Synthesis of sulfhydryl cross-linking poly(ethylene glycol)-peptides and glycopeptides as carriers for gene delivery

Sulfhydryl cross-linking poly(ethylene glycol) (PEG)-peptides and glycopeptides were prepared and tested for spontaneous polymerization by disulfide bond formation when bound to plasmid DNA, resulting in stable PEG-peptide and glycopeptide DNA condensates. A 20 amino acid synthetic peptide possessing a single sulfhydryl group on the N-terminal cysteine, with two or five internal acetamidomethyl (Acm)-protected cysteine residues, was reacted with either PEG vinyl sulfone or iodoacetamide tyrosinamide triantennary N-glycan. Following RP-HPLC purification, Acm groups were removed by silver tetrafluoroborate to generate sulfhydryl cross-linking PEG-peptides and glycopeptide that were characterized by either (1)H NMR or LC-MS. Sulfhydryl cross-linking PEG-peptides and glycopeptides were found to bind to plasmid DNA and undergo disulfide cross-linking resulting in stable DNA condensates with potential utility for in vivo gene delivery.     

Selection of continuous epitope sequences and their incorporation into poly(ethylene glycol)-peptide conjugates for use in serodiagnostic immunoassays: application to Lyme disease

Continuous epitope sequences were selected from immunogenic Bb proteins by epitope mapping. The identified epitope sequences were synthesized by solid phase peptide synthesis and purified by high performance liquid chromatography. Each epitope was conjugated individually to a multifunctional poly(ethylene glycol) (PEG) carrier. The result PEG-peptide conjugates were used as antigens in ELISA for diagnosis of Lyme disease. The results showed that the defined epitope peptides were Lyme disease specific and could be used in a format of PEG-peptide conjugate as the antigen to achieve improved sensitivity and specificity.     

Synthesis of polyethylene glycol (PEG) derivatives and PEGylated-peptide biopolymer conjugates

We synthesized a library of 50 poly(ethylene glycol) (PEG) derivatives to expand the extent of conjugation with biologically active molecules (biopolymers, peptides, drugs, etc.) and biomaterial substrates. The formation of PEG derivatives was confirmed with HPLC, (1)H and (13)C NMR. PEG derivatives were polymerized into networks in order to study the role of PEG and terminal functional groups in modulating the hydrophilicity of biomaterials and cell-biomaterial interaction. The resulting surface hydrophilicity and the number of adhered fibroblasts were primarily dependent on the PEG concentration with the molecular weight and the terminal functional group of PEG derivatives being less important. One of PEG derivatives, PEG-bis-glutarate, was utilized to link peptide sequences to gelatin backbone in the formation of novel biomedical hydrogels. PEG-peptide conjugates were characterized by mass spectroscopy. PEG-peptide modified gelatins were characterized by gel permeation chromatography.     

Synthetic PEGylated glycoproteins and their utility in gene delivery

PEGylated glycoproteins (PGPs) were synthesized by copolymerizing a Cys-terminated PEG-peptide, glycopeptide, and melittin peptide. Compositionally unique PGPs were prepared by varying the ratio of PEG-peptide (20-90%) and melittin (0-70%) with a constant amount of glycopeptide (10%). The PGPs were purified by RP-HPLC, and characterized for molecular weight and polydispersity by GPC-HPLC and SDS-PAGE and for composition by RP-HPLC following reduction to form monomeric peptides. PGPs formed DNA condensates of 200-300 nm in diameter that were administered to mice via the tail vein. Biodistribution studies confirmed their primary targeting to liver hepatocytes with a DNA metabolic half-life of 1 h. Upon stimulation by hydrodynamic dosing with saline, PGP DNA (5 microg) mediated luciferase expression in the liver detected by bioluminescence imaging (BLI) after 24 h. The level of gene expression mediated by PGP DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP. The results establish the ability to prepare three-component gene delivery polymers that function in vivo. Further design improvements in fusogenic peptides for gene delivery and for the simultaneous use of a nuclear targeting strategy will be necessary to approach levels of expression mediated by the direct hydrodynamic dosing of DNA.     

PEGylation enhances the therapeutic potential of peptide antagonists of the neonatal Fc receptor, FcRn

Peptides targeting the human neonatal Fc receptor (FcRn) were conjugated to poly(ethylene glycol) (PEG) polymers to study their effect on inhibition of the IgG:FcRn protein-protein interaction both in vitro and in mice. Both linear (5-40kDa) and branched (20, 40kDa) PEG aldehydes were conjugated to an amine-containing linker of a homodimeric anti-FcRn peptide using reductive alkylation chemistry. It was found that conjugation of PEG to the peptide compromised the in vitro activity, with larger and branched PEGs causing the most dramatic losses in activity. The conjugates were evaluated in transgenic mice for their ability to accelerate the catabolism of human IgG. Optimal pharmacodynamic properties were observed with PEG-peptide conjugates that contained 20-40kDa linear PEGs and a 20kDa branched PEG. The optimal PEG-peptide conjugates were more effective in vivo than the unconjugated peptide control on a mole:mole and mg/kg basis, and represent potential new longer-acting peptide therapeutics for the treatment of humorally-mediated autoimmune disease.     

