α,β-Poly(l-aspartate-graft-PEI): A pseudo-branched PEI as a gene carrier with low toxicity and high transfection efficiency

The aim of this research is to develop a novel branched polyethylenimine (PEI)-like polycation as a potential gene carrier with high gene transfection efficiency and low toxicity. In particular, α,β-poly(l-aspartate-graft-PEI) (Asp-g-PEI), a pseudo-branched PEI, was synthesized by the ring-opening reaction of poly(l-succinimide) (PSI) with low molecular weight branched PEI (LMW PEI, MW = 600 and 1200). Good plasmid condensation and protection ability of Asp-g-PEI were confirmed by agarose gel electrophoresis assay. Asp-g-PEI/DNA complexes showed high positive zeta potential, narrow size distribution, good dispersity and a compact spherical shape with size below 250 nm when the N/P ratio was above 5, suggesting that they can be endocytosed. Cytotoxicity of Asp-g-PEI/DNA complexes was rather lower than that of PEI25K/DNA complexes, especially at high N/P ratio. The most efficient gene transfection of Asp-g-PEI/DNA complexes was similar or a little higher than that of PEI25K in 293T, HeLa and HepG2 cell lines, while almost 4 and 6 times higher than that of parent PEI1200 and PEI600, respectively, in HeLa cell line; as the molecular weight of parent PEI in Asp-g-PEI was increased from 600 to 1200, the transfection efficiency showed a tendency to decrease. The mechanism of Asp-g-PEI-mediated gene transfection was attributed to the “proton sponge effect” due to PEI in the copolymer.     

A biodegradable poly(ester amine) based on polycaprolactone and polyethylenimine as a gene carrier

The aim of research was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biodegradable polymers, in particular, poly(ester amine)s (PEAs), suitable for non-viral gene therapy. Poly(ester amine)s were successfully synthesized by Michael addition reaction between polycaprolactone (PCL) diacrylate and low molecular weight polyethylenimine (PEI). PEA/DNA complexes showed effective and stable DNA condensation with the particle sizes below 200nm, implicating its potential for intracellular delivery. PEAs showed controlled degradation and were essentially non-toxic in all three cells (293T: Human kidney carcinoma, HepG2: Human hepatoblastoma and HeLa: Human cervix epithelial carcinoma cell lines) at higher doses in contrast to PEI 25K. PEAs also revealed much higher transfection efficiencies in three cell lines as compared to PEI 25K. The highest reporter gene expression was observed for PCL/PEI-1.2 (MW 1200) complex having transfection efficiency 15-25 folds higher than PEI 25K in vitro. Also PEA/DNA complexes successfully transfected cells in vivo after aerosol administration than PEI 25K. These PEAs can be used as most efficient polymeric vectors which provide a versatile platform for further investigation of structure property relationship along with the controlled degradation, significant low cytotoxicity and high transfection efficiency.     

Polyethyleneimine-mediated gene delivery into human adipose derived stem cells

In this study, we examined the use of polyethyleneimine (PEI) as a carrier for gene delivery in human adipose tissue-derived stem cells (hADSCs). These multipotent cells can form bone, cartilage, adipose, and other connective tissues. In primary culture, hADSCs are fibroblastic in appearance in primary culture, and they show a high rate of proliferation for at least five passages. Immunophenotyping showed that these cells are positive for the mesenchymal stem cell markers CD29 and CD44 but negative for the hematopoietic cell surface markers CD34, CD45, and c-kit. PEI and Lipofectamine were compared as gene carriers for hADSCs. DNA completely bound PEI at a negative-to-positive (N/P) charge ratio of 4. The PEI-DNA complexes were spherical with smooth surfaces. As the proportion of PEI was increased, the size of the PEI-DNA complexes decreased from 990 to 130nm, the positive surface charge decreased, and the cytotoxicity increased. Flow cytometry revealed that the transfection efficiency using PEI at N/P charge ratios of 4 and 8 was higher than that of Lipofectamine. The highest transfection efficiency (19%) was obtained at an N/P charge ratio of 8. After transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of hADSCs at 4h 30m and localize over cytoplasm from 9h 30m. In conclusion, PEI acts as an effective gene carrier for hADSCs.     

