Hu-Lin Jiang

Galactosylated poly(ethylene glycol)-chitosan-graft-polyethylenimine as a gene carrier for hepatocyte-targeting

Chitosan and chitosan derivatives have been proposed as alternative and biocompatible cationic polymers for non-viral gene delivery. However, the low transfection efficiency and low specificity of chitosan is an aspect of this approach that must be addressed prior to any clinical applications. In the present study a chitosan derivative, galactosylated poly(ethylene glycol)-chitosan-graft-polyethylenimine (Gal-PEG-CHI-g-PEI), was investigated as a potential hepatocyte-targeting gene carrier. The composition of Gal-PEG-CHI-g-PEI was characterized using (1)H nuclear magnetic resonance ((1)H NMR), and the particle size and zeta potential of Gal-PEG-CHI-g-PEI/DNA complexes were measured using dynamic light scattering (DLS). The Gal-PEG-CHI-g-PEI exhibited lower cytotoxicity compared to PEI 25K as a control. Likewise, Gal-PEG-CHI-g-PEI/DNA complexes showed good hepatocyte specificity. Furthermore, Gal-PEG-CHI-g-PEI/DNA complexes transfected liver cells more effectively than PEI 25K in vivo after intravenous (i.v.) administration. Together, these results suggest that Gal-PEG-CHI-g-PEI, which has improved transfection efficiency and hepatocyte specificity both in vitro and in vivo, may be useful for gene therapy.

The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization

A vaccine delivery system based on mannosylated chitosan microspheres (MCMs) was studied in vitro and in vivo. Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) were loaded in MCMs or chitosan microspheres (CMs). Fluorescence confocal microscopy indicated that BBD-loaded MCMs (BBD-MCMs) bound with mannose receptors on murine macrophages (RAW264.7 cells). In vitro experiments using macrophages demonstrated that BBD-MCMs had more effective immune-stimulating activity than BBD-loaded CMs (BBD-CMs). Mice intranasally immunized with BBD-MCMs showed significantly higher BBD-specific IgA antibody responses in saliva and serum than mice immunized with BBD-CMs (p<0.05). After challenge with B. bronchiseptica via the nasal cavity, groups treated with BBD-MCMs or BBD-CMs showed similar patterns with a high survival rate even though there was no significant difference between those groups. These results suggested that mannose moieties in the MCMs enhanced immune-stimulating activities through mucosal delivery due to a specific interaction between mannose groups in the MCMs and mannose receptors on the macrophages.

The suppression of lung tumorigenesis by aerosol-delivered folate-chitosan-graft-polyethylenimine/Akt1 shRNA complexes through the Akt signaling pathway

RNA interference (RNAi) represents a promising new approach to the inhibition of gene expression in vitro and in vivo, and has therapeutic potential for human diseases. Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNAi studies. Here we report the development of a new polymeric gene carrier for cancer cell-targeting, designed to enhance the intracellular delivery of shRNA and reduce cytotoxicity. Folate-chitosan-graft-polyethylenimine (FC-g-PEI) copolymer was prepared by an imine reaction between periodate-oxidized folate-chitosan (FC) and low molecular weight polyethylenimine (PEI). FC-g-PEI copolymer was investigated as a potential cancer cell-targeting gene carrier. The composition of FC-g-PEI was characterized using (1)H nuclear magnetic resonance ((1)H NMR), and particle size and zeta potential of FC-g-PEI/shRNA complexes were measured using dynamic light scattering (DLS). FC-g-PEI showed good shRNA condensation ability and high protection of shRNA from nuclease attack. It also exhibited lower cytotoxicity compared to PEI 25K control, and showed good cancer cell-targeting ability. Furthermore, aerosol delivery of FC-g-PEI/Akt1 shRNA complexes suppressed lung tumorigenesis in a urethane-induced lung cancer model mouse through the Akt signaling pathway. Together, these results suggest that FC-g-PEI may be useful for shRNA-based gene therapy.

Degradable polyethylenimines as DNA and small interfering RNA carriers

Gene therapy is a powerful approach in the treatment of a wide range of both inherited and acquired diseases. Nonviral delivery systems have been proposed as safer alternatives to viral vectors because they avoid the inherent immunogenicity and production problems that are seen when viral systems are used. Many cationic polymers, including high-molecular-weight polyethylenimine (PEI) have been widely studied as gene-delivery carriers, both, in vitro and in vivo. However, interest has recently developed in degradable polymeric systems. The advantage of degradable polymer is its low in-vivo cytotoxicity, which is a result of its easy elimination from the cells and body. Degradable polymer also enhances transfection of DNA or small interfering RNA (siRNA) for efficient gene expression or silencing, respectively. This review paper summarizes and discusses the recent advances with degradable PEIs, such as cross-linked and grafted PEIs for DNA and siRNA delivery.

