Shirong Pan

A Biodegradable Low Molecular Weight Polyethylenimine Derivative as Low Toxicity and Efficient Gene Vector

Polyethylenimine (PEI) is a class of cationic polymers proven to be effective for gene delivery. However, PEI is nondegradable and the molecular weight of PEI affects the cytotoxicity and gene transfer activity. Aiming to prepare a biodegradable gene vector with high transfection efficiency and low cytotoxicity, we conjugated low molecular weight (LMW) PEIs to the biodegradable backbone polyglutamic acids derivative (PEG-b-PBLG) by aminolysis to form PEIs combined PEG-b-PLG-g-PEIs (GGI). Two copolymers, GGI 30 and GGI 40, were synthesized. The chemistry of GGI was characterized using IR, 1H NMR and 13C NMR, GPC, and CD, respectively. The degradation behaviors of copolymer GGI in papain solution were investigated. GGIs showed good DNA condensation ability and high protection of DNA from nuclease degradation. The zeta potential of the GGI/pDNA polyplexes was approximately 15 mV, and the particle size was in the range 102-138 nm at N/P ratios between 10 and 30. The particle size and the morphology of the polyplex was further confirmed by transmission electron microscope (TEM). In cytotoxicity assay, GGIs were significantly less toxic than PEI 25k. The degradation product of GGI exhibited negligible effects on cells even at high copolymer concentration. The results of GFP flow cytometry and fluorescence imaging showed that the trasnfection efficiencies of GGIs were all markedly higher than PEI 25k in Hela, HepG2, Bel 7402, and 293 cell lines. Importantly, the presence of serum had a lower inhibitive effect on the transfection activity of GGI in comparison to PEI 25k and Lipofectamine 2000. Therefore, PEG-b-PLG-g-PEI copolymers may be attractive cationic polymers for nonviral gene therapy.

A novel size-tunable nanocarrier system for targeted anticancer drug delivery

We have developed a nanocarrier drug-delivery system based on micelles formed by a new class of well-defined linear PEGylated two-arm oligomer of cholic acids in aqueous solution. By varying the length of the linear PEG chains and the configuration of cholic acid oligomer, one can easily fine-tune the physicochemical properties of the amphiphilic polymers and the resulting micelles. These include particle size, critical micelle concentration, and drug-loading capacity. High level of hydrophobic anticancer drugs such as PTX, etoposide and SN-38 can be readily loaded into such nanocarriers. The loading capacity of the nanocarrier for PTX (PTX) is extremely high (12.0mg/mL), which is equivalent to 37.5% (w/w) of the total mass of the micelle. PTX-loaded nanocarriers are much more stable than Abraxane (PTX/human serum albumin nanoaggregate) when stored in bovine serum albumin solution or dog plasma. PTX release profile from the micelles is burst-free and sustained over a period of seven days. The anti-tumor activity of PTX-loaded nanocarriers against ovarian cancer cell line in vitro, with continuous drug exposure, is similar to Taxol (formulation of PTX dissolved in Cremophor EL and ethanol) or Abraxane. Targeted drug delivery to tumor site with these novel micelles was demonstrated by near infrared fluorescence (NIRF) imaging in nude mice bearing ovarian cancer xenograft. Furthermore, PTX-loaded nanocarriers demonstrated superior anti-tumor efficacy compared to Taxol at equivalent PTX dose in ovarian cancer xenograft model.

Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives

Aiming to aid polyamidoamine (PAMAM, generation 4, PG4) to overcome gene delivery barriers like extrinsic serum inhibition, intrinsic cytotoxicity and lysosome digestion, histidine motifs modified PAMAM was prepared. The histidine activated PAMAM generation 4 (HPG4) was synthesized via aminolysis reaction and characterized by 1H NMR spectrum and MALDI-TOF-MS. Cytotoxicity profiles of HPG4 on MD-MB-231 cells were significantly improved in the form of polymer and polymer/DNA complexes comparing to PG4. The luciferase protein expression level of HPG4 was 20-, 2.7- and 1.2- fold higher than that of PG4, SuperFect and PEI 25k. Most importantly, flow cytometry and gene transfection studies showed that histidine motifs of HPG4 not only acted as enhancer for faster cellular uptake, but also played an important role on enhancing serum tolerance of the system on cellular uptake and transfection. Among the serum concentrations of 10%-50%, HPG4 showed 10-100 folds higher transfection efficiency than PG4. Intracellular fate observation conducted by confocal microscope provided visual and quantitative evidence that endsomal escape efficiency of HPG4 system was higher than that of PG4. Lastly, the endosomal escape mechanism of HPG4 system was analyzed by endosome destabilization and proton pump inhibition treatment. Collectively, compared to PG4/pDNA, HPG4/pDNA showed improvement on cellular uptake, serum tolerance, cytotoxicity profile, and endosomal escape.

