Qinglian Hu

Cationic microRNA-delivering nanovectors with bifunctional peptides for efficient treatment of PANC-1 xenograft model

Therapeutic strategies based on modulation of microRNA activity possess much promise in cancer therapy, but the in vivo delivery of microRNA to target sites and its penetration into tumor tissues remain great challenge. In this work, miR-34a-delivering therapeutic nanocomplexes with a tumor-targeting and -penetrating bifunctional CC9 peptide were proposed for efficient treatment of pancreatic cancers. In vitro study indicated that the nanoparticle-based miR-34a delivery systems could effectively facilitate cellular uptake and greatly up-regulate the mRNA level of miR-34a in PANC-1 cell lines. The up-regulation of miR-34a remarkably induced cell cycle arrest and apoptosis, suppressed the tumor cell migration and inhibited the target gene expressions such as E2F3, Bcl-2, c-myc and cyclin D1. More importantly, the in vivo systemic administration of the developed targeting miR-34a delivery systems in a pancreatic cancer model significantly inhibited tumor growth and induced cancer cell apoptosis. Such bifunctional peptide-conjugated miRNA-delivering nanocomplexes should have great potential applications in cancer therapy.

Intracellular pathways and nuclear localization signal peptide-mediated gene transfection by cationic polymeric nanovectors

Polyethylenimine (PEI) - based polymers are promising cationic nanovectors. A good understanding of the mechanism by which cationic polymers/DNA complexes are internalized and delivered to nuclei helps to identify which transport steps may be manipulated in order to improve the transfection efficiency. In this work, cell internalization and trafficking of PEI-CyD (PC) composed of β-cyclodextrin (β-CyD) and polyethylenimine (PEI, Mw 600) are studied. The results show that the PC transfected DNA is internalized by binding membrane-associated proteoglycans. The endocytic pathway of the PC particles is caveolae- and clathrin-dependent with both pathways converging to the lysosome. The intracellular fate of the PC provides visual evidence that it can escape from the lysosome. Lysosomal inhibition with chloroquine has no effect on PC mediated transfection implying that blocking the lysosomal traffic does not improve transfection. To improve the nuclear delivery of PC transfected DNA, nuclear localization signal (NLS) peptides are chosen to conjugate and combine with the PC. Compared to PC/pDNA, PC-NLS/pDNA, and PC/pDNA/NLS can effectively improve gene transfection in dividing and non-dividing cells.

Engineering Nanoparticle-Coated Bacteria as Oral DNA Vaccines for Cancer Immunotherapy

Live attenuated bacteria are of increasing importance in biotechnology and medicine in the emerging field of cancer immunotherapy. Oral DNA vaccination mediated by live attenuated bacteria often suffers from low infection efficiency due to various biological barriers during the infection process. To this end, we herein report, for the first time, a new strategy to engineer cationic nanoparticle-coated bacterial vectors that can efficiently deliver oral DNA vaccine for efficacious cancer immunotherapy. By coating live attenuated bacteria with synthetic nanoparticles self-assembled from cationic polymers and plasmid DNA, the protective nanoparticle coating layer is able to facilitate bacteria to effectively escape phagosomes, significantly enhance the acid tolerance of bacteria in stomach and intestines, and greatly promote dissemination of bacteria into blood circulation after oral administration. Most importantly, oral delivery of DNA vaccines encoding autologous vascular endothelial growth factor receptor 2 (VEGFR2) by this hybrid vector showed remarkable T cell activation and cytokine production. Successful inhibition of tumor growth was also achieved by efficient oral delivery of VEGFR2 with nanoparticle-coated bacterial vectors due to angiogenesis suppression in the tumor vasculature and tumor necrosis. This proof-of-concept work demonstrates that coating live bacterial cells with synthetic nanoparticles represents a promising strategy to engineer efficient and versatile DNA vaccines for the era of immunotherapy.

A gene nanocomplex conjugated with monoclonal antibodies for targeted therapy of hepatocellular carcinoma.

To enhance tumor-targeting abilities and therapeutic efficiency, a monoclonal antibody-conjugated gene nanocomplex was herein designed. The biodegradable cationic polyethylenimine-grafted-α,β-poly(N-3-hydroxypropyl)-DL-aspartamide (PHPA-PEI) was used for complexing pDNA to form the PHPA-PEI/pDNA nanoparticle, and then 9B9 mAb, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, was conjugated to produce the PHPA-PEI/pDNA/9B9 mAb (PP9mN) complex. The PP9mN complex with the diameter of around 300 nm at its optimal weight ratio could be uptaken effectively by SMMC-7721 cells. The cytotoxicity of the PP9mN complex was much lower than that of PEI 25 kD in SMMC-7721, HepG2, Bel-7404 and COS-7 cell lines. The PP9mN complex possessed the highly efficient in vitro gene delivery ability to the hepatocellular carcinoma cells. The in vivo gene expression indicated that PP9mN could target to the tumor tissues effectively. By using the therapeutic AChE gene, it was found that the PP9mN complexes significantly enhanced the anti-tumor effect on tumor-bearing nude mice. Such monoclonal antibody-conjugated gene complex should have great potential applications in liver cancer therapy.

Targeted Co‐delivery of PTX and TR3 siRNA by PTP Peptide Modified Dendrimer for the Treatment of Pancreatic Cancer

A new type of tumor-targeted nanovehicle peptide-conjugated PSPG (PSPGP) is successfully synthesized for co-delivery of paclitaxel (PTX) and TR3 small interfering RNA (siRNA). In vitro and in vivo investigations demonstrate that the redox-responsive PSPGP exhibit enhanced endosomal escape and intracellular degradation, which facilitate PTX and TR3 siRNA release, effectively improving the antitumor efficacy.

