pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles. Poly(L-histidine) ?PLH? has been shown to induce membrane fusion at endosomal pH values, whereas PLL has a well documented efficacy in polyplex formation. Therefore, N-Ac-poly(L-histidine)-graft-poly(L-lysine) ?PLH-g-PLL? was synthesized by grafting poly(L-histidine) to poly(L-lysine) ?PLL?. PLH-g-PLL formed polyplex particles by electrostatic interactions with plasmid DNA ?pDNA?. The mean particle size of the polyplexes was in the range of 117 +/- 6 nm to 306 +/- 77 nm. PLH-g-PLL gene carrier demonstrated higher transfection efficacy in 293T cells than PLL at all equivalent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic agent enhanced transfection for both PLL and PLH-g-PLL gene carriers. PLH-g-PLL enhanced beta-galactosidase expression compared to PLL, but still increased in efficacy when chloroquine was included.     

Poly(ethylene oxide) grafted with short polyethylenimine gives DNA polyplexes with superior colloidal stability, low cytotoxicity, and potent in vitro gene transfection under serum conditions

Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80-245 nm) particles with moderate positive surface charges (+7.2 ～ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications.     

Branched polyethylenimine derivatives with reductively cleavable periphery for safe and efficient in vitro gene transfer

Twenty-five kDa polyethylenimine (PEI) is one of the most efficient nonviral gene transfer agents currently applied as a golden standard for in vitro transfection. In this study, novel 25 kDa PEI derivatives with reductively cleavable cystamine periphery (PEI-Cys) were designed to reduce carrier-associated cytotoxicity and to enhance further the transfection activity. The Michael-type conjugate addition of 25 kDa PEI with N-tert-butoxycarbonyl-N'-acryloyl-cystamine (Ac-Cys-(t)Boc) and N-tert-butoxycarbonyl-N'-methacryloyl-cystamine (MAc-Cys-(t)Boc) followed by deprotection readily afforded PEI-Cys derivatives, denoted as PEI-(Cys)x(Ac) and PEI-(Cys)x(MAc), with degree of substitution (DS) ranging from 14 to 34 and 13 to 38, respectively. All PEI-Cys derivatives had higher buffer capacity than the parent 25 kDa PEI (21.2 to 23.1% versus 15.1%). Gel retardation and ethidium bromide exclusion assays showed that cystamine modification resulted in largely enhanced interactions with DNA. PEI-(Cys)x(Ac) could condense DNA into small-sized particles of 80-90 nm at and above an N/P ratio of 5/1, which were smaller than polyplexes of 25 kDa PEI (100-130 nm). In comparison, PEI-(Cys)x(MAc) condensed DNA into somewhat larger particles (100-180 nm at N/P ratios from 30/1 to 5/1). Gel retardation and dynamic light scattering (DLS) measurements showed that PEI-Cys polyplexes were quickly unpacked to release DNA in response to 10 mM dithiothreitol (DTT). These PEI-Cys derivatives revealed markedly decreased cytotoxicity as compared with 25 kDa PEI with IC(50) values of >100 mg/L and 50-75 mg/L for HeLa and 293T cells, respectively (corresponding IC(50) data of 25 kDa PEI are ca. 11 and 3 mg/L). The in vitro transfection experiments in HeLa and 293T cells using pGL3 as a reporter gene showed that gene transfection activity of PEI-Cys derivatives decreased with increasing DS and PEI-(Cys)x(MAc) exhibited higher transfection activity than PEI-(Cys)x(Ac) at similar DS. Notably, polyplexes of PEI-(Cys)14(Ac) and PEI-(Cys)13(MAc) showed significantly enhanced gene transfection efficiency (up to 4.1-fold) as compared with 25 kDa PEI formulation at an N/P ratio of 10/1 in both serum-free and 10% serum-containing conditions. The modification of PEI with reductively cleavable periphery appears to be a potential approach to develop safer and more efficient nonviral gene vectors.     

Effective delivery of siRNA to transgenic rice cells for enhanced transfection using PEI-based polyplexes

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.     

Nonviral vector-based gene transfection of primary human skeletal myoblasts

Low-level transgene efficiency is one of the main obstacles in ex vivo nonviral vector-mediated gene transfer into primary human skeletal myoblasts (hSkMs). We optimized the cholesterol:N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate liposome (CD liposome) and 22-kDa polyethylenimine (PEI22)- and 25-kDa polyethylenimine (PEI25)-mediated transfection of primary hSkMs for angiogenic gene delivery. We found that transfection efficiency and cell viability of three nonviral vectors were cell passage dependent: early cell passages of hSkMs had higher transfection efficiencies with poor cell viabilities, whereas later cell passages of hSkMs had lower transfection efficiencies with better cell viabilities. Trypsinization improved the transfection efficiency by 20% to 60% compared with adherent hSkMs. Optimum gene transfection efficiency was found with passage 6 trypsinized hSkMs: transfection efficiency with CD lipoplexes was 6.99 +/- 0.13%, PEI22 polyplexes was 18.58 +/- 1.57%, and PEI25 polyplexes was 13.32 +/- 0.88%. When pEGFP (a plasmid encoding the enhanced green fluorescent protein) was replaced with a vector containing human vascular endothelial growth factor 165 (phVEGF(165)), the optimized gene transfection conditions resulted in hVEGF(165) expression up to Day 18 with a peak level at Day 2 after transfection. This study demonstrated that therapeutic angiogenic gene transfer through CD or PEI is feasible and safe after optimization. It could be a potential strategy for treatment of ischemic disease for angiomyogenesis.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Biodegradable cyclen-based linear and cross-linked polymers as non-viral gene vectors

