Ternary complexes of pDNA,polyethylenimine, and γ-polyglutamic acid for gene delivery systems

We discovered a vector coated by γ-polyglutamic acid (γ-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic–polycytidylic acid, α-polyaspartic acid, α-polyglutamic acid, and γ-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive ζ-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/γ-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/γ-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of γ-PGA significantly inhibited the uptake of pDNA/PEI/γ-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/γ-PGA complex was taken up by γ-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/γ-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.     

The effects of coating pDNA/chitosan complexes with chondroitin sulfate on physicochemical characteristics and cell transfection

pDNA/chitosan complexes have been investigated as promising non-viral vectors for gene delivery. However, an increase in transfection efficiency and enhancement of physicochemical stability are required for their practical use. In this study, chondroitin sulfate (CS) was employed as a coating agent to increase the stability and transfection efficiency of a pDNA/chitosan complex. The pDNA/chitosan/CS ternary complexes formed with six kinds of CSs having different limiting viscosities (0.2-1.6) and sulfation degrees (5.0-7.0%) showed considerable differences in particle size, surface charge, and morphology. Among them, CS having a medium limiting viscosity (0.5-0.6) and a high sulfation degree (6.9%) showed significant enhancements in cell transfection efficiency. Analyses of cellular uptake and intracellular trafficking revealed that increased cellular uptake via macropinocytosis, together with reduced entry into lysosomes, may explain the promotion of transfection efficiency of ternary complexes.     

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI-25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.     

Gene delivery of PEI incorporating with functional block copolymer via non-covalent assembly strategy

A novel functional diblock polymer P(PEGMA-b-MAH) is prepared and incorporated to improve the gene delivery efficiency of poly(ethyleneimine) PEI via non-covalent assembly strategy. First, P(PEGMA-b-MAH) is prepared from l-methacrylamidohistidine methyl ester (MAH) by reversible addition fragmentation chain transfer polymerization, with poly[poly(ethylene glycol) methyl ether methacrylate] (P(PEGMA)) as the macroinitiator. Then P(PEGMA-b-MAH) is assembled with plasmid DNA (pDNA) and PEI (M_w = 10 kDa) to form PEI/P(PEGMA-b-MAH)/pDNA ternary complexes. The agarose gel retardation assay shows that the presence of P(PEGMA-b-MAH) does not interfere with DNA condensation by the PEI. Dynamic light scattering tests show that PEI/P(PEGMA-b-MAH)/pDNA ternary complexes have excellent serum stability. In vitro transfection indicates that, compared to the P(PEGMA-b-MAH) free PEI-25k/pDNA binary complexes, PEI-10k/P(PEGMA-b-MAH)/pDNA ternary complexes have lower cytotoxicity and higher gene transfection efficiency, especially under serum conditions. The ternary complexes proposed here can inspire a new strategy for the development of gene and drug delivery vectors.     

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.     

Design,Synthesis and Evaluation of Cationic Poly(N-substituent acrylamide)s for In Vitro Gene Delivery

A series of poly(N-substituent acrylamide)s (PAms) that differ in alkylamine side-chain was synthesized via free radical polymerization. The PAms were designed to examine the effects of the methylene numbers (from 2 to 12) in the alkylamine side-chain on cytotoxicity, plasmid DNA (pDNA) binding affinity, cellular uptake efficiency and gene expression. The cytotoxicity of PAms evaluated in HEK293 cells using the MTT assay showed a trend of decreasing toxicity with increasing side-chain length and the IC₅₀ values of all PAms were lower than that of polyethylenimine (PEI) control. The primary amine-based polymers were able to efficiently condense pDNA to form complexes with size ranging from 100 to 350 nm. The gene transfection ability of PAms is dominantly determined by the specific side-chain length that P8Am (with an octylamine side-chain) reveals higher gene expression than other PAms containing the same backbone structure. Although the gene transfection efficiency of PEI was better than all of PAms, PAms were found not to be uptake-limited. This was supported by the effect of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. Especially, complexes formed from P8Am displayed high uptake level relative to PEI, which was attributed to the proper structure of P8Am to compact pDNA to form stable nanoparticles in the heparin replacement assay. The present study offers the understanding to polymer structure that influences the transfection ability and gives useful information when designs efficient polymeric gene carrier.     

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.     

Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers

The structure-activity relationships between hydrophobic and hydrophilic modification on chitosan and resultant physicochemical properties along with performances in dealing with critical gene delivery barriers were investigated through amphiphilic linoleic acid(LA) and poly (β-malic acid) (PMLA) double grafted chitosan (LMC)/plasmid DNA (pDNA) nanocomplexes. LMC polymers with various LA and PMLA substitution degrees were synthesized and their hydrophilicity/hydrophobicity was characterized. Compared to chitosan, LMC nanoparticles retained the pDNA binding ability at pH 5.5 when they formed nanocomplexes with pDNA encoding enhanced green fluorescence protein (pEGFP) and the resultant complexes showed diameters below 300 nm. Hydrophobic LA and hydrophilic PMLA substitution contributed to suppressed non-specific adsorption, reduced interactions inside LMC/pDNA nanocomplexes, and enhanced pDNA dissociation. However, enzymatic degradation resistance, cell adsorption, and cellular uptake through clathrin-mediated pathway were promoted by hydrophobic LA grafting while being inhibited by hydrophilic PMLA substitution. In vitro transfection assay suggested the optimal LMC/pEGFP nanocomplexes mediated an 8.0-fold improved transfection compared to chitosan/pEGFP nanocomplexes. The 4.2-fold and 2.2-fold higher intramuscular gene expression in mice compared to chitosan/pEGFP and polyethyleneimine (PEI)/pEGFP nanocomplexes further demonstrated the superiority of LMC/pDNA nanocomplexes. Therefore, amphiphilic chitosan derivates with appropriate combination of hydrophobic and hydrophilic modification would be promising gene delivery nanocarriers.     

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.     

壳聚糖脂质体复合载体的核酸递送

目的构建一种由脂质体Lipofectamine2000、低分子质量壳聚糖、pDNA组成的三元新型复合载体用于核酸递送能力研究。方法复合物形态采用原子力显微镜轻敲模式下表征、载体与核酸结合能力采用凝胶延滞法表征,Hep-2细胞报告基因表达利用倒置荧光显微镜检测。细胞毒性研究采用3-甲基-2-噻唑硫酮(MTT)法。结果复合载体与pDNA结合能力强,可完全延滞pDNA。脂质体/壳聚糖/pDNA复合载体形态呈现出未完全压缩的球形,短棒状和不规则的聚集块。新型载体转染Hep-2细胞提高了绿色荧光蛋白报告基因的表达效率。与脂质体对照载体比较,基因转染效率提高了2～4倍,对照壳聚糖载体无明显转染效果。细胞毒性表明壳聚糖降低了脂质体的细胞毒性。结论基于脂质体的壳聚糖新型复合载体具有核酸递送潜力。     

A facile preparation of novel multifunctional vectors by non-covalent bonds for co-delivery of doxorubicin and gene

In this study, novel multifunctional ternary complexes of biotinylated transferrin-avidin-biotin-poly(ethylene glycol)-poly(L-glutamate acid)/poly(2-(2-aminoethylamino) ethyl methacrylate)/doxorubicin-poly(L-aspartic acid)/pDNA (TAB/PIC-D/pDNA complexes) were prepared based on polyion complex micelles (PIC) and the avidin-biotin system, which aimed to target co-delivery of anti-cancer doxorubicin and gene. Cytotoxicity studies revealed that PIC-D could have anti-tumor effect on HeLa cells and HepG2 cells; TAB coating could increase the biocompatibility of PIC-D/pDNA complexes and the targeting delivery efficiency of doxorubicin. TAB/PIC-D/pDNA complexes possessed higher transfection efficiency than the unmodified complexes in serum, and transferrin could enhance luciferase expression in HeLa cells and HepG2 cells. Furthermore, confocal laser scanning microscopy showed that doxorubicin and gene could be delivered into HepG2 cells simultaneously by TAB/PIC-D/pDNA complexes. The formation of the ternary complexes provides a facile approach to constructing a multifunctional delivery system for targeted co-delivery of anticancer drugs and gene.     

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.     

Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs

Polymeric nanosphere-mediated gene delivery may sustain the duration of plasmid DNA (pDNA) administration. In this study, poly(lactic-co-glycolic acid) (PLGA) nanospheres were evaluated as a gene carrier. The pDNA-loaded PLGA nanospheres were formulated with high encapsulation efficiency (87%). The nanospheres sustained release of pDNA for 11 days. The released pDNA maintained its structural and functional integrity. Furthermore, the PLGA nanospheres showed lower cytotoxicity than polyethylenimine (PEI) in vitro and in vivo. The nanospheres with vascular endothelial growth factor (VEGF) gene were injected into skeletal muscle of ischemic limb model, and gene expression mediated by the PLGA nanospheres with VEGF gene was compared to that of PEI/pDNA or naked pDNA in vivo. PLGA nanosphere/pDNA had significantly higher VEGF expression levels in comparison to PEI/pDNA and naked pDNA at 12 days after administration. In addition, gene therapy using PLGA nanospheres resulted in more extensive neovascularization at ischemic sites than both naked pDNA and PEI/pDNA. These results indicated that PLGA nanosphere might be useful as a potential carrier for skeletal muscle gene delivery applications.     

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.     

A silica–polymer composite nano system for tumor-targeted imaging and p53 gene therapy of lung cancer

In our study, a silica–polymer composite nano system (MB-NSi–p53–CS ternary complexes) composed of methylene blue-encapsulated amine-terminated silica nanoparticles (MB-NSi) and chondroitin sulfate (CS) were successfully developed for tumor-targeted imaging and p53 gene therapy of lung cancer. MB was employed as a NIR probe for in vivo imaging, MB-NSi nanoparticles were served as gene vector, while CS was applied to be a coating and targeting polymer. MB-NSi–p53–CS ternary complexes displayed nanosized diameter, effective p53 condensation ability, efficient p53 protection profile, and superior bovine serum albumin stability in vitro. Experiments on A549 cell line further revealed low cytotoxicity, high p53 transfection, and anticancer efficacy of MB-NSi–p53–CS ternary complexes. In vivo imaging and tumor targetability assays demonstrated that MB-NSi–p53–CS ternary complexes were a preferable system with desirable imaging and tumor-targeting properties.     

N-亚甲基磷酸化壳聚糖基因纳米粒子的体外细胞毒性及基因转染实验研究

目的 考察N-亚甲基磷酸化壳聚糖( NMPCS)基因纳米粒子的体外细胞毒性及基因转染效率.方法 采用均相反应法制备了NMPCS,用复凝聚法制备了NMPCS/DNA纳米粒子;通过MTT实验考察了NMPCS及其与DNA复合物对HeLa细胞的细胞毒性,以荧光索酶质粒为报告基因考察了NMPCS及NMPCS-CaZ+载体介导的体外基因转染效率.结果 NMPCS及其与DNA的复合物在体外表现出很小的细胞毒性,远远低于同等浓度时聚乙烯亚胺(PEI)的毒性.通过对壳聚糖进行亚甲基磷酸化修饰后,可大幅提高载体的基因转染效率.结论 N-亚甲基磷酸化壳聚糖有望成为一种新型、安全、高效的非病毒基因载体.     

Gelatin coating to stabilize the transfection ability of nucleic acid polyplexes

Amphiphilic polymers are effective in complexing and delivering therapeutic nucleic acids, such as plasmid DNA (pDNA) and short interfering RNA (siRNA). However, long-term stability of the complexes is not desirable, as it may have an impact on the transfection efficiency in vivo. To develop a method to preserve complex stability we first showed that pDNA complexes formed with the amphiphilic polymer linoleic acid-substituted polyethylenimine (PEI–LA) and incubated at 37 °C lost ～90% of their transfection efficiency after only 24 h of complex formation. Polyethyleneglycol modification of complexes to control the increase in complex size and incubation in scaffolds used for implantation did not preserve the transfection ability of the complexes. Among a variety of approaches explored, gelatin coating of complexes was found to be the best at maintaining the original transfection efficiency. Mechanistic studies suggested that improved complex uptake, not size stability, was responsible for retention of the transfection efficiency. Similarly to the results with pDNA, gelatin coating also prevented the decreases in uptake and silencing efficiency of siRNA complexes observed following incubation at 37 °C. Gelatin-stabilized complexes were, furthermore, effective in vivo and led to subcutaneous transgene expression with a low pDNA dose that was otherwise ineffective. We conclude that a simple gelatin coating approach offers an efficient means to preserve the transfection efficiency of polyplexes.     