Chitosan-graft-polyethylenimine for Akt1 siRNA delivery to lung cancer cells

Efficient delivery of small interfering RNA (siRNA) remains a challenging task in RNA interference (RNAi) studies. In this study, we used chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer composed of chitosan and low molecular weight polyethylenimine (PEI) for the delivery of siRNA. The CHI-g-PEI carrier formed stable complexes with siRNA with compact spherical morphology. CHI-g-PEI delivered EGFP siRNA (siGFP) silenced EGFP expression nearly 2.5 folds higher than PEI25K at 50 pM siGFP concentration. Cell viability was found to be 2 folds high with CHI-g-PEI carrier than PEI25K. Also, our CHI-g-PEI carrier efficiently delivered Akt1 siRNA (siAkt) and thereby silenced onco-protein Akt1. Silencing of this crucial cell survival protein significantly reduced the lung cancer cell survival and proliferation. Additionally, Akt1 protein knock-down decreased A549 cell malignancy and metastasis. These findings suggest that the CHI-g-PEI carrier efficiently and safely delivered siRNA. Moreover, CHI-g-PEI mediated Akt1 siRNA delivery may emerge as a viable approach for lung cancer treatment.

Folate-PEG-superparamagnetic iron oxide nanoparticles for lung cancer imaging

While superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used in biomedical applications, rapid blood clearance, instability and active targeting of the SPIONs limit their availability for clinical trials. This work was aimed at developing stable and lung cancer targeted SPIONs. For this purpose firstly folic acid (FA)-conjugated poly(ethylene glycol) (FA-PEG) was synthesized, and FA-PEG-SPIONs were subsequently prepared by the reaction of FA-PEG with aminosilane-immobilized SPIONs. FA-PEG-SPIONs were labeled with Cy5.5 for optical imaging. The intracellular uptake of FA-PEG-SPIONs-Cy5.5 was evaluated in KB cells and lung cancer model mice to confirm active targeting. The sizes of the FA-PEG-SPIONs were little changed after up to 8 weeks at 4 °C, suggestive of very stable particle sizes. The results of fluorescent flow cytometry and confocal laser scanning microscopy suggest that the intracellular uptake of FA-PEG-SPIONs-Cy5.5 was greatly inhibited by pre-treatment with free folic acid, indicative of receptor-mediated endocytosis. Stronger optical imaging was observed in the lung cancer model mice for FA-PEG-SPIONs-Cy5.5 than PEG-SPIONs-Cy5.5 6 and 24 h post-injection through the tail vein, due to receptor-mediated endocytosis.

Biodegradable poly(ester amine) based on glycerol dimethacrylate and polyethylenimine as a gene carrier

Polyethylenimine (PEI) vectors are widely used in gene delivery because of their high transfection efficiency owing to a unique proton sponge effect. An increase in molecular weight increases transfection efficiency, but simultaneously results in increased toxicity. Therefore, the design and synthesis of new degradable gene delivery carriers having high transfection efficiencies and reduced cytotoxicity are necessary.

Efficient gene delivery using chitosan–polyethylenimine hybrid systems

Chitosan and chitosan derivatives have been investigated as non-viral vectors because they have several advantages, such as biocompatibility, biodegradability, low cytotoxicity and low immunogenicity. However, low transfection efficiency and low cell specificity must be solved for their use in clinical trials. In this paper, chitosan-polyethylenimine (PEI) hybrid systems such as chitosan/PEI blend and chitosan-graft-PEI are described for efficient gene delivery because the PEI has high transfection efficiency owing to a proton sponge effect and chitosan has biocompatibility. Also, hepatocyte specificity of the galactosylated chitosan is explained after combination with PEI.

Chitosan-graft-spermine as a gene carrier in vitro and in vivo

Chitosan has been proposed as a non-viral gene carrier because of its biodegradable and biocompatible cationic polymeric properties. However, the transfection efficiency of chitosan-DNA complexes is still too low for clinical trials. To improve transfection efficiency, we prepared a chitosan-graft-spermine (CHI-g-SPE) copolymer by an imine reaction between periodate-oxidized chitosan and spermine. The CHI-g-SPE copolymer was complexed with plasmid DNA in various copolymer-DNA weight ratios, and the complexes were characterized. The CHI-g-SPE copolymer showed good DNA binding ability and high protection of DNA from nuclease attack. The CHI-g-SPE/DNA complexes had well-formed spherical shapes and a nanoscale size with homogenous size distribution. The CHI-g-SPE copolymer had low cytotoxicity and CHI-g-SPE/DNA complexes showed transfection efficiency that was enhanced over that of chitosan-DNA. Furthermore, aerosol delivery of CHI-g-SPE/GFP complexes showed higher GFP expression compared with chitosan/GFP complexes, without toxicity. Our results indicate that the CHI-g-SPE copolymer has potential as a gene carrier.

Application of Recombinant Fusion Proteins for Tissue Engineering

Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Also, the development of new synthetic ECMs is very important because ECMs facilitate the localization and delivery of cells to the specific sites in the body. Therefore, the development of synthetic ECMs to replace the natural ECMs is increasingly essential and promising in tissue engineering. Recombinant genetic engineering method has enabled the synthesis of protein-based polymers with precisely controlled functionalities for the development of new synthetic ECMs. In this review, the design and construction of structure-based recombinant fusion proteins such as elastin-like polymers (ELPs) and silk-like polymers (SLPs), cell-bound growth factor-based recombinant fusion proteins such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), hybrid system composed of recombinant protein and synthetic polymer, and E-cadherin-based fusion protein by recombinant genetic engineering were explained for application of the synthetic ECMs. Modulation of mechanical properties, stimuli-sensitivity, biodegradation and cell recognition can be achieved through precise control of sequence, length, hydrophobicity and cell binding domain by recombinant genetic engineering.