Preparation and characterization of chitosan and trimethyl-chitosanmodified poly-(ε-caprolactone) nanoparticles as DNA carriers

The purpose of this research was to prepare poly-(ε-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA. Published: August 10, 2005     

Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bcl-2 overexpression in CHO cells improves polyethylenimine-mediated gene transfection

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI) as a transfection reagent has been considered as an attractive method to produce recombinant proteins rapidly for pre-clinical studies. A high level of transfection efficiency, which is required for high-level TGE in CHO cells, can be achieved by increasing the PEI concentration. However, PEI induces cytotoxicity in a dose-dependent manner. To overcome this problem, Bcl-2 protein, an anti-apoptotic protein, was overexpressed in CHO cells (DG44). At a ratio of PEI to DNA (an N/P ratio) of 10, there were no significant differences in transfection efficiency and cell viability between Bcl-2 overexpressing and non-overexpressing cells. The transfection efficiency and cell viability were 2–11% and 83–92%, respectively. However, there were significant differences (P < 0.05) in the transfection efficiency and cell viability between them at a higher N/P ratio. At an N/P ratio of 40, the transfection efficiency and cell viability of Bcl-2 non-overexpressing cells were 24–38% and 35–40%, respectively, while those of Bcl-2 overexpressing cells were 48–53% and 43–56%, respectively. Furthermore, compared with Bcl-2 non-overexpressing cells, more DNAs entered the Bcl-2 overexpressing cells, resulting in a higher rate of TGE per cell. PE-Annexin V apoptosis revealed that Bcl-2 overexpression suppressed PEI-induced apoptotic cell death at high N/P ratios. Taken together, Bcl-2 overexpression in CHO cells suppresses apoptotic cell death during PEI-mediated transient transfection, resulting in enhanced transfection efficiency and TGE.     

Polyethylenimine‐mediated gene delivery into human bone marrow mesenchymal stem cells from patients

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post‐infarction left ventricular (LV) dysfunction. However, age‐related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non‐viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI‐mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm². The average transfection efficiency for all tested samples, middle‐age group (<65 years), old‐age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI‐mediated therapeutic gene VEGF transfer could significantly enhance the expression level.     

Enhancing transfection efficiency using polyethylene glycol grafted polyethylenimine and fusogenic peptide

This study presents a new formulation method for improving DNA transfection efficiency using a fusogenic peptide and polyethylene glycol grafted polyethylenimine. Succinimidyl succinate polyethylene glycol (PEG-SSA) was conjugated with polyethylenimine (PEI). PEI is well known for a good endosomal escaping and DNA condensing agent. The positively charged synthetic fusogenic peptide, KALA, was coated on the negatively charged PEG-g-PEI/DNA and PEI/DNA complexes. The KALA/PEI/DNA complexes exhibited aggregation behavior at higher KALA coating amounts with an effective diameter of around 1,000 nm. However, the KALA/PEG-g-PEI/DNA complexes were 100–300 nm in size with a surface zeta-potential (ζ) value of about +20 mV. The conjugated PEG molecules suppressed any KALA-mediated inter-particle aggregation, and thereby improved the transfection efficiency. Consequently, the transfection efficiency of the KALA/PEG-g-PEI/DNA complexes was obtained by utilizing both the fusogenic activity of KALA and the steric repulsion effect of PEC.     

Blood compatible N-maleyl chitosan-graft-PAMAM copolymer for enhanced gene transfection

To improve transfection efficiency, we prepared N-maleyl chitosan-graft-polyamidoamine (NMCTS-graft-PAMAM) copolymer. Self-assembled NMCTS-graft-PAMAM/pDNA complexes were prepared by complex coacervation method at different N/P (nitrogen to phosphate ratio) ratios. The copolymer effectively formed complexes with pDNA at lower N/P ratio (N/P ratio 1.0) than that of unmodified chitosan (N/P ratio 2.0) and the complexes were spherical with particle size of 100–150 nm. The copolymer showed significant protection of DNA from nuclease attack with lower toxicity against HeLa cell. The copolymer also showed no noticeable hemolytic effects up to 10 mg/mL indicating no detectable disturbance of the red blood cell membranes. The transfection efficiency of the copolymer was increased significantly compared to that of chitosan and reached up to 36 ± 2% at N/P ratio 7.0 which was higher than that of PEI (30 ± 3% at N/P ratio 10). Therefore, the copolymer may be a strong alternative candidate as effective nonviral vector.     