Diaminododecane-based cationic bolaamphiphile as a non-viral gene delivery carrier

The advancement in gene therapy relies upon the discovery of safe and efficient delivery agents and methods. In this study, we report the design and synthesis of a cationic bolaamphiphile as a non-viral gene delivery agent. The bolaamphiphile is composed of 1,12-diaminododecane as the central hydrophobic unit linked to the hydrophilic pentaethylenehexamine via thioether-based glycidyl units. This bolaamphiphile condensed DNA efficiently into nanoparticles of sizes around 150-200 nm with positive zeta potential of 30-35 mV. In vitro luciferase expression levels and percentage of GFP expressing cells induced by the bolaamphiphile/DNA complexes were higher than those mediated by the often used "golden" standard of non-viral systems, polyethyleneimine (PEI, branched, 25 kDa) at its optimal N/P ratio in HEK293, HepG2, NIH3T3, HeLa and 4T1 cells. In vitro cytotoxicity testing revealed that the DNA complexes fabricated from this cationic bolaamphiphile displayed marginal toxicity towards all the cell lines tested. In addition, in vivo transfection studies carried out in a 4T1 mouse breast cancer model showed that the cationic bolaamphiphile delivered DNA more efficiently than PEI. This cationic bolaamphiphile may make a promising gene delivery vector for future gene therapy.     

All-trans-retinoic acid (ATRA)-grafted polymeric gene carriers for nuclear translocation and cell growth control

Polyethyleneimine (PEI)-g-All-trans-retinoic acid (ATRA) (designated as PRA) was synthesized as a gene carrier. ATRA at its low concentration is known to be linked to nuclear translocation and cell cycle control (either proliferation or growth arrest) depending on its binding protein in cells. The cytotoxicity of PRA conjugates was lower than that of PEI and was gradually reduced as increasing ATRA graft ratios. The resulting nanosized and positively charged PRA/pDNA complexes showed lower transfection efficiency than the PEI/pDNA complexes (N/P = 10) against NIH3T3 which is less sensitive to ATRA in cell growth and more sensitive HeLa cells. However, when a mixed gene complex of PEI and PRA was applied in an effort to reduce the ATRA contents, their NIH3T3 transfection evidenced effective nuclear translocation and induced 2- to 4-fold better transfection efficiency as compared with the PEI/pDNA complexes. When the PEI/pDNA complexes were utilized to transfect HeLa cells, free ATRA treatment reduced their cellular uptake and transfection efficiency. These findings show that the NIH3T3 cells against ATRA-mediated growth arrest would not damage the PRA-mediated transfection enhancement resulting from the facilitated nuclear translocation of polyplexes or pDNA. The more ATRA-sensitivity in growth arrest of HeLa cells would reduce the transfection efficiency of ATRA-incorporated polyplexes. The transfection capability of gene by newly synthesized PRA conjugates to cells is differentiated by their ATRA-sensitivity to nuclear translocation and cell growth control.     

Characterization of lactoferrin as a targeting ligand for nonviral gene delivery to airway epithelial cells

In this study lactoferrin (Lf) was investigated as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells. A high number of lactoferrin receptors (LfRs) were detected on bronchial epithelial (BEAS-2B), but not on alveolar epithelial (A549) cells by fluorescence microscopy and FACS measurements, suggesting potential targeting selectivity for bronchial epithelial cells. Molecular conjugates with ratios of Lf to branched polyethylenimine 25 kDa (PEI) ranging from 4:1 to 1:40 (mol/mol) were synthesized and analyzed for complexation of plasmid DNA (pDNA), transfection efficiency, and cytotoxicity. Whereas particle size increased with the degree of Lf coupling from 45 to 225 nm, surface charge was not significantly influenced. Transfection studies on BEAS-2B cells revealed that Lf-PEI 1:20 exhibited the highest luciferase gene expression which was 5-fold higher at an N/P ratio (molar ratio of PEI nitrogen to pDNA phosphate) of 4 than PEI and could be inhibited by an excess of free Lf. With A549 cells, no significant enhancement in transfection efficiency between Lf-PEI/pDNA and PEI/pDNA complexes could be observed. Increasing the degree of Lf coupling to PEI resulted in reduced transfection efficiency in both alveolar and bronchial epithelial cells. Cell viability assays resulted in significantly lower cellular toxicity of Lf-PEI/pDNA compared with PEI/pDNA complexes. We suggest that Lf represents a potent targeting ligand for receptor-mediated gene delivery to bronchial epithelial cells and might be a promising candidate for lung gene transfer in vivo.     

