Ya-Nan Xue

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.

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.

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.

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.

Poly(amidoamine)s with pendant primary amines and flexible backbone for enhanced nonviral gene delivery: transfection and intracellular trafficking.

We synthesized poly(amidoamine)s with pendant primary amines and flexible backbone (polymers 1-3) by Michael polyaddition of N-tert-butyloxycarbonyl (N-Boc) protected diamine to 1,6-Bis(acrylamido)hexane, followed by the deprotection of N-Boc under acidic conditions. The physicochemical properties of polymers 1-3, including buffer capacity, DNA-binding capacity, cytotoxicity, particle sizes, and zeta potentials of polycation/DNA complexes, were explored. All the three polymers possess high buffer capacity and excellent DNA-binding capacity. In vitro MTT assay revealed that these synthesized poly(amidoamine)s were less cytotoxic than commercial branched PEI (25 kDa). These poly(amidoamine)s with pendant primary amines and flexible backbone were evaluated as in vitro nonviral gene delivery vectors for 293T and COS-7 cells. All the three polymers exhibited high transfection efficiencies, which were even higher than branched PEI (25 kDa) at optimized conditions. Further evidences from confocal laser scanning microscope (CLSM) demonstrated that the high transfection efficiencies of polymers 1-3 were due to the efficient uptake and intracellular trafficking of plasmid DNA in the cells during the transfection.

The effectiveness, cytotoxicity, and intracellular trafficking of nonviral vectors for gene delivery to bone mesenchymal stem cells:

Nonviral gene delivery that enables exogenous gene expression in bone mesenchymal stem cells could accelerate clinical application of cell-based gene therapy. This study systematically investigated and compared the potential of polyethylenimine and Lipofectamine 2000 as gene carriers to modify bone mesenchymal stem cells including transfection efficiency, cytotoxicity, intracellular trafficking as well as cell membrane damage and apoptosis/necrosis. Polyethylenimine at its optimal N/P ratio of 10 demonstrated the same toxic effects but lower transfection efficiency (17.1% vs 39.5%) compared to Lipofectamine. Intracellular trafficking resulted in over 80% of bone mesenchymal stem cells that were able to take up polyethylenimine polyplexes, but only 20.69% showed nuclear uptake; however, for Lipofectamine, about half bone mesenchymal stem cells were found to uptake lipoplexes but about 30% displayed nuclear localization. Moreover, the percentages of nuclear localization of both vectors were in close relationship with their transfection efficiency. We concluded that for bone mesenchymal stem cell transfection, polyethylenimine displayed high cellular uptake but Lipofectamine was more effective in delivering genes into the nucleus, which was likely the underlying basis for a more efficient gene expression. Further structure modification of polyethylenimine such as improving its nuclear entry ability will eventually make it a better candidate for bone mesenchymal stem cells’ in vitro gene delivery.

Biocleavable Polycationic Micelles as Highly Efficient Gene Delivery Vectors

An amphiphilic disulfide-containing polyamidoamine was synthesized by Michael-type polyaddition reaction of piperazine to equimolar N, N′-bis(acryloyl)cystamine with 90% yield. The polycationic micelles (198 nm, 32.5 mV), prepared from the amphiphilic polyamidoamine by dialysis method, can condense foreign plasmid DNA to form nanosized polycationic micelles/DNA polyelectrolyte complexes with positive charges, which transfected 293T cells with high efficiency. Under optimized conditions, the transfection efficiencies of polycationic micelles/DNA complexes are comparable to, or even higher than that of commercially available branched PEI (Mw 25 kDa).