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.     

Methylated N-aryl chitosan derivative/DNA complex nanoparticles for gene delivery: Synthesis and structure–activity relationships

In this study, three kinds of methylated chitosan containing different aromatic moieties were synthesized by two steps, reductive amination and methylation, respectively. The chemical structures of all methylated derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMChC), methylated N-(4-N,N-dimethylaminobenzyl) chitosan chloride (MDMBzChC), and methylated N-(4-pyridinylmethyl) chitosan chloride (MPyMeChC) were characterized by ATR–FTIR and ¹H NMR spectroscopy. The complexes between the chitosan derivatives and plasmid DNA at different N/P ratios were characterized by gel electrophoresis, dynamic light scattering, and atomic force microscopic techniques. The smallest particle sizes of these complexes were obtained at N/P ratio of 5 and ranged from 95 to 124 nm while the zeta-potentials were in the range of 18–27 mV. Transfection efficiencies of these complexes were investigated by expression of the plasmid DNA encoding green fluorescence protein (pEGFP-C2) on human hepatoma cells (Huh 7 cells) compared to N,N,N-trimethyl chitosan chloride (TMChC). The rank of transfection efficiency was MPyMeChC > MDMBzChC > TMChC > MDMCMChC, respectively. The cytotoxicity of these complexes was also studied by MTT assay where the MPyMeChC complex exhibited less toxicity than other derivatives even at high N/P ratios. Therefore, MPyMeChC demonstrated potential as its safe and efficient gene carrier.     

Water-soluble lipopolymer for gene delivery

The use of biocompatible polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. Nontoxic water-soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl) ethyleneimin]-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine) was synthesized using branched poly(ethylenimine) (PEI, mw 1800) and cholesteryl chloroformate. Following synthesis and purification, the structure and molecular weight of WSLP were confirmed by (1)H NMR and MADI-TOF mass spectrometry, respectively. The percentage of cholesterol conjugated to PEI was about 47%, and the average molecular weight of WSLP was approximately 2000 Da. WSLP/pDNA complexes were prepared at different N/P (nitrogen atoms of WSLP/phosphate of plasmid DNA) ratios and characterized in terms of particle size, zeta potential, osmolarity, surface morphology, and cytotoxicity. WSLP condensed plasmid DNA when N/P ratio reached 2.5/1 and no free DNA was detected at N/P ratio of 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was in the range of 25.9 to 148.5 nm and was dependent on N/P ratios. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of approximately 50 nm in diameter. WSLP/pDNA complexes or WSLP itself were nontoxic to CT-26 colon adenocarcinoma and 293 T human embryonic kidney transformed cells when formulated at the N/P ratio of 10/1 and below as determined by MTT assay. In contrast, PEI25000/pDNA complexes were toxic to these cells. Erythrocytes aggregated when incubated with PEI25000/pCMV-Luc complexes at high DNA concentrations, but there was little aggregation with WSLP/pCMV-Luc complexes. WSLP/pCMV-Luc complexes demonstrated higher transfection efficiency in both CT-26 and 293 T cells compared to PEI25000- or PEI1800-based formulations. WSLP/pCMV-Luc complexes are nontoxic and showed enhanced in vitro transfection. Thus, WSLP will be a suitable carrier for in vivo gene delivery.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo

Polyethylenimine-graft-chitosan (PEI-g-chitosan) was synthesized by performing cationic polymerization of aziridine in the presence of water-soluble oligo-chitosan (M(n) = 3400). The absolute molecular weight and chemistry of the PEI-g-chitosan obtained were characterized using GPC, 13C and 1H NMR, respectively. The results indicated that all the amines of chitosan were grafted with oligo-PEI, and the average length of the oligo-PEI side chains was determined by the feed molar ratio of aziridne/amine in chitosan. PEI-g-chitosan of M(n) = 7400 with a polydispersity index (PDI) of 1.50, and PEI side chains of M(n) = 206 was prepared for gene delivery. Gel electrophoresis showed that DNA migration was retarded completely at a N/P ratio of 2.5/1, indicating good DNA condensation capability of PEI-g-chitosan. The sizes and the zeta-potentials of the complexes of PEI-g-chitosan/DNA were characterized. The cytotoxicity of PEI-g-chiotsan was evaluated, and the results reflected that PEI-g-chitosan had a lower cytotoxicity than PEI (25 K). Gene transfection efficiency of PEI-g-chitosan in HepG2, HeLa, and primary hepatocytes cells and after administration in the common bile duct of rat liver was determined. Remarkably, PEI-g-chitosan showed a higher transfection efficiency than that of PEI (25 K) both in vitro and in vivo. The systematic distribution and the distribution in liver of the gene expression of the complexes of PEI-chitosan/DNA were determined as well.     

Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions

BACKGROUND: Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion 1. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions. METHODS: PEG (Mw: 5 kDa) was grafted onto chitosan (Mw: 47 kDa, deacetylation degree: 94%) with grafting degrees of 3.6% and 9.6% (molar percentage of chitosan monosaccharide units grafted with PEG). The stability of chitosan-g-PEG/DNA complexes was studied by measuring the change in particle size and by agarose gel electrophoresis against bile or serum challenge. The influence of PEG grafting on gene transfection efficiency was evaluated in HepG2 cells using luciferase reporter gene. Chitosan and chitosan-g-PEG/DNA complexes were delivered to the liver through bile duct and portal vein infusions with a syringe pump. Gene expression in the liver and the distribution of gene expression in other organs were evaluated. The acute liver toxicity of chitosan and chitosan-g-PEG/DNA complexes was examined by measuring serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST) activities as a function of time. RESULTS: Both chitosan and chitosan-g-PEG displayed comparable gene transfection efficiency in HepG2 cells. After challenge with serum and bile, chitosan-g-PEG/DNA complexes, especially those prepared with chitosan-g-PEG (GD = 9.6%), did not form large aggregates like chitosan/DNA complexes but remained stable for up to 30 min. In addition, chitosan-g-PEG prevented the degradation of DNA in the presence of serum and bile. On day 3 after bile duct infusion, chitosan-g-PEG (GD = 9.6%)/DNA complexes mediated three times higher gene expression in the liver than chitosan/DNA complexes and yielded background levels of gene expression in other organs. On day 1 following portal vein infusion, gene expression level induced by chitosan/DNA complexes was hardly detectable but chitosan-g-PEG (GD = 9.6%) mediated significant transgene expression. Interestingly, transgene expression by chitosan-g-PEG/DNA complexes in other organs after portal vein infusion increased with increasing grafting degree of PEG. The ALT and AST assays indicated that grafting of PEG to chitosan reduced the acute liver toxicity towards the complexes. CONCLUSION: This study demonstrated the potential of chitosan-g-PEG as a safe and more stable gene carrier to the liver.     

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (&lt; 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 10⁶ cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/10⁶ transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

     

Development of novel poly(ethylene glycol)-based vehicles for gene delivery

The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.     

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.     

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.     

High cell density transient transfection of CHO cells for TGF‐β1 expression

High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A linear correlation between fluorescence intensity and transfection efficiency was improved. The transfection efficiency of PEI was highly dependent on the transfection conditions and directly related to the level of recombinant protein. Several factors were required to optimize the transient transfection process; these factors included the media type (which is compatible with low or high cell density transfection), the preculture CHO‐K1 suspension cell density, and the pDNA to PEI level. Based on design of experiment (DoE) analyses, the optimal transfection conditions for 10 × 10⁶ cells/mL in the CHOMACS CD medium achieved 73% transfection efficiency and a cell viability of over 80%. These results were confirmed for the production of transforming growth factor‐beta 1 (TGF‐β1) in a shake flask. The purified TGF‐β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.