A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Background

We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods

POD peptide was modified using poly(ethylene) glycol (PEG‐POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post‐injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG‐TAT and PEG‐CK30.

Results

PEG‐POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG‐POD significantly increased expression of plasmid DNA by 215‐fold, PEG‐TAT by 56.52‐fold, and PEG‐CK30 by 24.73‐fold relative to DNA injected alone. In all cases β‐galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG‐POD transduced retina indicates an absence of PEG‐POD‐mediated toxicity. PEG‐POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice.

Conclusions

PEG‐POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG‐POD, as novel gene delivery vectors. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative analysis of free DNA delivery and expression into protoplasts of Panicum maximum Jacq. (Guinea grass) by electroporation and polyethylene glycol

Relative levels of gene expression were studied in protoplasts isolated from two cell lines of Panicum maximum following DNA delivery by electroporation and polyethylene glycol (PEG). Gene expression was evaluated by assaying for chloramphenicol acetyltransferase (CAT) activity expressed by the CaMV 35S promoter with a nopaline synthase 3' polyadenylation signal, approximately 48 hours after DNA delivery. The expression of the CAT gene was slightly higher in electroporated protoplasts in comparison to PEG mediated delivery. However, PEG treated protoplasts showed higher plating efficiency. The effect of different salts and the molecular weight of PEG used on gene expression was also studied.     

A potent new class of reductively activated peptide gene delivery agents

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.     

Synthesis and in vitro characterization of an ABC triblock copolymer for siRNA delivery

The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG-PPS-peptide). The diblock copolymer PEG(45)-PPS(5,10) was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys(9)). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression down-regulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% +/- 0.02% down-regulation.     

Multifunctional siRNA delivery system: Polyelectrolyte complex micelles of six‐arm PEG conjugate of siRNA and cell penetrating peptide with crosslinked fusogenic peptide

For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six‐arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG‐siRNA). The 6PEG‐siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG‐siRNA‐Hph1). The 6PEG‐siRNA‐Hph1 conjugate was electrostatically complexed with cationic self‐crosslinked fusogenic KALA peptide (cl‐KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG‐siRNA‐Hph1/cl‐KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA‐MB‐435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

(LYS)(16)-based reducible polycations provide stable polyplexes with anionic fusogenic peptides and efficient gene delivery to post mitotic cells

Extracellular stability, endocytic escape, intracellular DNA release and nuclear translocation of DNA are all critical properties of non-viral vector/DNA particles. We have evaluated a (Lys)(16)-based linear, reducible polycation (RPC) in combination with an acid-dependent, anionic fusogenic peptide for gene delivery to dividing and post-mitotic cells. The RPC was formed from Cys(Lys)(16)Cys monomers. Molecular weight was 24,000 Da, corresponding to an average of 10.5 peptide monomers per RPC. Non-reducible polylysine (PLL) (27,000 Da) and monomeric (Lys)(16) peptide were evaluated for comparison. (Lys)(16)/DNA particles were disrupted at fusogenic peptide concentrations well below those used for gene delivery. By contrast, RPC/DNA an PLL/DNA particles were stable in the presence of high concentrations of the anionic peptide. Addition of 10% serum virtually abolished the transfection ability of (Lys)(16)/DNA/fusogenic peptide particles, but had little effect on RPC/DNA/fusogenic peptide particles. RPC/DNA/fusogenic peptide particles were highly effective for gene delivery to both cell lines and post-mitotic corneal endothelium. PLL/DNA/fusogenic peptide particles were moderately effective on cell lines, but gave no gene delivery with corneal endothelial cells. We conclude that (Lys)(16)-based RPC/DNA/fusogenic peptide particles provide a gene delivery system which is potentially stable in the extracellular environment and, on reductive depolymerisation, can release DNA plasmids for nuclear translocation.     

