Shi-Wen Huang

Dendrimer modified magnetic iron oxide nanoparticle/DNA/PEI ternary magnetoplexes: a novel strategy for magnetofection

As a powerful technology to enhance the efficiency of gene delivery, magnetofection has attracted considerable attention in the past decade. In this work, we introduced 6 generation of PAMAM dendrimer modified superparamagnetic nanoparticles (DMSPION-G6) to PEI/DNA polyplexes by a two-step process and enhanced the transfection efficiency of PEI with the help of a magnetic field. We prepared DMSPION-G6/DNA/PEI ternary magnetoplexes by precondensing DMSPION-G6 with DNA at a low mass ratio to yield DMSPION-G6/DNA magnetoplexes with negative surface charge, followed by further coating with branched PEI (25 kDa) via electrostatic interactions. We measured the transfection efficiencies of DMSPION-G6/DNA/PEI ternary magnetoplexes in COS-7, 293T and HeLa cells in the presence or absence of a magnetic field. Compared with PEI/DNA polyplexes, DMSPION-G6/DNA/PEI ternary magnetoplexes exhibited enhanced transfection efficiencies in all the three cell lines when a magnetic field was applied, especially in the presence of 10% FBS. Time-resolved and dose-resolved transfection indicated that high-level transgene expression was achievable with a relatively short incubation time and low DNA dose when magnetofection was employed. Further evidence from Prussian blue staining, quantification of cellular iron concentration and cellular uptake of Cy-3 labelled DNA demonstrated that the magnetic field could quickly gather the magnetoplexes to the surface of target cells and consequently enhanced the uptake of magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitrogene delivery enhanced by a magnetic field.

Polyamidoamine-grafted multiwalled carbon nanotubes for gene delivery: synthesis, transfection and intracellular trafficking.

Functionalized multiwalled carbon nanotubes (f-MWNTs) are of great interest and designed as a novel gene delivery system. In this paper, we presented synthesis of polyamidoamine-functionalized multiwalled carbon nanotubes (PAA-g-MWNTs) and their application as a novel gene delivery system. The PAA-g-MWNTs, obtained from amide formation between PAA and chemically oxidized MWNTs, were stable in aqueous solution and much less toxic to cells than PAA and PEI 25KDa. More importantly, PAA-g-MWNTs showed comparable or even higher transfection efficiency than PAA and PEI at optimal w/w ratio. Intracellular trafficking of Cy3-labeled pGL-3 indicated that a large number of Cy3-labeled pGL-3 were attached to nucleus membrane, the majority of which was localized in nucleus after incubation with cells for 24 h. We have demonstrated that PAA modification of MWNTs facilitate higher DNA uptake and gene expression in vitro. All these facts suggest potential application of PAA-g-MWNTs as a novel gene vector with high transfection efficiency and low cytotoxicity.

Improving Gene Delivery Efficiency of Bioreducible Poly(amidoamine)s via Grafting with Dendritic Poly(amidoamine)s

Dendritic poly(amidoamine)s (PAMAM)s were introduced into the side chains of disulfide-containing poly(amidoamine)s via repetitive Michael addition and amidation. The bioreducible poly(amidoamine)s grafted with dendritic polyamidoamines showed high buffer capacity, low cytotoxicity and strong DNA binding ability at low N/P ratio. They were able to condense DNA into small sized polycation/DNA complexes, which degraded and released the incorporated DNA under reductive conditions. In comparison to the original disulfide-containing poly(amidoamine) with aminoethyl side chain, the grafting of the bioreducible poly(amidoamine) with dendrimer greatly improved the transfection efficiencies of 293T and HeLa cells with foreign DNA at various N/P ratios. The structure-gene delivery property relations of dendrimer-grafted polycations will provide valuable insight into the design of highly efficient and less toxic polycationic gene carriers.

A redox stimuli-responsive superparamagnetic nanogel with chemically anchored DOX for enhanced anticancer efficacy and low systemic adverse effects

