mg Fe, at which DM-ACMSs could exhibit the best hyperthermia performance. Under a remote AMF, DM-ACMs can quickly reach a 22.5% cumulative drug release in the tumor site within 10 min upon exposure under AMF, whereas only 0.2% DOX is released in the absence of AMF. Furthermore, a comparison study of AMF and water bath as heating source indicates that the cumulative drug release amount upon AMF exposure is twice that by water bath heating. Further analysis revealed that the AMF stimulated drug release is driven by both thermal and concentration gradient from inside to outside, which can be well-described by the coupling mechanism of mass and heat transfer using the Soret diffusion model. In vitro cytotoxicity tests on MCF-7 breast cancer cells show that the combined therapy based on DM-ACMSs leads to 95.5% cell death, about 1.5-fold and 1.1-fold higher than that of single magnetic hyperthermia or chemotherapy, respectively. The in vivo anti-tumor effect on tumor-bearing mice demonstrates that the residual tumor disappears in 12 days after chemo-thermal synergistic treatment using DM-ACMSs, and there is no recurrence in the entire experiment period (40 days) as compared to 25 days recurrence for single-modality treatment. Our results not only provide an innovative DM-ACMSs system as a stimuli-responsive, synergistic chemo-thermal therapy platform for efficient reduction in the recurrence of breast cancer, but also provide insight into the intricate interplay of the functional components in magnetic hydrogel microspheres.