A strategy to improve serum-tolerant transfection activity of polycation vectors by surface hydroxylation

The aim of this contribution is to develop a universal method to promote the serum-tolerant capability of polycation-based gene delivery system. A "hydroxylation camouflage" strategy was put forward by coating the polycation vectors with hydroxyl-enriched "skin". Branched polyethyleneimine (PEI) was herein used as the polycation model and modified via the catalyst-free aminolysis reaction with 5-ethyl-5-(hydroxymethyl)-1,3-dioxan-2-oxo (EHDO). PEI-g-EHDO, PEI and alkylated PEI derivative termed as PEI-g-DPA were comparatively explored with respect to the transfection efficiency in the serum-free and serum-conditioned medium. The resultant data indicate that the serum-tolerant capability largely depended on the surface composition and substitution degree. In addition to the reduced surface charge, the introduced function caused by hydroxyl coating is believed to play a crucial role for the improved properties of PEI-g-EHDOs. The EHDO modification can effectively inhibit the adsorption of BSA proteins onto polyplexes surface. And the polyplexes stability was remarkably enhanced in the presence of DNase and heparin after EHDO modification. Note that the transfection activity of PEI-g-EHDO(34.5%) in the serum-conditioned medium was even higher than that without serum addition. In contrast, serum addition led to appreciable reduction in the transfection efficiency mediated by PEI and PEI-g-DPAs. Specifically, as far as the transfection activity in the presence of serum is concerned, PEI-g-EHDO could be up to 30-fold higher than unmodified PEI25k. PEI-g-EHDO(34.5%) displayed little to no hemolytic effect and high cell-biocompatibility with nearly no cytotoxicity detected in 293T cells and HeLa cells. Taking into account the high biocompatibility and serum-tolerant transfection activity, PEI-g-EHDO(34.5%) holds great potential for the use as efficient gene vector. More importantly, it is expected that such "hydroxylation camouflage" strategy may be universally applicable for a majority of existing polycation vectors.     

Enhanced transfection efficiency of low generation PAMAM dendrimer conjugated with the nuclear localization signal peptide derived from herpesviridae

Abstract

Polyamidoamine (PAMAM) dendrimer is an extensively studied polymer in the biomedical research because of its low polydispersity, distinct molecular structure, and surface functionalities. Generally, a high-generational PAMAM dendrimer is used for gene delivery because transfection efficiency is dependent on charge density; however, an increase in charge density induces disruption of the cellular membrane, and damage to the membrane results in cytotoxicity. In this study, we selected PAMAM generation 2 to reduce the cytotoxic effect and conjugated RRILH and RRLHL sequences, nuclear localization signals (NLS) derived from herpesviridae to PAMAM generation 2. The transfection efficiency of RRILH-PAMAM G2 and RRLHL-PAMAM G2 was similar to that of polyethylenimine (PEI) in Neuro2A, HT22, and HaCaT cells, whereas their transfection efficiency was much higher than that of PEI in NIH3T3 cells. RRILH-PAMAM G2 showed relatively lower cytotoxicity than did RRLHL-PAMAM G2 in all cell lines, but the transfection capacity of the two polymers was similar. Our study shows that low-generational PAMAM dendrimer conjugated with NLS sequences has potential as an alternative to PEI in gene delivery.     

Non-viral gene transfection in vitro using endosomal pH-sensitive reversibly hydrophobilized polyethylenimine