Redox-Activated Light-Up Nanomicelle for Precise Imaging-Guided Cancer Therapy and Real-Time Pharmacokinetic Monitoring

Simultaneous tumor imaging, therapy, and pharmacokinetic monitoring can offer a safe and effective strategy for cancer therapy. This work describes the design of a fluorescence light-up nanomicelle that can afford precise imaging-guided drug delivery and pharmacokinetic monitoring in a real-time fashion for cancer chemotherapy. The nanomicelle, which contains a boron dipyrromethene based fluorescent probe as the hydrophobic core and a redox-triggered detachable poly(ethylene glycol) (PEG) shell, can accumulate at the tumor site via enhanced permeation and retention effect. The PEG detachment induced by tumoral and intracellular glutathione can destabilize the nanomicelle, leading to fluorescence light up and simultaneous drug release. Importantly, the fluorescence intensities generated by the nanomicelles in different organs are well-correlated with released drug concentrations in both temporal and spatial manners, suggesting its precise role for imaging-guided drug delivery and pharmacokinetic monitoring in vivo. The tumor growth can be effectively inhibited by the docetaxel-loaded nanomicelle formulation, and the nanomicelles are monitored to be excreted via hepatobiliary routes. This nanomicelle for precise imaging-guided chemotherapy provides a safe and robust theranostic strategy for the evaluation of cancer nanomedicine.

Targeting ETS1 with RNAi-based supramolecular nanoassemblies for multidrug-resistant breast cancer therapy

&NA; Overexpression of erythroblastosis virus E26 oncogene homolog 1 (ETS1) gene is correlated with both tumor progression and poor response to chemotherapy in cancer treatment, and the exploitation of RNA interference (RNAi) technology to downregulate ETS1 seems to be a promising approach to reverse multidrug‐resistant cancer cells to chemotherapy. Hence, the RNAi‐based nanomedicine which is able to simultaneously downregulate ETS1 expression and to deliver chemotherapeutic agents may improve multidrug‐resistant cancer therapy synergistically. In this study, we developed a supramolecular nanoassembly that could deliver siRNA targeting ETS1 (siETS1) and doxorubicin (DOX) as an effective nanomedicine to achieve successful chemotherapy towards multidrug‐resistant breast cancer. The nanotherapeutic system was prepared by loading adamantane‐conjugated doxorubicin (AD) into polyethyleneimine‐modified (2‐hydroxypropyl)‐&ggr;‐cyclodextrin (HP) through the supramolecular assembly to form AD‐loaded HP (HPAD), followed by electrostatically‐driven self‐assembly between siETS1 and HPAD. When the HPAD/siETS1 nanoassemblies were delivered into drug‐resistant MCF‐7/ADR cells, the drug efflux was significantly reduced as a result of simultaneous silencing of ETS1 and MDR1 genes. Importantly, the HPAD/siETS1 nanoassembly could enhance drug residence time at tumor site, and effectively inhibit drug‐resistant tumor growth due to the inhibition of angiogenesis and necrosis in tumor tissues. Western blot analysis indicated that the gene expression of both ETS1 and MDR1 in vivo was considerably downregulated after the drug‐resistant tumor‐bearing mouse was treated with HPAD/siETS1 nanoassemblies. This study offers a new therapeutic delivery strategy targeting ETS1 for the effective multidrug‐resistant chemotherapy. Graphical abstract Figure. No caption available.

Folate receptor mediated genetic modification of human mesenchymal stem cells via folic acid-polyethylenimine-grafted poly(N-3-hydroxypropyl)aspartamide

Mesenchymal stem cells (MSCs) are targeted as vehicles for cell mediated gene therapy. Here we report on a macromolecular carrier, which was designed aiming at successful targeted gene delivery into MSCs through the mediation of folate receptor and reduced cytotoxicity compared to established cationic polymer vector - polyethylenimine with a weight average molecular weight (Mw) of 25,000 Dalton (PEI25K). The carrier PHPA-PEI1800-FA was synthesized in a two-step procedure. PHPA-PEI1800 was prepared by grafting polyethylenimine with a Mw of 1800 Dalton (PEI1800) onto the α,β-poly(N-3-hydroxypropyl)-D,L-aspartamide (PHPA) backbone. PHPA-PEI1800-FA was obtained by chemically conjugating folic acid onto PHPA-PEI1800. The grafting degree of PEI1800 was 3.9±0.2% in relation to the CH groups of PHPA and the molar ratio of folic acid conjugated to PEI1800 (χFA) was 1.8±0.1 as calculated by NMR spectroscopy. The copolymers were biodegradable and exhibited lower cytotoxicity than PEI25K. Compared to PHPA-PEI1800, PHPA-PEI1800-FA led to a significantly higher transfection efficiency in human MSCs, which could be attributed to the mediation of folate receptor during the transfection process as confirmed by folic acid competition assay. Both marker gene (GFP) and therapeutic gene (VEGF) were delivered into human MSCs from multi-donors using PHPA-PEI1800-FA. The percentage of GFP+ MSCs showed an average value of 2.85±1.60% but a large variation for different samples. The VEGF expression level of the PHPA-PEI1800-FA transfected cells was significantly higher than that of either untransfected or naked DNA transfected cells. Conclusively, PHPA-PEI1800-FA is a suitable vector to deliver genes into human MSCs through the interaction with folate receptor.