Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex

Safe and efficient systems capable of specifically targeting brain tumour cells represent a promising approach for the treatment glioblastoma multiforme. Neuropilin-1 (NRP-1) is over-expressed in U87 glioma cells. In the current study, the tumour specific peptide RGERPPR, which binds specifically to NRP-1, was used as a targeting ligand in a gene delivery strategy for glioblastoma. The RGERPPR peptide was coupled to branched polyethylenimine (PEI, 25 kDa) using heterobifunctional Mal–PEG–NHS, resulting in a novel gene delivery polymer. Polymer/plasmid DNA (pDNA) complexes were formed and their sizes and zeta potentials were measured. Compared with the unmodified mPEG–PEI/pDNA complexes, the RGERPPR–PEG–PEI/pDNA complex led to a significant enhancement in intracellular gene uptake and tumour spheroid penetration. Furthermore, the RGERPPR–PEG–PEI/pDNA complex facilitated enhanced transfection efficiency levels, as well as a reduction in cytotoxicity when tested in U87 glioma cells in vitro. Most significantly of all, when complexes formed with pDsRED-N1 were injected into the tail vein of intracranial U87 tumour-bearing nude mice, the RGERPPR–PEG–PEI complexes led to improved levels of red fluorescence protein expression in the brain tissue. Taken together, the results show that RGERPPR–PEG–PEI could be used as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene delivery.     

Gene delivery system of pDNA using the blood glycoprotein fetuin

Fetuin is a biocompatible plasma protein and strongly enhances phagocytosis of bacteria, DNA and apoptotic cells by peripheral blood cells such as monocytes, macrophages and dendritic cells. We developed a novel gene delivery system: ternary complexes constructed with pDNA, polyethylenimine (PEI) and fetuin. Without covalent binding, fetuin was able to coat pDNA–PEI complexes, and stable anionic nanoparticles formed at a weight ratio greater than 30. Optimised pDNA–PEI–fetuin complexes significantly decreased the cytotoxicity of pDNA–PEI complexes in the melanoma cell line B16F10. Furthermore, the pDNA–PEI–fetuin complexes had higher transgene efficiency compared to that of commercial lipofectin previously reported in B16F10 cells despite an anionic surface. The pDNA–PEI–fetuin complexes did not agglutinate with erythrocytes. The pDNA–PEI–fetuin complexes had high gene expression in the spleen after intravenous administration in mice. Thus, the pDNA–PEI–fetuin complexes were a useful in vivo gene delivery system with tropism for the spleen.     

Nanoparticles electrostatically coated with folic acid for effective gene therapy

We developed a novel vector, electrostatically coated poly(ethylenimine) (PEI)/pDNA complexes with folic acid (FA). Without covalent binding, the FA molecules could coat the PEI/pDNA complexes, and stable anionic nanoparticles were formed at a charge ratio greater than 60. The addition of FA markedly decreased the cytotoxicity of the cationic PEI/pDNA complexes to the melanoma cell line, B16-F10 cells, which regularly expressed FA-specific receptor (FR). Furthermore, the anionic FA60/PEI/pDNA complexes showed high transgene efficiency via the FR-mediated pathway in B16-F10 cells. The FA60/PEI/pDNA complexes did not show agglutination with erythrocytes. After the intravenous injection of FA60/PEI/pDNA complexes into mice, a higher transgene efficiency than PEI/pDNA complexes was observed in the liver, kidney, spleen, and lung with FR. The gene expressions of FA60/PEI/pDNA complexes were significantly inhibited by preadministration of FA. Thus, the FA60/PEI/pDNA complexes were useful for effective gene therapy.     