Reversibly hydrophobilized 10 kDa polyethylenimine (PEI) based on rapidly acid-degradable acetal-containing hydrophobe was designed for nontoxic and highly efficient non-viral gene transfer. Water soluble PEI derivatives with average 5, 9 and 14 units of pH-sensitive 2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME) hydrophobe per molecule, denoted as PEI-g-(TMB-THME)(n), were readily obtained by treating 10 kDa PEI with varying amounts of TMB-THME-nitrophenyl chloroformate. Gel retardation assays showed that all PEI-g-(TMB-THME)(n) derivatives could effectively condense DNA at an N/P ratio of 5/1. Notably, polyplexes of PEI-g-(TMB-THME)(n) derivatives had smaller sizes (about 100～170 nm) and higher surface charges (+25 ～ +43 mV) than the parent 10 kDa PEI at the same N/P ratios ranging from 10/1 to 40/1. MTT assays revealed that these PEI-g-(TMB-THME)(n) derivatives were practically non-toxic at polymer concentrations used in transfection experiments. The acetal degradation of PEI-g-(TMB-THME)(9) was shown to be highly pH dependent in which half lives of 1.3, 2.8 and 11 h were determined for pH 4.0, 5.0 and 6.0, respectively, while negligible hydrolysis (<12%) was observed after 24 h at pH 7.4. Gel electrophoresis, dynamic light scattering (DLS) and zeta potential analyses indicated that polyplexes formed at an N/P ratio of 10/1 were dissociated following 5 h incubation at pH 5.0, highlighting the importance of hydrophobic TMB-THME moieties in DNA condensation and supporting that acetal hydrolysis in endosomes would facilitate DNA release. Notably, in vitro transfection experiments performed at N/P ratios of 10/1 and 20/1 in HeLa, 293T, HepG2 and KB cells using plasmid pGL3 expressing luciferase as the reporter gene showed that reversibly hydrophobilized PEIs had superior transfection activity to 25 kDa PEI control. For example, polyplexes of PEI-g-(TMB-THME)(14) showed about 235-fold and 175-fold higher transfection efficiency as compared to 10 kDa PEI in HeLa cells in serum-free and 10% serum media, respectively, which were approximately 7-fold and 16-fold higher than 25 kDa PEI formulation at its optimal N/P ratio under otherwise the same conditions. Confocal laser scanning microscope (CLSM) studies confirmed that PEI-g-(TMB-THME)(14) efficiently delivered Cy5-labeled DNA to the nuclei of HeLa cells. These endosomal pH-sensitive reversibly hydrophobilized PEIs have great potentials for safe and efficient non-viral gene transfection.     

Tyroserleutide-based gene vector for suppressing VEGF expression in cancer therapy

A small interfering RNA (siRNA) plasmid DNA (pYr-1.1-hU6-EGFP-siVEGF) was constructed and used for suppressing vascular endothelial growth factor (VEGF) expression and inhibiting tumor growth. Then, a (tyrosyl-seryl-leucine)-polyethyleneimine-poly(ethylene glycol) (YSL-PEI-PEG) conjugate was designed and synthesized as a gene carrier for the delivery of pYr-1.1-hU6-EGFP-siVEGF plasmid. The therapeutic peptide YSL was conjugated to PEI to improve the anti-cancer efficiency, and the PEG chain was introduced to reduce the serum protein adsorption and improve the stability of the complex in the systemic circulation. It was found that YSL-PEI-PEG could efficiently condense plasmid DNA when the vector/DNA weight ratio was higher than 2. Compared with PEI 25?kDa, YSL-PEI-PEG exhibited higher transfection efficiency and lower cytotoxicity. More importantly, the results showed that the gene delivery system owned strong ability to inhibit cancer cell proliferation in?vitro and tumor growth in?vivo. YSL-PEI-PEG has great potential as a gene vector for clinical applications.     

New cationic biodegradable poly(urethane-co-ester): synthesis, structural characterization, modification and gene delivery

To improve the transfection efficiency of poly(urethane-co-ester) and the cytotoxicity of PEI25k with DNA, we synthesized a new poly(urethane-co-ester), PUE, bearing ester linkages and amino groups in the backbone and urethane linkages in the side-chain, and then prepared a binary mixture, PUE-PEI25k, using a physical blending method. The structure of PUE was confirmed by FT-IR and NMR spectra. Both poly(urethane-co-ester), PUE, and binary mixture PUE-PEI25k, readily self-assembled with plasmid DNA (pCMV-βgal) in a HEPES buffer, were characterized by dynamic light scattering. The results revealed that PUE and PUE-PEI25k were able to self-assemble plasmid DNA into PUE/DNA and PUE-PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PUE and PUE-PEI25k. The COS-7 cell viability in the presence of PEI25k, PUE and PUE-PEI25k was studied. At low mass ratio of PUE/PEI25k (150:1), it was found that the PUE-PEI25k/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier, PEI25k/DNA. The results indicate that the binary mixture PUE-PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.     

