Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.     

Positive hyaluronan/PEI/DNA complexes as a target-specific intracellular delivery to malignant breast cancer

In the present study, The PEI/DNA (PD) complexes was first prepared with positive surface charge under a suitable N/P ratio of 10. The redundant positive charge was partially and excessively shielded by a polysaccharide, hyaluronic acid (HA), in aqueous solution. The HA/PEI/DNA ternary complexes were characterized by assessing the zeta potential and size, then transferred to MDA-MB-435, MDA-MB-231, and MCF-7 cell lines with different amounts of HA-specific CD44 receptors on the surface. Consequently, The transfection efficiency of all the prepared complexes show a little increased to MCF-7 (low CD44 level) while a large increased to MDA-MB-231 and MDA-MB-435 cells (high CD44 level) with adding HA. Also, when HA:PEI charge ratio was 7.5%, the ternary complexes show the highest transfection efficiency. The prepared ternary complexes exhibited increased 2–13-fold fluorescence intensity and lower cell toxicity compared to the PD (N/P, 10). These results indicated that the positive HA/PEI/DNA ternary complexes (HA:PEI charge ratio, 7.5%) can target malignant breast cancer cells with high CD44 level and might be a promising candidate vector for gene therapy.     

A novel application of indolicidin for gene delivery

A bovine derived antimicrobial peptide, indolicidin (IL), was studied of its new application for gene transfer. Plasmid DNA was complexed with both IL and polyethylenimine (PEI) as ternary particles. Compared to DNA/IL complexes, the DNA/IL/PEI particles demonstrated high zeta potentials, small particle sizes, and superior loading efficiencies, suggesting the incorporation of polycations can support IL for gene delivery. For in vitro experiments, these ternary particles significantly improved gene transfection efficiencies over the sole administrations of IL or PEI. This synergistic effect revealed that IL and PEI may play different roles for gene transfer. Our results suggest that IL should be a potential carrier for gene delivery. As our knowledge, our study should be the first article indicating the carrier ability of IL for gene transfer.     

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.     

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

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

Transfection by Polyethyleneimine-Coated Microspheres

Polyethyleneimine (PEI) can be used as a DNA delivery mechanism in cell culture and in vivo. Cells can be transfected by using surface-bound PEI, as well as by PEI/DNA microparticles. In the present experiments we extended these observations by preparing microspheres with covalently attached PEI. Blends of poly(?-CBZ-L-lysine) mixed with poly(D,L-lactic-co-glycolic acid) were formed into microspheres using a double-emulsification/solvent evaporation procedure. CBZ (carbobenzoxy) groups on the surface of microspheres were removed by Li⁰/liquid ammonia reduction. Surface amino groups were used for covalent attachment of PEI and other molecules. Silica microspheres with bonded-phase PEI were also used. Microspheres were mixed with plasmid DNA encoding green fluorescent protein and added to cultured cells. PEI-coated microspheres transfected cultured Caco cells and MH-S alveolar macrophages. Expression of the transfected DNA increased over several days. MH-S cells phagocytosed PEI-coated silica microspheres, which were shown to reside in an acidic subcellular compartment. This was demonstrated by conjugating a pH-sensitive fluorescent dye (seminaphthofluorescein, SNAFL) to the microsphere surface. Transfection of MH-S cells was increased when plasmid DNA was complexed with histone on the surface of the microspheres. Conclusions PEI-coated microspheres have potential as a DNA delivery device with advantages of the unique properties of PEI and ease of surface chemical modification.     

Polyethylenimine–DNA solid particles for gene delivery

Polyethylenimine (PEI), a cationic polymer, was used to develop a non-viral vector for gene delivery. A simple, reproducible process is described with which to condense plasmid DNA with PEI. When prepared at the optimum charge ratio of 6.3 ( ± ; PEI:DNA, 5:1 w/w), PEI–DNA complexes were 30–60 nm in diameter and excluded intercalating dyes from the plasmid DNA. The particles were stable for more than one month at 4°C with respect to size and transfection activity. PEI–condensed DNA transfected a broad range of murine and human tumor cell lines (B16, Lewis Lung, SK-OV-3 and LS180) in vitro in the presence of fetal calf serum. Intraperitoneal administration of PEI–condensed DNA resulted in significant gene expression in a human ovarian cancer peritoneal xenograft model.     

Ternary Complexes with Core-Shell Bilayer for Double Level Targeted Gene Delivery: In Vitro and In Vivo Evaluation

Purpose

Hyaluronic acid (HA)/polyethyleneimine-dexamethasone (PEI-Dex)/DNA ternary complexes with “core-shell” bilayer were developed for double level targeted gene delivery. A PEI₁₈₀₀-Dex, as a core, was applied to compact DNA into a nano-sized structure and facilitate the nuclear translocation of DNA after endocytosis into tumor cells, and a polyanion HA, as the outer corona, was employed to improve targeted tumor delivery and reduce cytotoxicity.

Methods

PEI-Dex was synthesized and characterized by ¹H NMR. The characterizations of ternary complexes were investigated. Their biological properties, including transfection efficiency, cytotoxicity, cellular uptake and in vivo efficacy were evaluated systemically.