Enhancement of gene transfection into human dendritic cells using cationic PLGA nanospheres with a synthesized nuclear localization signal

Effective delivery of DNA encoding antigen into the dendritic cells (DCs), which are non-dividing cells, is very important for the development of DNA vaccines. In a previous study, we developed the PLGA nanospheres that contained a cationic nanomaterial and showed high transfection efficiency in COS7 cells, which divide. In the present study, to produce an effective vector for the DNA vaccines, the gene expression and intracellular trafficking of pDNA complexed with PLGA/PEI nanospheres, in combination with an NF-κB analog as a nuclear localization signal (NLS) and electroporation were evaluated in human monocyte-derived DCs (hMoDCs). Cellular uptake of pDNA both in COS7 cells and hMoDCs was enhanced using the PLGA/PEI nanospheres. On the other hand, the PLGA/PEI nanospheres significantly promoted the transfection in COS7 cells, but had almost no effect on transfection in hMoDCs. The intranuclear transport of pDNA by PLGA/PEI nanospheres in COS7 cells was significantly higher than that in hMoDCs. These results indicate that pDNA complexed with PLGA/PEI nanospheres cannot enter into the nuclei of non-dividing cells. However, PLGA/PEI nanospheres combinated with NLS and electroporation (experimental permeation enhancer) greatly elevated the transfection efficiency by improvement of not only intracellular uptake but also intranuclear transport of pDNA in the hMoDCs. Thus, this delivery system using nanospheres combined with synthesized NLS might be applicable to DC-based gene vaccines when much non-invasive application such as needle-free injector should be required.     

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ∼2.0–7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.     

Biocompatible complex coated with glycosaminoglycan for gene delivery

The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with dendrigraft poly-l-lysine (DGL) and biodegradable glycosaminoglycan for effective and secure gene delivery. High gene expression of pDNA/DGL complex was confirmed with slight cytotoxicity and erythrocyte agglutination. Anionic ternary complexes of 55.4–223.8?nm were formed by the addition of a glycosaminoglycan such as chondroitin sulfate A (CS-A), chondroitin sulfate B (CS-B), chondroitin sulfate C (CS-C) or hyaluronic acid (HA). Using the cell line B16-F10, most of the ternary complexes showed only weak gene expression and little cytotoxicity, although the pDNA/DGL/CS-A complexes maintained a certain level of gene expression. In particular, the pDNA/DGL/CS-A8 complexes showed significantly higher gene expression than pDNA/DGL complexes in the presence of fetal bovine serum. Gene expression from the pDNA/DGL/CS-A8 complex was inhibited by a high concentration of CS-A and endocytosis inhibitors. After intravenous administration of the pDNA/DGL/CS-A8 complex and the pDNA/DGL complex into ddY mice, high gene expression was observed in the reticuloendothelial systems, the pDNA/DGL/CS-A complex is expected to be useful for gene therapy.     

Intracellular uptake and release of poly(ethyleneimine)-co-poly(methyl methacrylate) nanoparticle/pDNA complexes for gene delivery

Our previous studies demonstrated that cationic nanoparticles composed of well-defined poly(methyl methacrylate) (PMMA) cores surrounded by a hairly poly(ethyleneimine) (PEI) shells have comparative advantages over the PEI system for gene delivery. In this study, we focused on the intracellular uptake and release of PEI-PMMA nanoparticle/pDNA complexes. The behavior of the nanoparticle/pDNA complexes in recipient cells was monitored by using confocal laser scanning microscopy. We found that the nanoparticle/pDNA complexes were internalized very effectively by endocytosis. In the recipient cells the nanoparticles were found localized in the cytoplasm. At the same time, the pDNA carried by the nanoparticles successfully detached from the nanoparticles and localized in the nucleus of the HeLa cells.     

PEG conjugation of a near-infrared fluorescent probe for noninvasive dual imaging of lung deposition and gene expression by pulmonary gene delivery

Dual imaging of lung deposition and gene expression following the pulmonary delivery of a gene formulation is useful for a precise analysis of gene transfection efficiency in vivo. As a novel probe for evaluating lung deposition, in this study, a poly(ethylene glycol)-conjugated near-infrared fluorescent probe (PEG-NIRF) was newly synthesized, and compared with indocyanine green (ICG), for application to pDNA/polyethyleneimine (PEI) complex. PEG-NIRF had superior characteristics including a larger Stokes shift (absorption maximum, 662?nm; emission maximum, 772?nm) and relatively equivalent fluorescence intensity compared with ICG. ICG affected the physicochemical properties of pDNA/PEI complex with a loss of fluorescence intensity, while PEG-NIRF did not. Experiments in mice demonstrated that PEG-NIRF showed greater lung localization than ICG following pulmonary co-delivery with pDNA/PEI complex, indicating the possibility of accurately evaluating lung deposition. Moreover, it was clarified that the evaluation of lung deposition by PEG-NIRF even at 60?min could be significantly correlated with gene expression in each mouse following pulmonary co-delivery with pDNA/PEI complex. These results suggest that PEG-NIRF is widely applicable to the dual imaging of the lung deposition and gene expression of inhaled gene formulations.     