Chitosan/TPP-Hyaluronic Acid Nanoparticles: A New Vehicle for Gene Delivery to the Spinal Cord

Abstract

Gene delivery offers therapeutic promise for the treatment of neurological diseases and spinal cord injury. Several studies have offered viral vectors as vehicles to deliver therapeutic agents, yet their toxicity and immunogenicity, along with the cost of their large-scale formulation, limits their clinical use. As such, non-viral vectors are attractive in that they offer improved safety profiles compared to viruses. Poly(ethylene imine) (PEI) is one of the most extensively studied non-viral vectors, but its clinical value is limited y its cytotoxicity. Recently, chitosan/DNA complex nanoparticles have een considered as a vector for gene delivery. Here, we demonstrate that DNA nanoparticles made of hyaluronic acid (HA) and chitosan have low cytotoxicity and induce high transgene expression in neural stem cells and organotypic spinal cord slice tissue. Chitosan-TPP/HA nanoparticles were significantly less cytotoxic than PEI at various concentrations. Additionally, chitosan-TPP/HA nanoparticles with pDNA induced higher transgene expression in vitro for a longer duration than PEI in neural stem cells. These results suggest chitosan-TPP/HA nanoparticles may have the potential to serve as an option for gene delivery to the spinal cord.     

A chitosan-graft-PEI-candesartan conjugate for targeted co-delivery of drug and gene in anti-angiogenesis cancer therapy

A multifunctional copolymer–anticancer conjugate chitosan-graft-polyethyleneimine-candesartan (CPC) containing low molecular weight chitosan (CS) backbone and polyethyleneimine (PEI) arms with candesartan (CD) conjugated via an amide bond was fabricated as a targeted co-delivery nanovector of drug and gene for potential cancer therapy. Here, CD was utilized to specifically bind to overexpressed angiotensin II type 1 receptor (AT₁R) of tumor cells, strengthen endosomal buffering capacity of CPC and suppress tumor angiogenesis. The self-assembled CPC/pDNA complexes exhibited desirable and homogenous particle size, moderate positive charges, superior stability, and efficient release of drug and gene in vitro. Flow cytometry and confocal laser scanning microscopy analyses confirmed that CD-targeted function and CD-enhanced buffering capacity induced high transfection, specific cellular uptake and efficient intracellular delivery of CPC/pDNA complexes in AT₁R-overexpressed PANC-1 cells. In addition, CPC/wt-p53 complexes co-delivering CD and wild type p53 (wt-p53) gene achieved synergistic angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor (VEGF) mRNA and protein via different pathways in vitro, as compared to mono-delivery and mixed-delivery systems. In vivo investigation on nude mice bearing PANC-1 tumor xenografts revealed that CPC/wt-p53 complexes possessed high tumor-targeting capacity and strong anti-tumor activity. Additional analysis of microvessel density (MVD) demonstrated that CPC/wt-p53 complexes significantly inhibited tumor-associated angiogenesis. These findings suggested that CPC could be an ideal tumor-targeting nanovector for simultaneous transfer of drug and gene, and a multifunctional CPC/wt-p53 co-delivery system with tumor-specific targetability, enhanced endosomal buffering capacity and synergistic anti-angiogenesis efficacy might be a new promising strategy for effective tumor therapy.     

Lipid substitution on low molecular weight (0.6-2.0 kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression

Cationic polymers are desirable gene carriers because of their better safety profiles than viral delivery systems. Low molecular weight (MW) polymers are particularly attractive, since they display little cytotoxicity, but they are also ineffective for gene delivery. To create effective carriers from low MW polymers palmitic acid (PA) was substituted on 0.6-2.0 kDa polyethylenimines (PEIs) and their efficiency for plasmid DNA (pDNA) delivery was evaluated. The extent of lipid substitution was dependent on the lipid/PEI feed ratio and the polymer MW. While the hydrodynamic size of the polymer/pDNA complexes (polyplexes) increased or decreased depending on the extent of lipid substitution, the ζ potential of the assembled complexes was consistently higher as a result of lipid substitution. Lipid substitution generally increased the in vitro toxicity of the PEIs, but it was significantly lower than that of the 25 kDa branched PEI. The in vitro transfection efficiency of the lipid-substituted polymers was higher than that of native PEIs, which were not at all effective. The delivery efficiency was proportional to the extent of lipid substitution as well as the polymer MW. This correlated with the increased uptake of lipid-substituted polyplexes, based on confocal microscopic investigations with FITC-labeled pDNA. The addition of chloroquine further increased the transfection efficiency of lipid-substituted PEIs, indicating that endosomal release was a limiting factor affecting the efficiency of these carriers. This study indicates that lipid substitution on low MW PEIs makes their assembly more effective, resulting in better delivery of pDNA into mammalian cells.