Genuine DNA/polyethylenimine (PEI) Complexes Improve Transfection Properties and Cell Survival

Polyethylenimine (PEI) has been described as one of the most efficient cationic polymers for in vitro gene delivery. Systemic delivery of PEI/DNA polyplexes leads to a lung-expression tropism. Selective in vivo gene transfer would require targeting and stealth particles. Here, we describe two strategies for chemically coupling polyethylene glycol (PEG) to PEI, to form protected ligand-bearing particles. Pre-grafted PEG–PEI polymers lost their DNA condensing property, hence their poor performances. Coupling PEG to pre-formed PEI/DNA particles led to the expected physical properties. However, low transfection efficacies raised the question of the fate of excess free polymer in solution. We have developed a straightforward a purification assay, which uses centrifugation-based ultrafiltration. Crude polyplexes were purified, with up to 60% of the initial PEI dose being removed. The resulting purified and unshielded PEI/DNA polyplexes are more efficient for transfection and less toxic to cells in culture than the crude ones. Moreover, the in vivo toxicity of the polyplexes was greatly reduced, without affecting their efficacy.     

Biodegradable, cationic methacrylamide-based polymers for gene delivery to ovarian cancer cells in mice

A series of cationic, methacrylamide polymers was tested for use as a biodegradable gene carrier in ovarian cancer. Tumor transfection activity of polyplexes consisting of a reporter gene and different methacrylamide polymers was assessed, after intraperitoneal injection in mice bearing an ovarian cancer xenograft. In this model, polyplexes based on poly(HPMA-DMAE) showed transfection activity similar to polyplexes based on the nondegradable and rather toxic polyethylenimine (PEI22). The tumor transfection activity of the pHPMA-DMAE polyplexes was remarkable considering their poor transfection activity in in vitro assays. Polyplexes based on pHPMA-DMAE were devoid of any cytotoxicity and mediated highest transfection activity at the highest N/P ratio investigated. Tumor cell gene expression after a single administration of these polyplexes rapidly declined within time, at a similar rate to that observed after injection with polyplexes based on PEI22. Incubation of the polyplexes with hyaluronic acid (HA), a polyanion accumulating in the ascitic fluid of ovarian cancer bearing mice, changed the physical characteristics of the pHPMA-DMAE and PEI22 polyplexes. The transfection activity of PEI22-based polyplexes, but not that of pHPMA-DMAE based polyplexes, was strongly impaired by HA. Differences in HA sensitivity might have contributed to the in vivo gene expression activities of pHPMA-DMAE- and PEI22-based polyplexes. pHPMA-DMAE-based polyplexes have potential for use in ovarian cancer therapy due to their considerable transfection activity, their low cytotoxicity, and their HA resistance.     

Enhanced gene expression and reduced toxicity in mice using polyplexes of low-molecular-weight poly(ethylene imine) for pulmonary gene delivery

The aim of this study was to elicit improved gene expression and decreased cytotoxicity for pulmonary gene therapy by replacing the commonly used carrier 25?kDa branched poly(ethylene imine) (BPEI) by two PEI derivatives, low-molecular-weight PEI (LMWPEI) and polyethylene glycol?grafted PEI (PEGPEI). All polymers were shown to condense DNA to spherical particles of approximately 100?nm. Biocompatibility was investigated in vitro and in vivo. Although transfection was less efficient with LMWPEI-DNA in vitro, this polyplex caused the highest luciferase expression in the mouse lung after intratracheal instillation. While PEGPEI luciferase expression in vitro was approximately three times higher when compared to BPEI, a transfection rate at the level of naked DNA was observed in vivo. LMWPEI polyplexes were located in both the bronchial and alveolar cells, whereas BPEI polyplexes were mainly detected in bronchial cells. LMWPEI combines low cytotoxicity with high transfection efficiency in the mouse lung in vivo, rendering it a promising strategy for pulmonary gene delivery.     