Poly(L-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 approximately 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 ～ 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Insights into the mechanism of magnetofection using MNPs-PEI/pDNA/free PEI magnetofectins

Magnetofection is an efficient new physical gene transfection technology. Despite its effective gene delivery capability, till now relatively little work has been conducted on the mechanism of magnetofection, especially the intracellular fates of the components of magnetofectins and their effects on magnetofection. In this study, we investigated the mechanism of magnetofection using magnetofectins that were prepared via electrostatic self-assembly of the three components: polyethyleneimine (PEI)-coated magnetic nanoparticles (MNPs-PEI), plasmid DNA (pDNA) and PEI in the free form (free PEI). TEM observation and agarose gel electrophoresis assays have indicated MNPs play the role of driving magnetofectins to the cell surface without entering into the nucleus. Confocal microscopic tracking of fluorescence-labeled PEI has shown that the free PEI (green) can be found in the nucleus but almost all of the MNPs-PEI (red) are confined in the cytoplasm in COS-7 cells 30 min post-transfection or in SPC-A1 cells 90 min post-transfection, implying that the pDNA/PEI complex must separate from MNPs-PEI before entering into the nucleus. In addition, reporter gene assays showed the magnetofectins, in which the free PEI was absent, failed to transfect SPC-A1 or COS-7 cell lines; and there was an optimal ratio of the constituents of magnectofectins to achieve optimal transfection efficiency by balancing stable complex formation and facile release of PEI/pDNA from the complex. In summary, our findings further the knowledge of magnetofection and can be helpful for the design and preparation of gene delivery vehicles for effective magnetofection.     

Physicochemical characterization of poly(L-lactic acid) and poly(D,L-lactide-co-glycolide) nanoparticles with polyethylenimine as gene delivery carrier

Polymer nanoparticles have been used as non-viral gene delivery systems and drug delivery systems. In this study, biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) and poly(D,L-lactide-co-glycolide) (PLGA)/PEI nanoparticles were prepared and characterized as gene delivery systems. The PLA/PEI and PLGA/PEI nanoparticles, which were prepared by a diafiltration method, had spherical shapes and smooth surface characteristics. The size of nanoparticles was controlled by the amount of PEI, which acted as a hydrophilic moiety, which effectively reduced the interfacial energy between the particle surface and the aqueous media. The nanoparticles showed an excellent dispersive stability under storage in a phosphate-buffered saline solution for 12 days. The positive zeta-potentials for the nanoparticles decreased and changed to negative values with increasing plasmid DNA (pDNA) content. Agarose gel electrophoresis showed that the complex formation between the nanoparticles and the pDNA coincided with the zeta-potential results. The results of in vitro transfection and cell viability on HEK 293 cells indicated that the nanoparticles could be used as gene delivery carriers.     

Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.     

Poly(L-lactic acid)/polyethylenimine nanoparticles as plasmid DNA carriers

Non-viral vectors such as liposomes, polycations, and nanoparticles have been used as gene delivery systems. In this study, we prepared and characterized biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) nanoparticles as gene carriers. pCMV/β-gal and pEGFP-C1 were utilized as model plasmid DNAs (pDNA). Nanoparticles were prepared using a double emulsion-solvent evaporation technique, and their pDNA binding capacity was assessed by agarose gel electrophoresis. Transfection was studied in HEK 293 and HeLa cell lines, and the transfection efficiencies were determined by β-galactosidase assay or flow cytometry. Three kinds of PLA/PEI systems were studied by varying the molecular weight of PEI. The PLA/PEI 25K system had a higher transfection efficiency than the PLA/PEI 0.8K or PLA/PEI 750K systems. The transfection efficiency was found to be dependent on the ratio of PLA/PEI nanoparticles to pDNA with an optimum ratio of 60:1 (w/w). The cytotoxicity was dependent on the quantity of PLA/PEI nanoparticles used, but it was comparable to that of commercial Lipofectin™. These results demonstrate the potential of PLA/PEI nanoparticles as gene carriers.     