A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection

A serum tolerant polycation gene vector, G(2) PAMAM-PGlu-G(1) PAMAMs (ALA), was designed, synthesized, characterized and evaluated. A honeycomb-like molecular structure model for mechanistic explanation of ALA was postulated and discussed. Designed as a star-shaped polyamidoamine (PAMAM)-based polypeptide dendrimer through peptide bond linkages, ALA was with non-toxic low generation G(2) PAMAM (G(2)) as its central core, polyglutamate (PGlu)s as its star-shaped backbone branches and G(1) PAMAM (G(1))s as its branch grafts and peripheral terminals. IR, (1)H NMR demonstrated its successful combination. As a gene carrier, ALA exhibited good DNA binding and condensation capacity with particle size (approximately 87 nm for N/P 40, approximately 170 nm for N/P 30) and ζ-potential (approximately 16 mV for N/P 30-40), negligible cytotoxicity, exciting serum tolerant capacity and significant serum-promoted (serum-containing 56.6%>serum-free 32.7%), cell line dependent (Hek 293 > Bel 7402 > Hela), incubation period dependent (38 h > 18 h > 12 h > 9 h > 4 h > 2 h > 1 h) and sustained (peak transfection appeared at 30 h incubation) transfection efficiency. The presence of serum had not only no inhibition on, but also prominent promotion to, the transfection activity of ALA. All above features differentiated ALA clearly from most other serum-inhibitive nonviral gene carriers, and proved ALA the promising and challenging potential efficient gene vector for practical clinical application.

Novel thermo-sensitive core–shell nanoparticles for targeted paclitaxel delivery

Novel thermo-sensitive nanoparticles self-assembled from poly(N,N-diethylacrylamide-co-acrylamide)-block-poly(gamma-benzyl L-glutamate) were designed for targeted drug delivery in localized hyperthermia. The lower critical solution temperature (LCST) of nanoparticles was adjusted to a level between physiological body temperature (37 degrees C) and that used in local hyperthermia (about 43 degrees C). The temperature-dependent performances of the core-shell nanoparticles were systemically studied by nuclear magnetic resonance (NMR), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and atom force microscopy (AFM). The mean diameter of the nanoparticles increased slightly from 110 to 129 nm when paclitaxel (PTX), a poorly water-soluble anti-tumor drug, was encapsulated. A stability study in bovine serum albumin (BSA) solution indicated that the PTX loaded nanoparticles may have a long circulation time under physiological environments as the LCST was above physiological body temperature and the shell remained hydrophilic at 37 degrees C. The PTX release profiles showed thermo-sensitive controlled behavior. The proliferation inhibiting activity of PTX loaded nanoparticles was evaluated against Hela cells in vitro, compared with Taxol (a formulation of paclitaxel dissolved in Cremophor EL and ethanol). The cytotoxicity of PTX loaded nanoparticles increased obviously when hyperthermia was performed. The nanoparticles synthesized here could be an ideal candidate for thermal triggered anti-tumor PTX delivery system.

Charge shielding effects on gene delivery of polyethylenimine/DNA complexes: PEGylation and phospholipid coating

Polyethylenimine (PEI) is an efficient cationic polymer for gene delivery, but defective in biocompatibility. In this study, we developed two different strategies to shield the positively charged PEI/DNA complexes: PEGylation and lipid coating. The physicochemical properties, cytotoxicity and transfection efficiency of the two gene delivery systems were investigated. Both PEGylation and lipid coating succeeded in reducing the zeta-potential of the complexes. Lipid-coated PEI/DNA complexes (LPD complexes) and PEI/DNA complexes exhibited similar cytotoxicity, whereas PEG–PEI/DNA complexes showed lower cytotoxicity, especially at high N/P ratios. LPD complexes were less efficient in transfection compared to PEG–PEI/DNA complexes. The transfection efficiency was influenced remarkably by cytotoxicity and surface charge of the complexes. Intracellular processes studies revealed that endosomal release might be one of the rate-limiting steps in cell transfection with PEI as a gene delivery carrier.

A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.