Exploration of peptide motifs for potent non-viral gene delivery highly selective for dividing cells

BACKGROUND: The immunogenicity of viral DNA vectors is an important problem for gene therapy. The use of peptide motifs for gene delivery would largely overcome this problem, and provide a simple, safe and powerful approach for non-viral gene therapy. METHODS: We explored the functional properties of two motifs: the (Lys)(16) motif (for binding and condensing DNA, and probably also nuclear translocation of plasmids) and the fusogenic peptide motif of influenza virus (for acid-dependent endocytic escape of peptide/DNA particles). The physical properties and gene delivery efficiencies of (Lys)(16)-containing peptides in combination with free fusogenic peptide were evaluated, and compared with a single composite peptide incorporating both moieties. Post-mitotic corneal endothelial cells and growth-arrested HeLa were included, so as not to neglect the question of nuclear translocation of plasmids. RESULTS: The fusogenic moiety in the composite peptide was able to adopt an alpha-helical configuration unhindered by the (Lys)(16) moiety, and retained acid-dependent fusogenic properties. The composite peptide gave remarkably high levels of gene delivery to dividing cell lines. However, in marked contrast to (Lys)(16)/DNA complexes plus free fusogenic peptide, the composite peptide was completely ineffective for gene delivery to post-mitotic and growth-arrested cells. CONCLUSIONS: Attachment of the fusogenic peptide to (Lys)(16) appears to block (Lys)(16)-mediated nuclear translocation of plasmid, but not fusogenic peptide mediated endocytic escape. This strengthens the experimental basis for (Lys)(16)-mediated nuclear translocation of plasmids, and provides a single peptide with potent gene delivery properties, restricted to dividing cells. This property is potentially useful in experimental biology and clinical medicine.     

Novel intracellular delivery system of antisense oligonucleotide by self-assembled hybrid micelles composed of DNA/PEG conjugate and cationic fusogenic peptide

An antisense oligonucleotide (ODN), c-myb, was covalently conjugated to poly(ethylene glycol) (PEG) via an acid-cleavable phosphoramidate linkage to form a diblock copolymer-like structure. The phosphoramidate linkage between ODN and PEG was completely cleaved within 5 h in an endosomal acidic condition (pH 4.7). When complexed with a cationic fusogenic peptide, KALA, the ODN/PEG conjugate self-associated to form polyelectrolyte complex micelles in an aqueous solution. The anionic ODN segments were ionically interacted with cationic KALA peptide to form an inner polyelectrolyte complex core, while the PEG segments constituted a surrounding corona. Effective hydrodynamic volume of the micelles was ca. 70 nm with a very narrow size distribution. The polyelectrolyte complex micelles, composed of c-myb ODN-PEG conjugate and KALA, were transported into cells far more efficiently than c-myb ODN itself. They also exhibited higher antiproliferative activity against smooth muscle cells. This study demonstrates that the DNA/PEG hybrid micelles system can be applied for the delivery of antisense oligonucleotide.     

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.     

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).     

A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.

A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.     

Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

Poly(DMAEMA-NVP)-b-PEG-galactose as gene delivery vector for hepatocytes

A block copolymer composed of cationic polymer and poly(ethylene glycol) (PEG) was used as a DNA carrier. Poly(2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-N-vinyl-2-pyrrolidone (NVP)) having a terminal carboxylic group was synthesized by free radical polymerization using an initiator, 4,4'-azobis(4-cyanovaleric acid). The terminal carboxylic acid was activated by N-hydroxysuccinimide (NHS) with dicyclohexylcarbodiimide (DCC) and then conjugated with PEG-bis(amine). For specific gene targeting to asialoglycoprotein receptor of hepatocytes, a galactose moiety was incorporated into the PEG terminal end of poly(DMAEMA-NVP)-b-PEG by reductive coupling using lactose and sodium cyanoborohydride. RSV luciferase plasmid was used as a reporter gene, and in vitro gene transfection efficiency was measured in HepG2 human hepatocarcinoma cells. Poly(DMAEMA-NVP)-b-PEG-galactose/DNA complexes formed at 0.5-2 polymer/plasmid weight ratio had compacted structures around 200 nm particle size and exhibited slightly negative surface charge. These complexes were coated with a cationic, pH sensitive, endosomolytic peptide, KALA, to generate positively charged poly(DMAEMA-NVP)-b-PEG-galactose/DNA/KALA complex particles. In the presence of serum proteins, both the PEG block and the galactose moiety of poly(DMAEMA-NVP)-b-PEG-galactose greatly enhanced the gene transfection efficiency, which was very close to that of Lipofectamine plus. Irrespective of the presence of serum proteins, as the KALA/DNA weight ratio increased, the transfection efficiency of poly(DMAEMA-NVP)-b-PEG-galactose was enhanced due to the pH dependent endosomal disruptive property of KALA. This study demonstrates that sufficient transfection efficiency as high as that of commercial agent could be attained by judicious formulation of molecular engineered poly(DMAEMA-NVP)-b-PEG-galactose in combination with an endosomolytic peptide, KALA.     

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.