Combinational therapy of ischemic brain stroke by delivery of heme oxygenase-1 gene and dexamethasone

Combinational therapies using genes and drugs are promising therapeutic strategies for various diseases. In this research, a co-delivery carrier of dexamethasone and plasmid DNA (pDNA) was developed by conjugation of dexamethasone to polyethylenimine (2 kDa, PEI2k) for combinational therapy of ischemic brain. Dynamic light scattering, atomic force microscopy and flow cytometry studies showed that the pDNA/dexamethasone-conjugated PEI2k (PEI2k-Dexa) complex was 150 nm in size and was taken up by cells more easily than PEI2k-Dexa only. The tumor necrosis factor-α (TNF-α) level was decreased more efficiently by pDNA/PEI2k-Dexa complex than dexamethasone only in hypoxia activated Raw 264.7 macrophage cells, suggesting that pDNA/PEI2k-Dexa complex increased the delivery efficiency and therapeutic effect of dexamethasone. In in vitro transfection assay, PEI2k-Dexa had higher transfection efficiency than PEI2k and lipofectamine. However, the simple mixture of PEI2k and dexamethasone did not show this effect, suggesting that the conjugation of dexamethasone to polyethylenimine increased DNA delivery efficiency of PEI2k. To evaluate the effects of combinational therapy in vivo, pDNA/PEI2k-Dexa complex was applied to a transient focal ischemia animal model. At 24 h after the injection, mean infarction volume and the TNF-α level were reduced more efficiently in the pDNA/PEI2k-Dexa injection group, compared with the control, pDNA/PEI2k, or dexamethasone injection group. The infarction volume and inflammatory cytokines were further decreased by delivery of pSV-HO-1 using PEI2k-Dexa. Magnetic resonance imaging and microPET studies confirmed the therapeutic effect of pSV-HO-1/PEI2k-Dexa complex at 10 days after the injection. Therefore, pSV-HO-1/PEI2k-Dexa complexes may be useful in combinational therapy for ischemic diseases such as stroke.     

Cationic star polymers consisting of alpha-cyclodextrin core and oligoethylenimine arms as nonviral gene delivery vectors

A series of novel cationic star polymers were synthesized by conjugating multiple oligoethylenimine (OEI) arms onto an alpha-cyclodextrin (alpha-CD) core as nonviral gene delivery vectors. The molecular structures of the alpha-CD-OEI star polymers, which contained linear or branched OEI arms with different chain lengths ranging from 1 to 14 ethylenimine units, were characterized by using size exclusion chromatography, 13C and 1H NMR, and elemental analysis. The alpha-CD-OEI star polymers were studied in terms of their DNA binding capability, formation of nanoparticles with plasmid DNA (pDNA), cytotoxicity, and gene transfection in cultured cells. All the alpha-CD-OEI star polymers could inhibit the migration of pDNA on agarose gel through formation of complexes with pDNA, and the complexes formed nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The star polymers displayed much lower in vitro cytotoxicity than that of branched polyethylenimine (PEI) of molecular weight 25K. The alpha-CD-OEI star polymers showed excellent gene transfection efficiency in HEK293 and Cos7 cells. Generally, the transfection efficiency increased with an increase in the OEI arm length. The star polymers with longer and branched OEI arms showed higher transfection efficiency. The best one of the star polymers for gene delivery showed excellent in vitro transfection efficiency that was comparable to or even higher than that of branched PEI (25K). The novel alpha-CD-OEI star polymers with OEI arms of different chain lengths and chain architectures can be promising new nonviral gene delivery vectors with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.     

New Cationic Biodegradable Poly(Urethane-co-Ester): Synthesis,Structural Characterization,Modification and Gene Delivery

To improve the transfection efficiency of poly(urethane-co-ester) and the cytotoxicity of PEI25k with DNA, we synthesized a new poly(urethane-co-ester), PUE, bearing ester linkages and amino groups in the backbone and urethane linkages in the side-chain, and then prepared a binary mixture, PUE-PEI25k, using a physical blending method. The structure of PUE was confirmed by FT-IR and NMR spectra. Both poly(urethane-co-ester), PUE, and binary mixture PUE-PEI25k, readily self-assembled with plasmid DNA (pCMV-βgal) in a HEPES buffer, were characterized by dynamic light scattering. The results revealed that PUE and PUE-PEI25k were able to self-assemble plasmid DNA into PUE/DNA and PUE-PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PUE and PUE-PEI25k. The COS-7 cell viability in the presence of PEI25k, PUE and PUE-PEI25k was studied. At low mass ratio of PUE/PEI25k (150:1), it was found that the PUE-PEI25k/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier, PEI25k/DNA. The results indicate that the binary mixture PUE-PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.     