Skin photoprotection improvement: synergistic interaction between lipid nanoparticles and organic UV filters

The main objective of this study was to prepare two types of nanoparticles with poly(d,l-lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) polymers. Plasmid DNA (pDNA) was adsorbed either on PLGA/PEI nanoparticles, or as PEI/DNA complex onto the surface of PLGA nanoparticles. Both types of nanoparticles were prepared by the double emulsion method. The nanoparticles were characterized by their size, zeta potential and pDNA or PEI/DNA complex adsorption. The PEI/DNA complex adsorption was confirmed with ethidium bromide assay. pDNA adsorption onto PLGA/PEI nanoparticles (PLGA/PEI-DNA) was studied by electrophoresis on agarose gel. Cytotoxicity and transfection efficiency of both types of nanoparticle and PEI/DNA complexes formulations were studied in head and neck squamous carcinoma cell line (FaDu). To improve endosomal release, photochemical internalization (PCI) was used. The zeta potential increased when the PEI/DNA complex adsorbed onto PLGA nanoparticles (PLGA-PEI/DNA). Optimal pDNA adsorption efficiency was achieved for nitrogen/phosphorous ratio≥20/1. In vitro transfection and cells viability on FaDu cells with or without PCI were found to be variable depending on the type and concentration of nanoparticles. The results showed that transfection efficiency for PLGA/PEI-DNA or PLGA-PEI/DNA nanoparticles ranged between 2 and 80%, respectively. PCI was found to slightly improve the transfection efficiency for all formulations.     

PEG修饰对PEI聚电解质复合物的药学特性及人乳腺癌细胞的摄取效率影响的研究

荧光素阴离子葡聚糖（DFA）和寡核苷酸（ODN）可以和带正电的聚乙亚胺（PEI）通过自组装的方式形成聚电解质复合物。为了调查PEG修饰对PEI聚电解质复合物的药学特性及细胞摄取效率的影响，我们进行了此项研究。DFA和ODN与PEI及PEI—PEG通过吹吸混匀形成聚电解质复合物。我们分别采用动态光散射法及琼脂糖凝胶电泳法对聚电解质复合物的表面性质（粒径，ξ电位），PEI及PEI—PEG延滞ODN的能力等进行了评价。MCF-7对DFA／PEI及DFA／PEI-PEG的摄取效率通过流式细胞术进行测量，并通过激光扫描聚焦显微镜（CLSM）法来观察DFA／PEI及DFA／PEI—PEG聚电解质复合物的细胞内摄作用。N／P比对ODN／PEI聚电解质复合物的粒径和ξ电位影响很大，而ODN／PEI—PEG复合物的粒径受N／P比的影响要小一些，在N／P=2～16范围内，ODN／PEI—PEG复合物的粒径在30～100nm左右。PEI和PEI—PEG在N／P比等于4时就开始出现延滞ODN的现象，彻底延滞在N／P比等于8时出现。MCF-7的细胞摄取效率与DFA浓度及转染时间呈正相关，在适宜条件下，细胞摄取率可超过99％。本实验结果显示通过隐形修饰的PEI自组装形成的聚电解质复合物可以提高其作为基因载体递送基因进入细胞的能力。     

Receptor-mediated, tumor-targeted gene delivery using folate-terminated polyrotaxanes

Safe and effective gene delivery is essential to the success of gene therapy. We synthesized and characterized a novel nonviral gene delivery system in which folate (FA) molecules were functioned as blockers on cationic polyrotaxanes (PR) composed of poly(ethylenimine) (PEI)(600)-grafted α-cyclodextrin rings linearized on polyethylene glycol to form FA-terminated PR-PEI(600) (FPP). The FA terminal caps of FPP target cell surfaces abundant in FA receptor (FR), a common feature of tumor cells. The structure of FPP was characterized by using (1)H nuclear magnetic resonance ((1)H NMR). The delivery particle was composed of chemically bonded PEG (4000), α-cyclodextrins (CD), and PEI (600 Da) at a molar ratio of 1:17:86.7, and the particle size and zeta potential of FPP/pDNA polyplexes were measured using dynamic light scattering. FPP/pDNA exhibited a lower cytotoxicity, strong specificity to FR, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. Furthermore, the FPP/DNA complex showed an enhanced antitumor effect in the nude mice compared with other delivery systems, such as PEI-25K. Together, these results suggest that FPP may be useful for gene therapy.     

