Xuan Zeng

The influence of RGD addition on the gene transfer characteristics of disulfide-containing polyethyleneimine/DNA complexes

Arginine-glycine-aspartic acid (RGD) ligand is often chemically attached to polycation vector to improve the transfection efficiency. However, the chemical reaction may reduce or even inactivate the biological activities of peptides. In order to retain the targeting ability and biological activities, the RGD peptide was noncovalently introduced into polycations as gene delivery systems. In this paper, the tripeptide sequence RGD was added to disulfide-containing polyethyleneimine (SS-PEI)/DNA binary complexes to evaluate the influence of RGD addition for the particle size, zeta potential, morphology, and transfection efficiency. GelRed was used as a molecular probe to show the effect of RGD addition on the cellular uptake of complexes. In vitro transfection experiments showed that SS-PEI exhibited comparable transfection efficiency, but lower cytotoxicity in comparison with 25kDa PEI. The transfection efficiency of complexes with RGD in HeLa cells was reduced statistically significantly with the increasing content of RGD peptide, but that in 293T cells was not altered significantly with the increasing content of RGD peptide. The reduced transfection efficiency of SS-PEI/DNA complexes with RGD in HeLa cells was attributed to the targeted binding interactions between the surplus RGD and the alpha(nu)beta(3) and alpha(nu)beta(5) integrins in HeLa cells, which would prevent the binding between RGD in complexes and integrin receptor on the surface of cells as well as nonspecific endocytosis of SS-PEI/DNA complexes mediated by proteoglycan in HeLa cells.

Targeted gene delivery mediated by folate-polyethylenimine-block-poly(ethylene glycol) with receptor selectivity.

The folate receptor (FR) is a tumor marker overexpressed in large numbers of cancer cells. Folic acid has high affinity to the FR and retains its binding affinity upon derivatization via its gamma-carboxyl. Therefore, in this article, folate-polyethylenimine-block-poly(ethylene glycol) (FOL-PEI-b-PEG) was designed for specific receptor targeted gene delivery. Physicochemical characterizations of resulting FOL-PEI-b-PEG/DNA complexes in terms of agarose gel electrophoresis, particle size, and zeta potential measurements were investigated. The results indicated that FOL-PEI-b-PEG was able to condense plasmid DNA tightly with a suitable particle size. The cytotoxicity study indicated that the copolymer exhibited less toxicity in comparison with that of 25 kDa PEI. Luciferase assay and green fluorescent protein (GFP) detections were also used to confirm that FOL-PEI-b-PEG could be an effective gene vector. Importantly, transfection efficiency of FOL-PEI-b-PEG with free folic acid was much lower than that of the copolymer without free folic acid on FR-positive HeLa cells, suggesting that FOL-PEI-b-PEG has great potential as a targeting gene vector.

Effect of Molecular Weight and Degree of Substitution of Quaternized Cellulose on the Efficiency of Gene Transfection

Quaternized celluloses (QCs) with different molecular weight (M(w)) and degree of cationic substitution (DS) were homogeneously synthesized in NaOH/urea aqueous solutions and were studied as gene carriers. QCs were evaluated for efficacy of nanoparticle formation, DNA binding efficiency, morphology, and in vitro gene transfection efficiency. The factors affecting the transfection efficiency, e.g., M(w), DS, and N/P ratios, have been evaluated. The cytotoxicity of QCs and QC/DNA complexes were evaluated in 293T cells and were found to be relatively low compared with 25 kDa PEI and PEI/DNA complex, which increased slightly with increasing of M(w) and DS. All QCs obtained could bind DNA efficiently, and QC/DNA complexes exhibited effective transfection in comparison to the naked DNA. More importantly, the QC/DNA complexes, were stable and the transfection efficiency was not inhibited in the presence of serum. The results revealed an important combined effect between M(w) and DS of QCs in determining transgene expression, and QCs with M(w) of about 8 x 10(4) g/mol and DS of about 0.6 displayed relatively higher transfection efficiencies attributed to the intermediate stability.