Results

Ternary complexes with the size of about 160 nm exhibited the lowest cytotoxicity and the highest transfection efficiency in B16F10 cells among investigated complexes. The sub-cellular localization study confirmed that ternary complexes could facilitate more efficient cell uptake and nuclear transport of DNA than binary complexes. Moreover, Cy7-labeled ternary complexes obviously accumulated in the tumor after i.v. administration, indicating that ternary complexes could assist the DNA targeting to the tumor. In in vivo studies, HA/PEI₁₈₀₀-Dex/DNA ternary complexes showed confirmed anti-inflammation activity, and could significantly suppress tumor growth of tumor-bearing nude mice.

Conclusions

HA/PEI-Dex/DNA ternary complexes might be a promising targeted gene delivery system.     

聚乙烯亚胺-DNA复合物的形成和聚集行为研究

聚乙烯亚胺 (polyethyleneimine,PEI) 是一种优良的非病毒基因传输载体材料,本文对PEI/DNA复合物粒子的形成机制进行了初步探讨,电泳阻滞实验和紫外测定实验表明, 复合物的形成过程中存在着某种过渡状态即珠串样结构,透射电镜的结果提供了相应的例证。此外通过离子强度实验,作者认为在PEI与DNA的复合过程尽管以静电作用为主要作用力,同时也可能存在着其他类型的非静电作用力。PEI/DNA复合物粒子的表面电荷随着N/P的增加逐步增加,但由于DNA的分子质量较大,在N/P为8和12时表面电荷的绝对值较小,容易聚集成葡萄串样聚集体,离子强度实验表明该聚集过程的支配作用力可能是疏水作用力。EI/DNA复合物在N/P为12时的细胞转染效果与阳性对照组相当,表明聚集的PEI/DNA复合物也具有一定的细胞转染能力。     

Chitosan enhances transfection efficiency of cationic polypeptides/DNA complexes

The aim of this research was to investigate the effect of cationic polypeptides mixed with chitosan (CS) on in vitro transfection efficiency and cytotoxicity in human cervical carcinoma cells (HeLa cells). The polypeptides/DNA complexes and ternary complexes (CS, polypeptides and DNA) at varying weight ratios were formulated and characterized by using gel electrophoresis. Their particle sizes and charge were evaluated. The effect of the type and molecular weight (MW) of polypeptides, the weight ratio, order of mixing, the pH and serum on transfection efficiency and cytotoxicity were evaluated in HeLa cells. Three types of polypeptides (poly-L-lysine; PLL, poly-L-arginine; PLA and poly-L-ornithine; PLO) were able to form complete complex with DNA at weight ratio above 0.1. The PLA MW >70 kDa showed the highest transfection efficiency. The order of mixing between CS, PLA and DNA affected the transfection efficiency. The highest transfection efficiency was observed in ternary complexes of PLA/DNA/CS (2:1:4) equal to PEI/DNA complex. For cytotoxicity studies, over 80% the average cell viabilities of the complexes were observed by MTT assay. This study suggests that the addition of CS to PLA/DNA is easy to prepare, safe and exhibits significantly improved DNA delivery potential in vitro.     

Carboxymethyl poly(L-histidine) as a new pH-sensitive polypeptide to enhance polyplex gene delivery

Carboxymethyl poly( l-histidine) (CM-PLH) as a new pH-sensitive polypeptide has enhanced polyplex gene delivery. Agarose gel retardation assay and zeta potential measurement proved that the anionic CM-PLH at physiological pH coated the PEI/DNA binary complexes. The resulting CM-PLH/PEI/DNA ternary complexes showed the gene expression value 300 times higher than that of the PEI/DNA binary complexes. These results suggest that the synergistic effect of the pH-sensitive imidazole groups at endosomal pH and the anionic carboxymethyl groups at physiological pH in the CM-PLH enhanced polyplex gene delivery.     

PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy

Background: Gene targeting by RNA interference (RNAi) is mediated through small interfering RNA (siRNA), which, as plasmid DNA molecules, can be delivered into cells by polyethylenimines (PEI). Grafting with poly(ethylene glycol) has been introduced previously to improve PEI biocompatibility; however, data on the effects of PEGylation have been somewhat contradictory and various PEI(-PEG) need to be evaluated independently for DNA transfection and siRNA gene targeting efficacies.

Aim: We directly compare plasmid DNA transfection and siRNA-mediated gene targeting efficacies, employing a larger set of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG; PEI(-PEG)) with different molecular weights and degrees of PEG substitution.

Method: We performed tissue culture-based bioassays on DNA transfection and siRNA-mediated targeting efficacies as well as on toxicity and cellular nucleic acid uptake, and, using sensitive assays based on radioactive labelling, physicochemically characterize the complexes regarding the degree of nucleic acid complexation and complex stabilities under various conditions.