A reduction-triggered superparamagnetic nanogel was designed for enhancing targeting release but minimizing leakage of drug in the drug-transportation pathway. Doxorubicin (DOX) was first conjugated onto sodium alginate (SA) with a disulfide linker (SA-SS-DOX). SA-SS-DOX was then electrostatically assembled with aminated superparamagnetic iron oxide nanoparticles for the preparation of nanogel. The nanogel was estimated with a size of 122 ± 15 nm, a polydispersity index of 0.178, a surface charge of around -36.0 mV, a DOX loading of 7.2 wt%, and a saturation magnetization of 40.0 emu g-1 Fe. In vitro release profiles showed a significantly high accumulative release at pH 5.5/10 mM Glutathione (GSH) but pretty low release at pH 7.4/pH 5.5 without GSH, exhibiting apparent reduction responsiveness. In vitro cytotoxicity tests clearly illustrated that the effective selectivity of killing the human cervical cancer cells (HeLa) with a IC50 of 0.30 μg mL-1, significantly enhanced the cytotoxicity of the African green monkey Sv40-transformed kidney fibroblast cells (COS-7). Prussian blue staining and quantification of cellular iron and protein concentration revealed apparent iron uptake by HeLa cells. Confocal laser scanning microscopy observation demonstrated that DOX was efficiently internalized into HeLa cells, released into the cytoplasm, and then principally entered the nuclei. In vivo investigation exhibited that nanogel treatment induced obvious shrinkage in tumor volume but a stable growth in body weight and a healthy appearance. Hematoxylin-eosin (H&E) staining indicated remarkable necrosis in the tumor area and histologic examination revealed lower toxicity in vital organs. Thus this kind of nanogel expressed high targeted release but low systemic toxicity. It is thus believed that this nanogel will offer a promising candidate for highly effective cancer therapy.

Polyaspartamide-based oligo-ethylenimine brushes with high buffer capacity and low cytotoxicity for highly efficient gene delivery.

Polyaspartamide-based oligo-ethylenimine brushes (PASP-EDA, PASP-TEPA, PASP-PEHA, and PASP-PEI 423) were synthesized from polysuccinimide (PSI) via a ring-opening reaction with N-Boc protected ethylenediamine, tetraethylenepentamine, pentaethylenehexamine, and linear polyethylenimine (Mn 423), respectively. PASP-TEPA, PASP-PEHA, and PASP-PEI 423 possess high buffer capacity between pH 5 and pH 7, which is comparable to that of branched PEI 25000. The cytotoxicity assay indicated that they all are less toxic than PEI 25000. At an N/P ratio of above 2, all of the four synthetic polycation brushes can condense plasmid DNA to form small sized (160-400 nm) polyelectrolyte complexes with positive surface charge. The transfection of HEK 293 cells with oligo-ethylenimine brush/pRE Luc polyplexes indicated that the transfection efficiencies increased with increasing the length of oligo-ethylenimine side chains. The luciferase expression with PASP-PEHA and PASP-PEI 423 were as high as or even a little higher than that of PEI 25000. The results demonstrate that polyaspartamide-based oligo-ethylenimine brushes are a very promising class of novel polycations for highly efficient and less toxic gene delivery.

Transfection and intracellular trafficking characteristics for poly(amidoamine)s with pendant primary amine in the delivery of plasmid DNA to bone marrow stromal cells

Poly(amidoamine)s with pendant primary amine (polymer 1a-1c) were evaluated as in vitro non-viral gene delivery vectors for bone marrow stromal cells (BMSCs). The cytotoxicity of these poly(amidoamine)s, measured by MTT assay, increased with increasing length of side chain, however, they were less toxic than branched polyethylenimine (PEI) 25k Da. Using pGL-3 and pEGFP-C1 as luciferase gene and green fluorescent protein (GFP) gene, among all polycations including polymer 1a-1c and PEI, polymer 1b at optimal N/P ratio showed highest luciferase expression (1.92 x 10(8) RLU/mg protein) as well as percentage of cells expressing GFP (29.01+/-2.33%). For all polycations, intracellular trafficking of Cy3-labelled plasmid DNA (pDNA) was similar. Fluorescent particles attached to cell membrane at 0.5 h after adding the polycation/DNA complexes, aggregated in cytoplasm after 2h, and then stayed around the perinuclear region after 4 h. pDNA nuclear localization appeared at 4 h post-transfection, but much more pDNA entered into nucleus at 24 h. At high N/P ratio, polymer 1a-1c could deliver pDNA into 70-80% of BMSCs after 24 h transfection, however, labelled pDNA was observed in only 4-25% of cells at the same time. Compared to PEI, polymer 1b showed comparable or even higher percentage of pDNA uptake and nuclear localization. We concluded that poly(amidoamine)s with pendant primary amine, especially polymer 1b, are new kind of promising candidates of less toxic and highly efficient non-viral gene delivery vectors for BMSCs.

Folic acid-conjugated iron oxide porous nanorods loaded with doxorubicin for targeted drug delivery.