Protamine sulfate/poly(l-aspartic acid) polyionic complexes self-assembled via electrostatic attractions for combined delivery of drug and gene

In this study, a series of self-assembled polyionic complexes (PICs) were prepared via electrostatic attraction between protamine sulfate (PS) and poly(L-aspartic acid) (PASP) or doxorubicin (DOX)-conjugated PASP (DOX-PASP). The size of the PICs measured by Nano-ZS ZEN3600 was around 200-300 nm at different weight ratios of PS/PASP. Transmission electron microscopy (TEM) showed that PS/PASP PICs displayed a regular spherical shape and no aggregation was observed. The cytotoxicity study indicated that the PICs did not exhibit apparent cytotoxicity in comparison with that of 25 kDa polyethylenimine (PEI). Gel retardation assay indicated that the PICs were able to bind DNA completely when weight ratio of PS/PASP was higher than 2:1. Luciferase assay and green fluorescent protein (GFP) detection were used to confirm that the PICs could be used as efficient non-viral gene vectors and they exhibited comparable transfection efficiency with the one of 25 kDa PEI. Furthermore, confocal laser scanning microscopy as well as suppression activity of DOX-conjugated PICs (DOX-PICs) showed that they could quickly release the loaded DOX into HeLa cells, indicating that PICs can be also used as carriers for combined delivery of drug and gene.

A boronate-linked linear-hyperbranched polymeric nanovehicle for pH-dependent tumor-targeted drug delivery.

Advanced drug delivery systems, which possess post-functionalization feasibility to achieve targetability and traceability, favorable pharmacokinetics with dynamic but controllable stability, and preferable tumor accumulation with prolonged drug residence in disease sites, represent ideal nanomedicine paradigm for tumor therapy. To address this challenge, here we reported a dynamic module-assembly strategy based on reversible boronic acid/1,3-diol bioorthogonality. As a prototype, metastable hybrid nanoself-assembly between hydrophobic hyperbranched diol-enriched polycarbonate (HP-OH) and hydrophilic linear PEG terminated with phenylboronic acid (mPEG-PBA) is demonstrated in vitro and in vivo. The nanoconstruction maintained excellent stability with little leakage of loaded drugs under the simulated physiological conditions. Such a stable nanostructure enabled the effective in vivo tumor accumulation in tumor site as revealed by NIR imaging technique. More importantly, this nanoconstruction presented a pH-labile destruction profile in response to acidic microenvironment and simultaneously the fast liberation of loaded drugs. Accordingly at the cellular level, the intracellular structural dissociation was also proved in terms of the strong acidity in late endosome/lysosome, thus favoring the prolonged retention of remaining drug-loaded HP-OH aggregates within tumor cells. Hence, our delicate design open up a dynamical module-assembly path to develop site and time dual-controlled nanotherapeutics for tumor chemotherapy, allowing enhanced tumor selectivity through prolonged retention of delivery system in tumor cells followed by a timely drug release pattern.

Programmed Nanococktail for Intracellular Cascade Reaction Regulating Self-Synergistic Tumor Targeting Therapy.

In this work, a ZnO based nanococktail with programmed functions is designed and synthesized for self-synergistic tumor targeting therapy. The nanococktail can actively target tumors via specific interaction of hyaluronic acid (HA) with CD44 receptors and respond to HAase-rich tumor microenvironment to induce intracellular cascade reaction for controlled therapy. The exposed cell-penetrating peptide (R8) potentiates the cellular uptake of therapeutic nanoparticles into targeted tumor cells. Then ZnO cocktail will readily degrade in acidic endo/lysosomes and induce the production of desired reactive oxygen species (ROS) in situ. The destructive ROS not only leads to serious cell damage but also triggers the on-demand drug release for precise chemotherapy, thus achieving enhanced antitumor efficiency synergistically. After tail vein injection of ZnO cocktail, a favorable tumor apoptosis rate (71.2 ± 8.2%) is detected, which is significantly superior to that of free drug, doxorubicin (12.9 ± 5.2%). Both in vitro and in vivo studies demonstrate that the tailor-made ZnO cocktail with favorable biocompatibility, promising tumor specificity, and self-synergistically therapeutic capacity opens new avenues for cancer therapy.