Results: In contrast to the DNA transfection efficacy, siRNA-mediated gene targeting is much less dependent on the PEGylation of PEI or on the N/P (= PEI nitrogen/nucleic acid phosphate) ratio. A more detailed analysis reveals that, in order to define optimal N/P ratios for DNA transfection, complex toxicities and nucleic acid uptake are the most critical parameters. In contrast, at optimal N/P ratios, complex stabilities and complexation efficacies determine PEI(-PEG)/DNA transfection efficacies and the major differences between various PEI(-PEG) are observed. All these parameters are less critical for PEI(-PEG)/siRNA gene targeting efficacy. Thus, our data lead to the distinction between three PEI(-PEG) groups, which relies on the differences in transfection rather than gene targeting efficacies, and which is correlated with the molecular weights and degrees of PEG substitution.

Conclusion: In contrast to PEI(-PEG)/DNA complexes, a broader panel of PEI-PEG are capable of siRNA-mediated gene targeting. Thus, PEG grafting of PEI requires a separate evaluation of siRNA and DNA complexes, which expands the portfolio of available PEI(-PEG) for the preparation of non-toxic, biocompatible siRNA delivery reagents for the induction of RNAi.     

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

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

Chitosan-Based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability

Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. Methods. Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. Results. In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50–100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time, from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. Conclusions. Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.     

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

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

Influence of acyl chain length on transfection mediated by acylated PEI nanoparticles

Polyethylenimine (750 kDa) has been derivatized to influence the proton sponge mechanism and hydrophobic-hydrophilic balance. The polymer was acylated using acid anhydrides of varying carbon chain length, followed by cross-linking with PEG-bis-P to form compact nanoparticles. The chemical linkages in the particles were characterized by FTIR and NMR spectroscopy. The hydrodynamic diameter of nanoparticles was found to be in the range of 83.5-124 nm. AFM imaging of native and DNA-loaded nanoparticles revealed highly compact and spherical shape. The positive surface charge on particles decreased with the increase in percentage of acylation and also on complexing with DNA. The buffering capacity of PEI was reduced considerably on preparing acylated nanoparticles. The nanoparticles formed stable complexes with DNA and higher weight ratios were required for formation of electro-neutral complexes. Further, these nanoparticles were investigated for their gene delivery efficacy on COS-1 cells. It was found that acylated PEI nanoparticles were 5-12-fold more efficient transfecting agents as compared to native PEI (750 kDa) and commercially available transfecting agent lipofectin. The MTT colorimetric assay revealed of considerable reduction in toxicity of acylated PEI nanoparticles as compared PEI. Of all the systems prepared, nanoparticles with 30% acylation using propionic anhydride were found to be the most efficient in in vitro transfection.     

Preparation of Calcium Phosphate Nanocapsule Including Deoxyribonucleic Acid–Polyethyleneimine–Hyaluronic Acid Ternary Complex for Durable Gene Delivery

Our plasmid delivery systems comprising deoxyribonucleic acid (DNA), polyethyleneimine (PEI), and hyaluronic acid (HA) have already achieved the high‐extragene expression in tumor tissues. Repeated transfection with certain cytokine genes effectively induced tumor regression and complete disappearance of the tumor in some cases. Frequent injection is sometimes difficult depending on the tumor site. However, single injection often leads to an unsatisfactory efficacy owing to the short duration of the gene expression. In this study, we prepared calcium phosphate (CaP) nanocapsule including plasmid DNA complexes as a durable gene transfection system, which would be slowly degraded, and release DNA complex in the body. CaP nanocupsule including DNA complexes with a diameter of approximately 200 nm was prepared by immersing HA‐coated DNA–PEI complex in simulated body fluid. It showed gene expression in cultured cells with duration longer than 2 weeks. By this slow‐releasing system, significant tumor‐growth suppression and, finally, complete tumor disappearance were observed after single injection into the tumor. Capsulated DNA complex with Ca thus seems promising as a sustained gene expression device. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:179–184, 2014     

DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine

Some genetic diseases are associated with the defects of the mitochondrial genome. Direct DNA delivery to the mitochondrial matrix has been suggested as an approach for mitochondrial gene therapy for these diseases. We hypothesized that a mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites. PEI-LP was synthesized by the conjugation of LP to PEI using disulfide bond. The complex formation of PEI-LP with DNA was confirmed by a gel retardation assay. In this study, DNA was completely retarded at a 0.4/1 PEI-LP/DNA weight ratio. In vitro delivery tests into isolated mitochondria or living cells were performed with rhodamin-labeled DNA and PEI-LP. In vitro cell-free delivery assay with isolated mitochondria showed that PEI-LP/DNA complexes were localized at mitochondria sites. Furthermore, the PEL-LP/DNA complexes were localized at the mitochondrial sites in living cells. However, a control carrier, PEI, did not show this effect. In addition, MTT assay showed that PEI-LP showed lower cytotoxicity than PEI. These results suggest that PEI-LP can deliver DNA to the mitochondrial sites and may be useful for the development of mitochondrial gene therapy.     

Cross-linked Small Polyethylenimines: While Still Nontoxic,Deliver DNA Efficiently to Mammalian Cells <Emphasis Type="Italic">in Vitro</Emphasis> and <Emphasis Type="Italic">in Vivo</Emphasis>

No HeadingPurpose. Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycations effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated.Methods. The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing -galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs.Results. Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times m ore efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI.Conclusions. Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.