Stabilized Nonviral Formulations for the Delivery of MCP-1 Gene into Cells of the Vasculoendothelial System

PURPOSE: The purpose of this study was to develop a stabilized non-viral gene transfer system for the efficient delivery and expression of monocyte chemoattractant protein 1 (MCP-1) gene in cells of the vasculoendothelial system. METHODS: Plasmid DNA was condensed with polyethylenimine (PEI), conjugates of PEI with polyethylene glycol (PEG), and PEI conjugates with the membrane-active peptide melittin. Surface charge and particle size of the resulting gene transfer particles were analyzed by laser light scattering. Reporter gene studies and toxicity assays were conducted on smooth muscle cells and endothelial cells of human, porcine, or rat origin. RESULTS: Nonviral gene carriers containing PEI and PEG were developed that could be produced in batches of several milligrams and conveniently stored as frozen samples. Incorporation of PEG into the transfection complex significantly reduced cellular toxicity. The cryoconserved gene transfer particles mediated high expression of luciferase, enhanced green fluorescent protein (EGFP), or secreted alkaline phosphatase reporter genes. Highest reporter gene expression was achieved with PEI polyplexes containing PEG and melittin. The gene for MCP-1 was efficiently delivered into target cells and resulted in expression of up to 125 ng/ml secreted bioactive MCP-1 protein per 50,000 cells. CONCLUSIONS: Gene carriers based on PEI and PEG display reduced toxicity, can be stored in frozen form without loss of biological activity, and can efficiently transfect cells of the vasculoendothelial system. Such gene carriers hold a potential for use in arterial gene transfer and local secretion of MCP-1 as trigger of therapeutic arteriogenesis in arterial occlusion diseases.     

Plasmid CpG Depletion Improves Degree and Duration of Tumor Gene Expression After Intravenous Administration of Polyplexes

PURPOSE: Tumor gene expression after the intravenous (i.v.) administration of current polymer-based gene delivery systems is generally low and short-lived. Immune stimulatory CpG dinucleotides, present within the plasmid DNA of the polyplexes are likely to contribute to this. The effect of CpG replacement on the levels of transgene expression was studied, after the i.v. administration of polyethylenimine (PEI) polyplexes. METHODS: Tumor transfection and immune stimulation of PEI polyplexes containing plasmid DNA encoding for luciferase and rich in CpG motifs was monitored and compared to polyplexes containing the same gene but devoid of CpG motifs. Lipoplexes based on 1,2-dioleyl-3-trimethylammonium-propane/dioleoylphosphatidylethanolamine liposomes were included as a control. RESULTS: The replacement of CpGrich DNA by CpGfree DNA did neither affect the physical properties of the DNA complexes nor did it affect their in vitro transfection activity or cytotoxicity. The immune stimulation (interleukin-12) after i.v. administration of the PEI DNA complexes was low and unaffected by the presence of CpG motifs. The absence of CpG motifs within the different DNA complexes improved the degree and the duration of organ and tumor gene expression. CONCLUSION: The depletion of CpG dinucleotides within the plasmid DNA of polyplexes enhances the degree and duration of in vivo transgene expression.     

A one-step process in preparation of cationic nanoparticles with poly(lactide-co-glycolide)-containing polyethylenimine gives efficient gene delivery

A one-step preparation of nanoparticles with poly(lactide-co-glycolide) (PLGA) pre-modified with polyethylenimine (PEI) is better in requirements for DNA delivery compared to those prepared in a two-step process (preformed PLGA nanoparticles and subsequently coated with PEI). The particles were prepared by emulsification of PLGA/ethyl acetate in an aqueous solution of PVA and PEI. DLS, AFM and SEM were used for the size characteristics. The cytotoxicity of PLGA/PEI nanoparticles was detected by MTT assay. The transfection activity of the particles was measured using pEGFP and pβ-gal plasmid DNA. Results showed that the PLGA/PEI nanoparticles were spherical and non-porous with a size of about 0.2 μm and a small size distribution. These particles had a positive zeta potential demonstrating that PEI was attached. Interestingly, the zeta potential of the particles (from one-step procedure) was substantially higher than that of two-step process and is ascribed to the conjugation of PEI to PLGA via aminolysis. The PLGA/PEI nanoparticles were able to bind DNA and the formed complexes had a substantially lower cytotoxicity and a higher transfection activity than PEI polyplexes. In conclusion, given their small size, stability, low cytotoxicity and good transfection activity, PLGA/PEI-DNA complexes are attractive gene delivery systems.     

