Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.     

Biotin-triggered release of poly(ethylene glycol)-avidin from biotinylated polyethylenimine enhances in vitro gene expression

Covalently poly(ethylene glycol) (PEG)-ylated polyethylenimine (PEI)/pDNA complexes display prolonged blood circulation profiles compared with PEI/pDNA complexes, but such PEGylated particles may not be suitable for tumor targeting due to low interaction with cell membranes, low internalization, and low gene expression. Noncovalent PEGylation of cationic particles via PEG-avidin/biotin-PEI is an attempt to bridge the gap between the positive attributes of PEG (prolonged particle circulation) and the positive attributes of nontoxic cationic polymers (enhanced cell interactions) for greater gene expression. Our polymer, 2PEG-avidin/biotin-PEI8, forms salt-stable particles ( approximately 100 nm) under physiologic conditions with a minimum of two 2PEG-avidin molecules bound per polymer chain (biotin-PEI8, 8 biotins/PEI). Following 10 days of incubation with 3000-fold excess biotin, 2PEG-avidin completely dissociated from biotin-PEI8, and gene expression was increased 2.1-32-fold in various cell lines when the desirable transfection feature of the cationic polymer was retained. This new PEGylation approach has implications for generally improving the clinical aspect of gene delivery via a two-step therapeutic strategy: (1) intravenous injection of noncovalent PEG-avidin/biotin-polycation nanoparticles for prolonged circulation, followed by (2) temporal release of PEG-avidin from biotin-polycation through either endogenous biotin or intravenous injection of biotin.     

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25 kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.     

Formulation of a microparticle carrier for oral polyplex-based DNA vaccines

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.     

Development of a nonviral gene delivery vehicle for systemic application

Polycation vehicles used for in vitro gene delivery require alteration for successful application in vivo. Modification of polycations by direct grafting of additional components, e.g., poly(ethylene glycol) (PEG), either before or after DNA complexation, tend to interfere with polymer/DNA binding interactions; this is a particular problem for short polycations such as linear, beta-cyclodextrin-containing polycations (betaCDPs). Here, a new method of betaCDP polyplex (polycation/DNA composite structures) modification is presented that exploits the ability to form inclusion complexes between cyclodextrins and adamantane. Surface-PEGylated betaCDP polyplexes are formed by self-assembly of the polyplexes with adamantane-PEG conjugates. While unmodified polyplexes rapidly aggregate and precipitate in salt solutions, the PEGylated betaCDP polyplexes are stable at conditions of physiological salt concentration. Addition of targeting ligands to the adamantane-PEG conjugates allows for receptor-mediated delivery; galactosylated betaCDP-based particles reveal selective targeting to hepatocytes via the asialoglycoprotein receptor. Galactosylated particles transfect hepatoma cells with 10-fold higher efficiency than glucosylated particles (control), but show no preferential transfection in a cell line lacking the asialoglycoprotein receptor. Thus, surface modification of betaCDP-based polyplexes through the use of cyclodextrin/adamantane host/guest interactions endows the particles with properties appropriate for systemic application.     

Epidermal growth factor (EGF) receptor targeted delivery of PEGylated adenovirus

A biotin-polyethylene glycol (PEG)-epidermal growth factor (EGF) conjugate was immobilized onto the surface of avidin-modified adenovirus (ADV-Avi) via biotin-avidin interaction to deliver ADV specifically to EGF receptor over-expressing cancer cells. ADV-Avi/biotin-PEG-EGF complexes showed greatly enhanced intracellular uptake of ADV particles for an EGF receptor positive cell line (A431 cells), compared to naked or PEG alone immobilized ADV. ADV coding an exogenous GFP gene was used to quantitatively evaluate the level of GFP expression. ADV-Avi/biotin-PEG-EGF complexes also exhibited significantly increased extent of GFP expression for A431 cells, but not for MCF-7 cells (an EGF receptor deficient cell line), suggesting that retargeting of ADV to specific cells occurred by tethering of a cell-specific targeting ligand to the distal end of a PEG chain anchored onto the surface of ADV. This study demonstrates that ADV-Avi/biotin-PEG-EGF construct systems can be applied for cell-specific delivery of ADV with simultaneously reducing innate immune responses.     

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.     

Development of novel poly(ethylene glycol)-based vehicles for gene delivery

The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.     

Melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors

Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI.DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI.DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.     

Polyethylenimine-graft-poly(ethylene glycol) copolymers: influence of copolymer block structure on DNA complexation and biological activities as gene delivery system

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.     

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells.

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.     

Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 881-888, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.