Several 1,4,7,10-tetraazacyclododecane (cyclen)-based linear (3a-c) and cross-linked (8a-d) polymers containing biodegradable ester or disulfide bonds were described. These polymeric compounds were prepared by ring-opening polymerization from various diol glycidyl ethers. The molecular weights of the title polymers were measured by GPC. Agarose gel retardation assays showed that these compounds have good DNA-binding ability and can completely retard plasmid DNA (pDNA) at weight ratio of 20 for linear polymers and 1.2 for cross-linked polymers. The degradation of these polymers was confirmed by GPC. The formed polyplexes have appropriate sizes around 400 nm and zeta-potential values about 15-40 mV. The cytotoxicities of 8 assayed by MTT are much lower than that of 25 KDa PEI. In vitro transfection toward A549 and 293 cells showed that the transfection efficiency (TE) of 8c-DNA polyplex is close to that of 25 kDa PEI at 8c/DNA weight ratio of 4. Structure-activity relationships (SAR) of these linear and cross-linked polymers were discussed in their DNA-binding, cytotoxicity, and transfection studies. In addition, in the presence of serum, the TE of 8/DNA polyplexes could be improved by introducing chloroquine or Ca(2+) to pretreated cells.     

Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans

BACKGROUND: Here we report on studies that probe whether the intracellular kinetics of plasmid DNA (pDNA) and cell surface glycosaminoglycans (GAGs) are modified during the cell cycle in a way that can be correlated with changes in gene transfer efficiency with poly(ethyleneimine) (PEI) and poly-L-lysine (PLL) polyplexes. METHODS: Synchronized D407 retinal cells were transfected with PEI and PLL polyplexes using a luciferase reporter. The free and/or loosely complexed nuclear pDNA was determined by real-time PCR, and compared with transgene expression, the rate of pinocytosis by FITC-dextran uptake and the content of cell surface GAGs. RESULTS: The amount of free and/or loosely complexed nuclear pDNA between cell cycle phases varied approximately 4-20 times (G1 < S < G2/M). Both carriers delivered pDNA in a similar way into the nucleus (PLL vs. PEI < or = 3.5-fold), but PEI was approximately 10-100 times more efficient in gene expression than PLL (G1 < G2/M < S). The rate of pinocytosis increased up to 70-fold from G1 to middle S phase. Cell surface heparan and chondroitin sulfate increased 50-80%, and hyaluronan decreased 50% when the cells went from G1 through S to G2/M. CONCLUSIONS: The data obtained indicates that no single parameter (pinocytosis, cell surface GAGs, nuclear uptake) solely accounts for the differential pDNA uptake or expression during cell cycle, and that the main difference in PLL- and PEI-mediated transfections seems to be at the nuclear level.     

Optimising non-viral gene delivery in a tumour spheroid model

BACKGROUND: Our current understanding of how the unique tumour microenvironment influences the efficacy of gene delivery is limited. The current investigation systematically examines the efficiency of several non-viral gene transfer agents to transfect multicellular tumour spheroids (MCTS), an in vitro model that displays a faithful three-dimensional (3D) representation of solid tumour tissue. METHODS: Using a luciferase reporter assay, gene transfer to MCTS was optimised for 22 kDa linear and 25 kDa branched polyethyleneimine (PEI), the cationic lipids Lipofectamine(trade mark) and DCChol : DOPE, and the physical approach of tissue electroporation. Confocal microscopy was used to take optical tissue slices to identify the tissue localisation of green fluorescent protein (GFP) reporter gene expression and the distribution of fluorescently labelled complexes. A MCTS model of quiescent tumour regions was used to establish the influence of cellular proliferation status on gene transfer efficiency. RESULTS: Of the polyplexes tested, 22 kDa linear PEI provided optimal gene delivery, with gene expression peaking at 46 h. Despite being the optimal vector tested, PEI-mediated transfection was limited to cells at the MCTS periphery. Using fluorescent PEI, it was found that complexes could only penetrate the outer 3-5 proliferating cell layers of the MCTS, sparing the deeper quiescent cells. Gene delivery in an MCTS model comprised entirely of quiescent cells demonstrated that in addition to being inaccessible to the vector, quiescent tumour regions are inherently less susceptible to PEI-mediated transfection than proliferating regions. This 'resistance' to transfection observed in quiescent cells was overcome through the use of electroporation. Despite the improved efficacy of electroporation in quiescent tissue, the gene expression was still confined to the outer regions of MCTS. The results suggest that limited access to central regions of an MCTS remain a significant barrier to gene delivery. CONCLUSIONS: This data provides new insights into tumour-specific factors affecting non-viral gene transfer and highlights the difficulties in delivering genes to avascular tumour regions. The MCTS model is a useful system for the initial screening of future gene therapy strategies for solid tumours.     