Receptor-targeted gene delivery viafolate-conjugated polyethylenimine

A novel synthetic gene transfer vector was evaluated for tumor cell-specific targeted gene delivery. The folate receptor is a tumor marker overexpressed in more than 90% of ovarian carcinomas and large percentages of other human tumors. Folic acid is a high affinity ligand for the folate receptor that retains its binding affinity upon derivatization via its gamma carboxyl. Folate conjugation, therefore, presents a potential strategy for tumor-selective targeted gene delivery. In the current study, we investigated a series of folate conjugates of the cationic polymer polyethylenimine (PEI) for potential use in gene delivery. A plasmid containing a luciferase reporter gene (pCMV-Luc) and the folate receptor expressing human oral cancer KB cells were used to monitor gene transfer efficiency in vitro. Transfection activity of polyplexes containing unmodified polyethylenimine was highly dependent on the positive to negative charge (or the N/P) ratio. Folate directly attached to PEI did not significantly alter the transfection activity of its DNA complexes compared to unmodified PEI. Modification of PEI by polyethyleneglycol (PEG) led to a partial inhibition of gene delivery compared to unmodified PEI. Attaching folates to the distal termini of PEG-modified PEI greatly enhanced the transfection activity of the corresponding DNA complexes over the polyplexes containing PEG-modified PEI. The enhancements were observed at all N/P ratios tested and could be blocked partially by co-incubation with 200 μM free folic acid, which suggested the involvement of folate receptor in gene transfer. Targeted vectors based on the folate-PEG-PEI conjugate are potentially useful as simple tumor-specific vehicles of therapeutic genes.     

Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex

PURPOSE: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency. METHODS: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using (1)H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay. RESULTS: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI. CONCLUSIONS: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.     

Lipoic acid modified low molecular weight polyethylenimine mediates nontoxic and highly potent in vitro gene transfection

The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI-LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4-6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI-LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI-LA conjugates condensed DNA into much smaller sizes (183-84 nm) than unmodified 1.8 kDa PEI (444-139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa PEI-LA(2) polyplexes formed at an N/P ratio of 10/1 were stable against exchange with 12-fold excess of negatively charged dextran sodium sulfate (DSS) relative to DNA phosphate groups while 1.8 kDa PEI controls dissociated at 6-fold excess of DSS, indicating that lipoylation of 1.8 kDa PEI resulted in stronger binding with DNA. Importantly, DNA was released from 1.8 kDa PEI-LA(2) polyplexes upon addition of 10 mM dithiothreitol (DTT). Reduction-triggered unpacking of 1.8 kDa PEI-LA(2) polyplexes was also confirmed by DLS. MTT assays demonstrated that all PEI-LA conjugates and polyplexes were essentially nontoxic to HeLa and 293T cells up to a tested concentration of 50 μg/mL and an N/P ratio of 80/1, respectively. The in vitro gene transfection studies in HeLa and 293T cells showed that lipoylation of 1.8 kDa PEI markedly boosted its transfection activity. For example, 1.8 kDa PEI-LA(2) polyplexes displayed 400-fold and 500-fold higher levels of gene expression than unmodified 1.8 kDa PEI controls, which were ca. 2-fold and 3-fold higher than 25 kDa PEI controls, in serum-free and 10% serum media, respectively. The transfection efficiency decreased with increasing DS, following an order of 1.8 kDa PEI-LA(2) > 1.8 kDa PEI-LA(4) > 1.8 kDa PEI-LA(6) ? 1.8 kDa PEI. Confocal laser scanning microscopy (CLSM) studies corroborated that 1.8 kDa PEI-LA(2) delivered and released DNA into the nuclei of HeLa cells more efficiently than 25 kDa PEI. These nontoxic 1.8 kDa PEI-LA conjugates form a superb basis for the development of targeting, biocompatible and highly efficient carriers of gene delivery.     

Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex

Purpose: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency.

Methods: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using ¹H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay.

Results: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI.

Conclusions: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.     