Utilization of H-bond interaction of nucleobase Uralic with antitumor methotrexate to design drug carrier with ultrahigh loading efficiency and pH-responsive drug release

A novel Uralic (U)-rich linear-hyperbranched mono-methoxy poly (ethylene glycol)-hyperbranched polyglycerol-graft-Uralic (mPEG-HPG-g-U) nanoparticle (NP) was prepared as drug carrier for antitumor methotrexate (MTX). Due to the H-bond interaction of U with MTX and hydrophobic interaction, this NP exhibited high drug loading efficiency of up to 40%, which was significantly higher than that of traditional NPs based on U-absent copolymers (<15%). In addition, MTX-loaded mPEG-HPG-g-U NPs also demonstrated an acidity-accelerated drug release behavior.

Enhanced Immunotherapy Based on Photodynamic Therapy for Both Primary and Lung Metastasis Tumor Eradication

Metastasis and recurrence are two unavoidable and intractable problems in cancer therapy, despite various robust therapeutic approaches. Currently, it seems that immunotherapy is an effective approach to solve these problems, but the high heterogeneity of tumor tissue, inefficient presentation of tumor antigen, and deficient targeting ability of therapy usually blunt the efficacy of immunotherapy and hinder its clinical application. Herein, an approach based on combining photodynamic and immunological therapy was designed and developed. We synthesized a chimeric peptide, PpIX-1MT, which integrates photosensitizer PpIX with immune checkpoint inhibitor 1MT via a caspase-responsive peptide sequence, Asp-Glu-Val-Asp (DEVD), to realize a cascaded synergistic effect. The PpIX-1MT peptide could form nanoparticles in PBS and accumulate in tumor areas via the enhanced penetration retention effect. Upon 630 nm light irradiation, the PpIX-1MT nanoparticles produced reactive oxygen species, induced apoptosis of cancer cells, and thus facilitated the expression of caspase-3 and the production of tumor antigens, which could trigger an intense immune response. The subsequently released 1MT upon caspase-3 cleavage could further strengthen the immune system and help to activate CD8+ T cells effectively. This cascaded synergistic effect could inhibit both primary and lung metastasis tumor effectively, which may provide the solution for solving tumor recurrence and metastasis clinically.

Fluorescence light-up AIE probe for monitoring cellular alkaline phosphatase activity and detecting osteogenic differentiation

Alkaline phosphatase (ALP) is an important monophosphate hydrolase during cell mineralization and osteogenic differentiation. Though traditional methods are provided for evaluating the ALP expression in the fixed and lysed cells, at present it is still challenging to monitor the ALP activity in living cells. In this work, three phosphorylated tetraphenylethylene (TPE) probes (TPE-PA, TPE-2PA and TPE-4PA) with different numbers of -PO3H2 groups were synthesized for monitoring the ALP activity. It was found that in aqueous solution, both the TPE-PA and TPE-2PA probes were highly sensitive to ALP. In the presence of ALP, they could be quickly hydrolysed, resulting in an aggregation-induced emission (AIE) to light up ALP. While in living cells, only TPE-2PA showed good cell penetrability and high fluorescence signal-to-noise ratio during osteogenic differentiation. This probe provides us a new strategy to screen the ALP activity in living stem cells for detecting osteogenic differentiation.

Functional mesoporous silica nanoparticles (MSNs) for highly controllable drug release and synergistic therapy

Synergistic therapy involving two or more therapeutic agents with different anticancer mechanisms represents a promising approach to eradicate chemotherapy-refractory cancers. However, the preparation of a synergistic therapy platform generally involves complicated procedures to encapsulate different therapeutic agents and thereby increases the purification difficulty. In this work, we reported a simple but robust strategy to prepare a highly controllable drug delivery system (DDS) for synergistic cancer therapy. To construct this robust DDS, mesoporous silica nanoparticles (MSNs) were employed as a nanoplatform to encapsulate anticancer drug doxorubicin (DOX). After using a tumor-targeting cellular membrane-penetrating peptide (TCPP) and a mitochondria-targeting therapeutic peptide (TPP) to seal the surface pores via disulfide bonds, these newly developed MSNs can target cancer cells, penetrate cell membrane and rapidly release anticancer drug and mitochondria-targeted peptide in cytoplasm, inducing a remarkable synergistic anticancer effect. The new design concept reported here will promote the development of targeted and smart DDSs for synergistic cancer therapy.