Short multi-armed polylysine-graft-polyamidoamine copolymer as efficient gene vectors

Polyamidoamine-polylysine graft copolymers (PAMAM-g-PLL) were prepared by ring-opening polymerization of benzyloxycarbonyl lysine N-carboxyanhydride (Lys(Z)-NCA) initiated with primary amine of generation 4 polyamidoamine (PAMAM G4) and subsequent deprotection of polyamidoamine-poly-(benzyloxycarbonyl lysine) copolymer (PAMAM-PLL(Z)). The chemical structure and composition of the PAMAM-g-PLL with varying length of PLL arms were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy ((1)H NMR). Agarose gel electrophoresis test revealed that the PAMAM-g-PLL could completely combine DNA to form complexes. The scanning electronic microscopy (SEM) and atomic force microscopy (AFM) observation showed that the morphology of these complexes was spherical. Dynamic light scattering (DLS) measurement illustrated that the sizes of complexes were in range of 100-200 nm. The MTT assay demonstrated that cytotoxicity of PAMAM-g-PLL were lower than the either PAMAM G4 or the poly-L-lysine-15k (PLL-15k). The in vitro transfection test indicated that the PAMAM-g-PLL with 3.8 average polymerization degrees of PLL arms (PAMAM-PLL-3.8) displayed significantly higher transfection efficiency than that of PAMAM G4 and PLL-15k at the same N/P ratio, Furthermore, PAMAM-PLL-3.8 at the N/P of 40 or 80 displayed better serum-resistant capability than that of PEI-25k and Lipofectamine 2000. The DNA local delivery test in rabbit vessel exhibited that the restenosis was inhibited to a significant extent. The above facts revealed that PAMAM-PLL-3.8 is a promising gene vector with low cytotoxicity, high transfection efficiency and serum-resistant ability.

Interaction of DNA/nuclear protein/polycation and the terplexes for gene delivery

Nuclear transport of exogenous DNA is a major barrier to nonviral gene delivery that needs to be addressed in the design of new vectors. In this study, we prepared pDNA/HMGB1/PEG-PEI terplexes to promote nuclear import. HMGB1 in the terplexes was used to assist the transportation of pDNA into the nucleus of cells, since it contained nuclear localization signal (NLS); PEG chains were introduced to stabilize pDNA/vector terplexes and reduce the cytotoxicity. HMGB1/PEG-PEI combined vectors have been investigated specifically for their structure interaction by atomic force microscopy and circular dichroic spectroscopy. The results demonstrated that the HMGB1 molecule could bind with the pDNA chains, but not condense pDNA well. The PEG-PEI further compacted pDNA/HMGB1 complexes into nanosized spherical terplexes. The pDNA delivered by HMGB1/PEG-PEI combined vectors was significantly accumulated in the nucleus of cells, as observed by confocal laser scanning microscopy. The percentage of GFP-transfected cells and VEGF protein expression level induced by HMGB1/PEG-PEI were 2.6-4.9-fold and 1.4-2.8-fold higher, respectively, than that of a common cationic polymer PEI 25 kDa. Therefore, the HMGB1/PEG-PEI combined vector could be used as a versatile vector for promoting exogenous DNA nuclear localization, thereby enhancing its expression.

High mobility group box 1 protein enhances polyethylenimine mediated gene delivery in vitro

The relatively low efficiency of transgene expression is a major drawback of non-viral gene delivery systems despite it being a safer alternative for gene therapy. Modifications of non-viral carriers with peptides have been utilized to improve their gene transfer efficiency. In this study, we described a new combined carrier, which was comprised of a cationic polymer, polyethylenimine (PEI), and a nuclear protein, high mobility group box 1 (HMGB1) containing nuclear localization sequences (NLS). The HMGB1/branched or linear PEI (b-PEI or l-PEI) combined carriers have been investigated for their cytotoxicity and ability to condense and deliver plasmid DNA (pDNA) to mammalian cells in vitro. Both HMGB1 and PEI formed complexes with pDNA are revealed by a gel-retardation assay. Compared with pDNA/l-PEI complexes, a reduction in particle size was observed for pDNA/HMGB1/l-PEI ternary complexes. In MTT assay the results of cell viability suggested lower cytotoxicity for HMGB1/PEI combined carriers. Transfection efficiencies of pDNA/HMGB1/PEI ternary complexes were evaluated by green fluorescent protein expression level measured by fluorescence spectroscopy and flow cytometry. Transfection efficiencies for pDNA/HMGB1/l-PEI ternary complexes and pDNA/HMGB1/b-PEI ternary complexes showed more than 2.9-fold and 4.0-fold greater than that for pDNA/l-PEI complexes and pDNA/b-PEI complexes, respectively. Thus, our results showed that HMGB1/PEI combined carriers may be a versatile non-viral carrier for pDNA with high transfection efficiency.