藻酸盐/PEI/DNA复合载体作为一种新型基因递送系统

目的克服多聚乙烯亚胺(PEI，polyethlenimine)/DNA载体对细胞的毒性以及在含血清培养基里对癌细胞基因的转移率低的问题。方法利用具有水溶性、可生物降解的、并带有负电的藻酸盐(alginate)对PEI/DNA载体进行包衣，制备出复合载体，并在体外含50%血清培养基里，与PEI/DNA载体比较对C3癌细胞转染率。结果 在含50%血清的培养基里，藻酸盐包衣制备的复合体载体［alginate:DNA，0.15 (w/w);PEI:DNA，N:P=10］与PEI/DNA载体相比，对C3癌细胞基因转染率高出10～30倍，而且其表面正电荷数比PEI/DNA载体减少了一半，颗粒较小，并降低对细胞毒性和红血球集聚反应。结论作为新型的藻酸盐包衣制备的复合载体能提高在体外含高浓度血清培养基里对C3癌细胞的转染率，并能减少其对细胞毒性。     

Synthesis and characterization of mannosylated pegylated polyethylenimine as a carrier for siRNA

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45-13.3 PEG chains and 4.7-3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169 to 357 nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2h post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone.     

Cellular Uptake of Cationic Polymer-DNA Complexes Via Caveolae Plays a Pivotal Role in Gene Transfection in COS-7 Cells

Purpose Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. Materials and Methods The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. Results Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. Conclusions These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.     

Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression.

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.     

Imidazolyl-PEI modified nanoparticles for enhanced gene delivery

The derivatives of polyethylenimine (PEI 25 and 750kDa) were synthesized by partially substituting their amino groups with imidazolyl moieties. The series of imidazolyl-PEIs thus obtained were cross-linked with polyethylene glycol (PEG) to get imidazolyl-PEI-PEG nanoparticles (IPP). The component of hydrophobicity was introduced by grafting the lauryl groups in the maximal substituted IPP nanoparticles (IPPL). The nanoparticles were characterized with respect to DNA interaction, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection efficiency on model cell lines. The IPP and IPPL nanoparticles formed a loose complex with DNA compared to the corresponding native PEI, leading to more efficient unpackaging of DNA. The DNA loading capacity of IPP and IPPL nanoparticles was also lower compared to PEI. The imidazolyl substitution improved the gene delivery efficiency of PEI (750kDa) by nine- to ten-fold and PEI (25kDa) by three- to four-fold. At maximum transfection efficiency, the zeta potential of nanoparticles was positive after forming a complex with DNA. The maximum level of reporter gene expression was mediated by IPPL nanoparticles in both the series. The cytotoxicity, another pertinent problem with cationic polymers, was also negligible in case of IPP and IPPL nanoparticles.     

The fate of poly(2-dimethyl amino ethyl)methacrylate-based polyplexes after intravenous administration

Poly(2-dimethyl amino ethyl) methacrylate (pDMAEMA) cationic polymers have been shown to be efficient vectors for gene delivery in vitro. This contribution deals with the in vivo properties of polyplexes based on this polymer. In mice, pDMAEMA/[32P]-pLuc complexes distributed primarily to the lungs. The gene expression profile matched the biodistribution profile. In vitro turbidity experiments in serum showed severe aggregation upon addition of cationic polyplexes, pointing out the involvement of aggregates in the dominant lung uptake of the positively charged polyplexes. Incubations of polyplexes with albumin yielded a decline of the zeta potential of the complexes to negative values, making an electrostatic mechanism in the dominant lung uptake less likely. Hemagglutination experiments showed that the polyplexes induce the formation of extremely large structures when incubated with washed erythrocytes. Altogether, the present data indicate that aggregate formation and trapping of the formed aggregates in the lung capillary bed is probably responsible for the dominant lung uptake and transfection. Poly(ethylene)glycol (PEG) of the polymeric structures prevented the increase in the observed turbidity in serum seen with polyplexes and was also able to reduce interactions with erythrocytes. Currently, the in vivo fate of the PEGylated polyplexes is under investigation.     