Poly(aspartate-g-PEI800), a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery

High-molecular-weight polyethylenimine (25 kDa, PEI25k) is one of the most common cationic polymers utilized in non-viral gene therapy. However, its methylene backbone (-CH(2)CH(2)N(x)-) and high charge density can result in poor biodegradability and high toxicity to cells. We hypothesize that optimizing the polymer length and charge density of PEI analogues may result in decreased toxicity and higher transfection efficiency, and improved biocompatibility in vivo. A series of PEI analogues with controlled molecular weight and charge density were synthesized by grafting low-molecular-weight PEI800 (800 Da) to a polyaspartate peptide backbone of varying degrees of polymerization. The optimum polymer had a degree of polymerization of 65 with an average of 16 PEI800 groups conjugated to it. All of the polycations investigated in the study caused inflammation and apoptosis/necrosis in the liver and spleen of rodents 24h post-injection; however, by day 5, the optimized poly(aspartate-g-PEI800) polymer and PEI800 did not show tissue damage or apoptosis, whereas PEI25k exhibited evidence of apoptosis/necrosis in the kidneys and spleen. Our study points to the need to optimize gene carriers to minimize toxicity, especially important for the safe delivery of therapeutic genes to explicit organs.     

A mannosylated cell-penetrating peptide-graft-polyethylenimine as a gene delivery vector

Polyethylenimine (PEI) is widely applied in non-viral gene delivery vectors. PEI with high molecular weight is highly effective in gene transfection but is high cytotoxic. Conversely, PEI with low molecular weight displays lower cytotoxicity but less delivering efficiency. To overcome this issue, a novel copolymer with mannosylated, a cell-penetrating peptide (CPP), grafting into PEI with molecular weight of 1800 (Man-PEI₁₈₀₀-CPP) were prepared in this study to target antigen-presenting cells (APCs) with mannose receptors and enhance transfection efficiency with grafting CPP. The copolymer was characterized by ¹H NMR and FTIR. Spherical nanoparticles were formed with diameters of about 80–250 nm by mixing the copolymer and DNA at various charge ratios of copolymer/DNA(N/P). Gel retardation assays indicated that Man-PEI₁₈₀₀-CPP polymers efficiently condensed DNA at low N/P ratios. Cytotoxicity studies showed that Man-PEI₁₈₀₀-CPP/DNA complexes maintained in a high percentage of cell viability compared to the PEI with molecular weight of 25 k (PEI_25k). Laser scan confocal microscopy and flow cytometry confirmed that Man-PEI₁₈₀₀-CPP/DNA complexes resulted in higher cell uptake efficiency on DC2.4 cells than on Hela cells line. The transfection efficiency of Man-PEI₁₈₀₀-CPP was significantly higher than that of PEI_25k on DC2.4 cells. More importantly, the complexes were mainly distributed in the epidermis and dermis of skin and targeted on splenocytes after percutaneous coating based on microneedles in vivo. These results indicated that Man-PEI₁₈₀₀-CPP was a potential APCs targeted of non-virus vector for gene therapy.     

Palmitic acid substitution on cationic polymers for effective delivery of plasmid DNA to bone marrow stromal cells

Nonviral gene carriers are actively explored in gene therapy due to safety concerns of the viral carriers. To design effective gene carriers for modification of bone marrow stromal cells (BMSC), an important cell phenotype for clinical application of gene therapy, cationic polymers polyethyleneimine (PEI), and poly-L-Lysine (PLL) were substituted with palmitic acid (PA) via amide linkages. Depending on the reaction conditions, PEI and PLL was substituted with 2.2-5.2 and 13.4-16.2 PA per polymer chain. The PA substituted polymers displayed slightly lower binding efficiency towards a plasmid containing Enhanced Green Fluorescent Protein (pEGFP) in an agarose gel binding assay. The cell binding of PLL-PA, but not PEI-PA, was particularly enhanced, resulting in higher percentage of the cells displaying a significant polymer uptake. pEGFP delivery into the BMSC was also significantly increased with the PLL-PA (vs. PLL), but not PEI-PA (vs. PEI). The transfection efficiency of PLL-PA was significantly higher ( approximately fivefold) than the unmodified polymer. We conclude that PA substitution on PLL provides an effective carrier for transfection of primary cells derived from the bone marrow.     