Iron oxide porous nanorods (IOPNR) with lengths ranging from 40nm to 60nm and pore diameters ranging from 5nm to 10nm were prepared, and further modified with NH2-PEG-FA (FA-PEG-IOPNR) for ligand targeting and modified with NH2-PEG-OCH3 (PEG-IOPNR) as a control. Instead of chemical bonding, doxorubicin (DOX), a low water solubility anticancer drug, was loaded in the pores of the modified IOPNR because of their porous structure and high porosity. The release of DOX in acidic PBS solution (pH 5.3) was faster than that in neutral (pH 7.4) solution. The analysis results from TEM, inductively coupled plasma emission spectroscopy, confocal laser scanning microscopy, and flow cytometry analyses indicated that the presence of FA on the surface of the nanorods increase the cellular uptake of nanorods in the case of HeLa cells, a folate receptor (FR)-positive cell line. In contrast, for COS 7 cells, a FR-negative cell line, FA ligand on the surface of the nanorods showed no effect on the cellular uptake. MTT assay indicated that the cytotoxicity of DOX loaded in FA-PEG-IOPNR to HeLa cells was higher than that of DOX in PEG-IOPNR. In the case of COS 7 cells, no significant difference between the cytotoxicity of DOX loaded in FA-PEG-IOPNR and PEG-IOPNR was found. These results suggested that FA-PEG-IOPNR had the potential for target delivery of chemotherapeutic into cancer cells.

Poly(β-aminoester)s with Pendant Primary Amines for Efficient Gene Delivery

Three hydrolytically degradable poly(beta-aminoester)s containing ester bonds in the main chain and primary amines in the side chain, synthesized by Michael polyaddition, were applied to deliver foreign DNA into cells in vitro. These linear polycations can condense DNA into small-sized particles with positive surface charge at high N/P ratios. Their high buffer capacity at pH 5-7 facilitated the escape of DNA from the endosome and resulted in efficient gene expression. Under the optimal conditions, poly(beta-aminoester)s with a pendant aminoethyl group showed higher transfection efficiencies than branched poly(ethylenimine) (PEI) 25KDa in 293T cells. The effect of side chain structure of the poly(beta-aminoester) on transfection efficiency has been investigated, which indicated that the poly(beta-aminoester) containing the pendant aminoethyl group was the most efficient carrier for both of 293T cells and COS-7 cells. The combination of hydrolytical degradation, high buffer capacity, relatively low cytotoxicity, and high transfection efficiency suggested that this kind of poly(beta-aminoester)s are novel promising nonviral gene carriers.

Reduction-sensitive micelles with sheddable PEG shells self-assembled from a Y-shaped amphiphilic polymer for intracellular doxorubicine release

A new type of shell-sheddable micelles with disulfide linkages between the hydrophobic polyester core and hydrophilic poly(ethylene glycol) (PEG) shell was developed based on Y-shaped amphiphilic polymers mPEG-S-S-(PCL)2. The micelles were then used for the glutathione-mediated intracellular delivery of the anticancer drug doxorubicin (DOX) into tumor cells. The polymer could self-assemble into micelles with an average diameter of 135nm in aqueous solution and load DOX at a total content of 3.6%. The hydrophilic PEG shell of these micelles could be shed in the presence of reducing agent dithiothreitol (DTT), which resulted in size change of the micelles. In vitro release studies revealed that DOX-loaded mPEG-S-S-(PCL)2 micelles exhibited faster DOX release in the presence of DTT. MTT assay demonstrated that DOX-loaded mPEG-S-S-(PCL)2 micelles showed higher cytotoxicity against 10mM of glutathione monoester (GSH-OEt) pretreated HeLa cells than that of the non-pretreated ones. Confocal laser scanning microscopy and flow cytometry analyses indicated that DOX-loaded mPEG-S-S-(PCL)2 micelles were efficiently internalized into HeLa cells and exhibited faster DOX release in GSH-OEt-pretreated cells than in cells with no pretreatment. Endocytosis inhibition results proved that mPEG-S-S-(PCL)2 micelles entered the cells mainly through the clathrin-mediated endocytosis pathway, and caveolae-mediated endocytosis was involved to a small extent. These results indicate the great potential of the proposed Y-shaped reduction-sensitive polymer for application in effective intracellular anticancer drug delivery.

Poly(L‐aspartamide)‐Based Reduction‐Sensitive Micelles as Nanocarriers to Improve Doxorubicin Content in Cell Nuclei and to Enhance Antitumor Activity

A reduction-sensitive graft polymer PHEA-S-S-C16 with poly{α,β-[N-(2-hydroxyethyl)-L-aspartamide]} (PHEA) as a backbone and a disulfide-containing alkyl as a side chain (HOOC-S-S-C16 ) is synthesized and evaluated for intracellular DOX delivery. PHEA-S-S-C16 can self-assemble into micelles in aqueous media and load DOX at a total content of 7.3%. In vitro release studies reveal that the release rate of DOX from PHEA-S-S-C16 micelles is accelerated in the presence of DTT. The results of cell experiments indicate that DOX-loaded mPEG-S-S-C16 micelles can achieve rapid DOX release in HeLa cells, as compared with their reduction-insensitive counterparts. Endocytosis inhibition analysis indicates that PHEA-S-S-C16 micelles entered cells mainly via clathrin-mediated endocytosis.