Blending of polyethylenimine with a cationic polyurethane greatly enhances both DNA delivery efficacy and reduces the overall cytotoxicity

Three blending methods were introduced to combine a biodegradable cationic- polyurethane (PUg3) and polyethylenimine (PEI) together with DNA by different mixing sequences. Results of gel electrophoresis assays and particle size measurements show that complexes prepared by method 1 and 3 bear an ability to condense DNA into small nanoparticles. On the contrary, the use of method 2 in making complexes produces significantly large particles because of the weaker interaction with DNA and lack of DNA condensation. Moreover, cell proliferation assays show that no cytotoxicity of the DNA/blended-polymers complexes (exhibited by method 1) was found and due to a result of the outer coating of PUg3, reducing cytotoxic PEI exposure outside the complexes. With a new technique in pharmaceutics, the complexes prepared for DNA delivery by mixing of PEI and PUg3 with DNA in a sequence (method 1) could achieve an even better transfection efficiency (reaching 40% higher) than using PEI alone as well as reduce the cytotoxicity substantially. In conclusion, a new class of complexes (non-viral combo-system) made by a skillful blending sequence (method 1) has been designed and demonstrated to obtain the beneficial properties from two useful and individual polymers for gene delivery. This method can be used in greatly improving the transfection efficiency of polymer-based gene vectors. The blended polymers with DNA also have a better biocompatibility and no cytotoxicity, which are the requirements and critical points for great success in performing gene therapy in vivo.     

Effect of polyethylene glycol on gene delivery of polyethylenimine

Polyethylene glycol (PEG) has been coupled to many cationic polymers such as polyethylenimine (PEI) to improve the stability and transfection efficiency. We prepared PEG-grafted PEI with different lengths and amounts of PEG and used these graft copolymers as nonviral gene vectors. We measured the complex size and zeta-potential of polymer-DNA complexes in the presence of salt to estimate the stability of polymer-DNA complexes. We also investigated the cytotoxicity and transfection efficiency in C3 cells. In the case of graft copolymers, the stability of polymer-DNA complexes and transfection efficiency were affected by the graft length and amount of PEG side chain. PEG side chains stabilize the polymer-DNA complexes in the presence of salt, but the longer PEG side chains also interrupt the gene delivery in the cells due to the more efficient steric hindrance by longer PEG side chains, and therefore the transfection efficiency is decreased. Short PEG side chains with molecular weight of 350 kDa stabilized the polymer-DNA complexes without decreased transfection efficiency.     

Polyplexes based on cationic polymers with strong nucleic acid binding properties

Cationic polymers have been studied for nucleic acid delivery both in vitro and in vivo. However, many polymer-based formulations suffer from lack of stability in biologic fluids due to interactions with anionic biomacromolecules such as proteins and polysaccharides. Likely, the stronger the electrostatic interactions between a cationic polymer and nucleic acids, the higher the stability of the polyplexes in biologic fluids will be. To get evidence for this hypothesis, quaternized poly[3,5-bis(dimethylaminomethylene)-p-hydroxyl styrene] (QNPHOS) with two permanently charged cationic sites per monomer unit as well as its block copolymer with PEG were synthesized and compared with the standard transfectant pDMAEMA, in terms of nucleic acid binding strength, gene silencing and transfection activities of the complexes which these polymers form with siRNA and plasmid DNA, respectively. It was shown that siRNA complexes based on QNPHOS and QNPHOS-PEG dissociate in the presence of a fourfold higher heparin concentration than necessary to destabilize pDMAEMA complexes. Under the same conditions, complexes of DNA and QNPHOS or QNPHOS-PEG did not show any dissociation, in contrast to pDMAEMA polyplexes. The DNA polyplexes based on QNPHOS or QNPHOS-PEG did not show transfection activity, which might be ascribed to their high physicochemical stability. On the other hand, siRNA complexes based on QNPHOS and QNPHOS-PEG showed a low cytotoxicity and an improved siRNA delivery and high gene silencing activity, even higher than those based on pDMAEMA. This might be due to the excellent binding characteristics of QNPHOS and QNPHOS-PEG to siRNA which in turn is ascribed to the presence of two permanently charged cationic groups per monomer unit. Based on the results of this study, it is concluded that formation of strong siRNA complexes with polymers containing double charges per monomer is advantageous.