Reconstitutable charged polymeric (PLGA)(2)-b-PEI micelles for gene therapeutics delivery

This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA(36kDa))(2)-b-bPEI(25kDa) (PLGA MW 36 kDa, bPEI M(w) 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100-150 nm) and surface charge (30-40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50-100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for "reconstituted" micelle/pDNA/bPEI(1.8kDa) (WR 1) complexes was 16-fold higher than its "fresh" counterpart. Although transfection levels achieved using "reconstituted" micelle/pDNA/bPEI(1.8kDa) complexes were 3.6-fold lower than control "fresh" bPEI(25kDa)/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than "reconstituted" bPEI(25kDa)/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI(1.8kDa) system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA-b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.     

Enhanced gene delivery by chitosan-disulfide-conjugated LMW-PEI for facilitating osteogenic differentiation

Chitosan-disulfide-conjugated LMW-PEI (CS-ss-PEI) was designed to combine the biocompatibility of chitosan and the gene delivery ability of polyethylenimine (PEI) using bio-reducible disulfide for bone morphogenetic protein (BMP2) gene delivery in mediating osteogenic differentiation. It was prepared by conjugating low molecular weight PEI (LMW-PEI) to chitosan through oxidization of thiols introduced for the formation of disulfide linkage. The structure, molecular weight and buffer capacity were characterized by Fourier transform infrared (FTIR), light scattering and acid–base titration, respectively. The reduction in molecular weight of CS-ss-PEI by the reducing agent indicated its bio-reducible property. With the increment in the LMW-PEI component, the copolymer showed increased DNA binding ability and formed denser nanocomplexes. CS-ss-PEI exhibited low cytotoxicity in COS-1, HepG2 and 293T cells over the different weight ratios. The transfection efficiency of CS-ss-PEI4 was significantly higher than that of PEI 25k and comparable with Lipofectamine in mediating luciferase expression. Its application for BMP2 gene delivery was confirmed in C2C12 cells by BMP2 expression. For inducing in vitro osteogenic differentiation, CS-ss-PEI4 mediated BMP2 gene delivery showed a stronger effect in MG-63 osteoblast cells and stem cells in terms of alkaline phosphatase activity and mineralization compared with PEI25k and Lipofectamine. This study provides a potential gene delivery system for orthopedic-related disease.     

Amphiphilic peptide carrier for the combined delivery of curcumin and plasmid DNA into the lungs

In this study, the R7L10 peptide, which is composed of a 7-arginine stretch and a 10-leucine stretch, was evaluated as a carrier for the combined delivery of curcumin and plasmid DNA (pDNA) into the lungs. Curcumin is a natural product with anti-inflammatory and anti-tumor effects. Curcumin-loaded R7L10 (R7L10-curucmin) was prepared by an oil-in-water (O/W) emulsion/solvent evaporation method. In vitro transfection showed that R7L10-curcumin had higher transfection efficiency than R7L10. Although R7L10-curcumin had lower transfection efficiency than polyethylenimine (25 kDa, PEI25k) and lipofectamine, R7L10-curcumin had lower cytotoxicity. In gel retardation assays and heparin competition assays, R7L10-curcumin formed a more stable complex with pDNA than R7L10. The intracellular curcumin delivery efficiency of R7L10-curcumin was higher than that of curcumin only. Furthermore, R7L10-curcumin more efficiently decreased TNF-α level in lipopolysaccharide (LPS)-activated Raw264.7 macrophage cells than curcumin only. For in vivo evaluation, pDNA/R7L10-curcumin complexes were administered into mouse lungs by intratracheal instillation. The results revealed that R7L10-curcumin delivered pDNA more efficiently than R7L10, poly-L-lysine (PLL), or PEI25k. In addition, R7L10-curcumin decreased TNF-α level in lung tissues in an acute lung injury mouse model. In contrast to PEI25k, R7L10-curcumin did not show liver toxicity after intravenous injection. These results suggest that R7L10-curcumin is a useful carrier for the combined delivery of curcumin and pDNA into the lungs.