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.     

Polyethylenimine/oligonucleotide polyplexes investigated by fluorescence resonance energy transfer and fluorescence anisotropy

To advance knowledge on polyplex structure and composition, fluorescence resonance energy transfer (FRET) and anisotropy measurements were applied to polyplexes of rhodamine-labeled polyethylenimine (PEI) and fluorescein-labeled double-stranded oligodeoxynucleotide (ODN). About 25?kDa PEI was compared with low-molecular-weight PEI of 2.7?kDa. FRET reached maxima at amine to phosphate (N/P) ratios of 2 and 3 for 2.7?kDa and 25?kDa PEI, respectively, with similar average distances between donor and acceptor dye molecules in polyplexes. Anisotropy measurements allowed estimating the bound fractions of PEI and ODN. At N/P?=?6, all ODN was bound, but only 58% of PEI 25?kDa and 45% of PEI 2.7?kDa. In conclusion, the higher molecular weight of PEI may conformationally restrict the availability of amino groups for charge interaction with phosphate groups in ODN. Moreover, significant fractions of both types of PEI remain free in solution at N/P ratios frequently used for transfection. FRET and anisotropy measurements provide effective tools for probing polyplex compositions and designing optimized delivery systems.     

Role of biophysical parameters on ex vivo and in vivo gene transfer to the airway epithelium by polyethylenimine/albumin complexes

Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.     

Biodegradable cross-linked poly(amino alcohol esters) based on LMW PEI for gene delivery

Polyethylenimine (PEI, especially with M(w) of 25,000) has been known as an efficient gene carrier and a gold standard of gene transfection due to its high transfection efficiency (TE). However, high concomitant cytotoxicity limited the application of PEI. In this report, several cationic polymers derived from low molecular weight (LMW) PEI (M(w) 600) linked with diglycidyl adipate (DA-PEI) or its analogs (diglycidyl succinate, DS-PEI and diglycidyl oxalate, DO-PEI; D-PEIs for all 3 polymers) were prepared and characterized. GPC gave M(w)s of DA-PEI, DS-PEI and DO-PEI as 6861, 16,015 and 35,281, respectively. Moreover, degradation of the ester-containing DS-PEI was also confirmed by GPC. In addition, hydroxyls in these polymers could improve their water solubility. These polymers exhibited good ability to condense plasmid DNA into nanoparticles with the size of 120-250 nm. ζ-potentials of the polyplexes were found to be around +10-20 mV under weight ratios (polymer/DNA) from 0.5 to 32. Agarose gel retardation showed that DNA could be released from the polyplexes after being pre-incubated for 30 h. In vitro experiments were carried out and it was found that DS-PEI showed about 5 times of TE compared to that of the PEI/DNA polyplex under a weight ratio of 1 in A549 cells. Meanwhile, the cytotoxicity of D-PEIs assayed by MTT is lower than that of 25 kDa PEI in HEK293 cells. These results suggested that this series of PEI derivatives would be promising non-viral biodegradable vectors for gene delivery.     

Optimized lipopolyplex formulations for gene transfer to human colon carcinoma cells under in vitro conditions

BACKGROUND: Polycation (PC, polyplex), cationic lipid (CL, lipoplex), and a combination of PC/CL (lipopolyplex) formulations were investigated for gene transfer to slow-proliferating human colon carcinoma cell lines (COGA). METHODS: The luciferase reporter gene was complexed with either PC, CL, or PC/CL. PCs included linear (PEI22lin, 22 kDa) and branched polyethylenimine (PEI2k, 2 kDa; PEI25br, 25 kDa) and poly-L-lysine (PLL18 with 18 lysine monomers). CLs included DOCSPER, DOSPER and DOTAP. Lipopolyplexes were formed by either sequentially first mixing DNA with PC or CL, followed by addition of CL or PC, respectively, or simultaneously with both PC and CL. Particle size and zeta-potential were determined and gene transfer and cytotoxicity were quantified on COGA-3, -5, -12, HeLa and Sw480 cells. RESULTS: The highest gene transfer was achieved when DNA was first complexed with PC followed by CL. At low ionic strength, particles were small (50-130 nm) with a zeta-potential of +20-40 mV. At physiological ionic strength, only lipoplexes of DOCSPER or DOSPER and their respective lipopolyplexes with PEI25br were stable to aggregation (140-220 nm). Lipopolyplexes of PEI25br were between 5- to 400-fold more efficient compared to the corresponding lipoplexes or polyplexes in all cases. Chloroquine did not significantly affect lipopolyplex-mediated gene transfer. CONCLUSIONS: Lipopolyplex formulations of PEI25br in combination with multivalent CLs (DOCSPER, DOSPER) are promising tools for in vitro and potentially also in vivo gene transfer to colorectal cancer cells.     

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.     

The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells

The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9 kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06 nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5 nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1 h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.