Analysis of hepatic disposition of native and galactosylated polyethylenimine complexed with plasmid DNA in perfused rat liver

We studied the intrahepatic disposition characteristics of galactosylated polyethylenimine (Gal-PEI)/plasmid DNA (pDNA) complexes using rat liver perfusion experiment. After intraportal administration, transfection activity in liver of Gal-PEI complexes was approximately 26-fold higher than that of native PEI complexes. To evaluate the relationship between hepatic gene expression and disposition profiles, hepatic disposition of Gal-PEI complexes were pharmacokinetically analyzed by use of perfused rat liver, which enables uptake characteristics intrinsic to the liver to be elucidated. Moment analysis revealed that both complexes exhibited very high single-pass extraction. To characterize each kinetic process in hepatic uptake of Gal-PEI complexes, their outflow profiles were analyzed based on a two-compartment dispersion model. Consequently, the tissue binding affinity of Gal-PEI complexes was 3.0-fold larger than that of native PEI complexes, suggesting the increasing of hepatic binding affinity much enhanced the hepatic gene transfection efficiency. In contrast, galactosylation of PEI did not affected internalization (and/or sequestration) rate.     

Anti-Inflammatory Therapeutic Effect of Adiponectin Gene Delivery Using a Polymeric Carrier in an Acute Lung Injury Model

Purpose

Adiponectin (APN) is an adipokine with anti-inflammatory and cytoprotective effects. In this study, the therapeutic effect of APN gene delivery using a polymeric carrier was evaluated in an acute lung injury (ALI) model.

Methods

Polyethylenimine (2 kDa, PEI2K), PEI25K (25 kDa), polyamidoamine (generation 2, PAMG2), dexamethasone-conjugated PEI2k (PEI2K-Dexa), and dexamethasone-conjugated PAMG2 (PAMG2-Dexa) were evaluated in vitro and in vivo as gene carriers. Formation of plasmid DNA (pDNA)/carrier complexes was confirmed by gel retardation and heparin competition assays. Delivery efficiency was measured by a luciferase assay and fluorescence microscopy. In an ALI animal model, pAPN/carrier complexes were delivered by intratracheal administration. Therapeutic effects were evaluated by cytokine assays and hematoxylin and eosin (H&E) staining.

Results

Gel retardation assays showed that PEI2K-Dexa and PAMG2-Dexa formed complexes with pDNA. In L2 lung epithelial cells, PAMG2-Dexa yielded higher transfection efficiency than PEI2K, PAMG2, PEI25K, lipofectamine, and PEI2K-Dexa. In vivo experiments showed that PAMG2-Dexa delivered DNA more efficiently to lung tissue than PEI2K-Dexa and PEI25K. Delivery of pAPN/PAMG2-Dexa complexes upregulated APN expression in the lungs of mice with ALI. As a result, the levels of pro-inflammatory cytokines such as TNF-α and IL-1β were decreased. H&E staining showed that inflammation in the lungs of mice with ALI was reduced by delivery of the APN gene.

Conclusion

Delivery of the APN gene using PAMG2-Dexa reduced inflammation in the lungs of mice with ALI. The APN gene could be a useful tool in the development of gene therapy for ALI.

     

Biodegradable nanoparticles composed of dendrigraft poly-l-lysine for gene delivery

We developed novel gene vectors composed of dendrigraft poly-l-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-l-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, γ-polyglutamic acid (γ-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/γ-PGA complexes) were shown to be stable nanoparticles, and those with γ-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/γ-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/γ-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/γ-PGA complexes for clinical gene therapy.