Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

Biotin-triggered release of poly(ethylene glycol)-avidin from biotinylated polyethylenimine enhances in vitro gene expression

Covalently poly(ethylene glycol) (PEG)-ylated polyethylenimine (PEI)/pDNA complexes display prolonged blood circulation profiles compared with PEI/pDNA complexes, but such PEGylated particles may not be suitable for tumor targeting due to low interaction with cell membranes, low internalization, and low gene expression. Noncovalent PEGylation of cationic particles via PEG-avidin/biotin-PEI is an attempt to bridge the gap between the positive attributes of PEG (prolonged particle circulation) and the positive attributes of nontoxic cationic polymers (enhanced cell interactions) for greater gene expression. Our polymer, 2PEG-avidin/biotin-PEI8, forms salt-stable particles ( approximately 100 nm) under physiologic conditions with a minimum of two 2PEG-avidin molecules bound per polymer chain (biotin-PEI8, 8 biotins/PEI). Following 10 days of incubation with 3000-fold excess biotin, 2PEG-avidin completely dissociated from biotin-PEI8, and gene expression was increased 2.1-32-fold in various cell lines when the desirable transfection feature of the cationic polymer was retained. This new PEGylation approach has implications for generally improving the clinical aspect of gene delivery via a two-step therapeutic strategy: (1) intravenous injection of noncovalent PEG-avidin/biotin-polycation nanoparticles for prolonged circulation, followed by (2) temporal release of PEG-avidin from biotin-polycation through either endogenous biotin or intravenous injection of biotin.     

Gene delivery properties of end-modified poly(beta-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.     

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.     

Hyperbranched poly(amino ester)s with different terminal amine groups for DNA delivery

Hyperbranched poly(amino ester)s containing tertiary amines in the core and primary, secondary, and tertiary amines in the periphery, respectively, were evaluated for DNA delivery in vitro. The same core structure facilitated the investigation on the effects of the terminal amine type on the properties of hyperbranched poly(amino ester)s for DNA delivery. The hydrolysis of the poly(amino ester)s was monitored using (1)H NMR. The results reflected that the terminal amine type had negligible effects on the hydrolysis rate but was much slower than that of linear poly(amino ester)s, probably due to the compact hyperbranched spatial structure preventing the accessibility of water. In comparison with PEI 25 K, the hyperbranched poly(amino ester)s showed much lower cytotoxicity in Cos7, HEK293, and HepG2 cells. Gel electrophoresis indicated that poly(amino ester)s could condense DNA efficiently, and the zeta potentials and sizes of the complexes formed with different weight ratios of hyperbranched poly(amino ester)s and DNA were measured. Remarkably, all the hyperbranched poly(amino ester)s showed DNA transfection efficiency comparable to PEI 25 K in Cos7, HEK293, and HepG2 cells regardless of the terminal amine type. Therefore, the terminal amine type had insignificant effects on the hydrolysis rate, cytotoxicity, DNA condensation capability, and in vitro DNA transfection efficiency of the hyperbranched poly(amino ester)s.     

PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice

The influence of PEGylation on polyplex stability from poly(ethylene imine), PEI, and plasmid DNA was investigated both in vitro and after intravenous administration in mice. Polyplexes were characterized with respect to particle size (dynamic light scattering), zeta-potential (laser Doppler anemometry), and morphology (atomic force microscopy). Pharmacokinetics and organ accumulation of both polymers and pDNA were investigated using 125I and 32P radioactive labels, respectively. Furthermore gene expression patterns after 48 h were measured in mice. To elucidate the effect of different doses, all experiments were performed using ca. 1.5 microg and 25 microg of pDNA per mouse. Our studies demonstrated that both PEI and PEG-PEI form stable polyplexes with DNA with similar sizes of 100-130 nm. The zeta potential of PEI/pDNA polyplexes was highly positive, whereas PEG-PEI/pDNA showed a neutral surface charge as expected. The pharmacokinetic and organ distribution profiles after 2 h show similarities for both PEI and pDNA blood-level time curves from polyplexes at both doses indicative for significant stability in the bloodstream. A very rapid clearance from the bloodstream was observed and as major organs of accumulation liver and spleen were identified. PEG-PEI/pDNA complexes at a dose of approximately 25 microg exhibit similar profiles except a significantly lower deposition in the lung. At the lower dose of approximately 1.5 microg pDNA, however, for polyplexes from PEG-PEI, significant differences in blood level curves and organ accumulation of polymer and pDNA were found. In this case PEG-PEI shows a greatly enhanced circulation time in the bloodstream. By contrast, pDNA was rapidly cleared from circulation and significant amounts of radioactivity were found in the urine, suggesting a rapid degradation possibly by serum nucleases after complex separation. Regarding in vivo gene expression, no luciferase expression could be detected at approximately 1.5 microg dose in any organ using both types of complexes. At 25 microg only in the case of PEI/pDNA complexes were significant levels of the reporter gene detected in lung, liver, and spleen. This coincided with high initial accumulation of pDNA complexed with PEI and a high acute in vivo toxicity. For PEG-PEI, initial accumulation was much lower and no gene expression as well as a low acute toxicity was found. In summary, our data demonstrate that PEG-PEI used in this study is not suitable for low dose gene delivery. At a higher dose of approximately 25 microg, however, polyplex stability is similar to PEI/pDNA combined with a more favorable organ deposition and significantly lower acute in vivo toxicity. These findings have consequences for the design of PEG-PEI-based gene delivery systems for in vivo application.     

Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody

To achieve efficient systemic gene delivery to the lung with minimal toxicity, a vector was developed by chemically conjugating a cationic polymer, polyethylenimine (PEI), with anti-platelet endothelial cell adhesion molecule (PECAM) antibody (Ab). Transfection of mouse lung endothelial cells with a plasmid expression vector with cDNA to luciferase (pCMVL) complexed with anti-PECAM Ab-PEI conjugate was more efficient than that with PEI-pCMVL complexes. Furthermore, the anti-PECAM Ab-PEI conjugate mediated efficient transfection at lower charge plus-to-minus ratios. Conjugation of PEI with a control IgG (hamster IgG) did not enhance transfection of mouse lung endothelial cells, suggesting that the cellular uptake of anti-PECAM Ab-PEI-DNA complexes and subsequent gene expression were governed by a receptor-mediated process rather than by a nonspecific charge interaction. Conjugation of PEI with anti-PECAM Ab also led to significant improvement in lung gene transfer to intact mice after intravenous administration. The increase in lung transfection was associated with a decrease compared with PEI-pCMVL with respect to circulating proinflammatory cytokine (tumor necrosis factor-alpha) levels. These results indicate that targeted gene delivery to the lung endothelium is an effective strategy to enhance gene delivery to the pulmonary circulation while simultaneously reducing toxicity.     

Enzyme-linked immunosorbent assay of Escherichia coli O157:H7 in surface enhanced poly(methyl methacrylate) microchannels

A novel surface treatment method was developed to enhance polymer-based microchannel enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157:H7 detection. By applying an amine-bearing polymer, poly(ethyleneimine) (PEI), onto poly(methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups were introduced to PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that the alkali condition is preferable for PEI attachment onto the PMMA surface. The amine groups on the PMMA surface were then functionalized with glutaraldehyde, whose aldehyde groups served as the active sites for binding the antibody by forming covalent bonds with the amine groups of the protein molecules. This surface modification greatly improved antibody binding efficiency and the microchannel ELISA for E. coli O157:H7 detection. Compared with untreated PMMA microchannels, approximately 45 times higher signal and 3 times higher signal/noise ratio were achieved with the PEI surface treatment, which also shortened the time required for cells to bind to the microchannel surface to approximately 2 min, much less than that usually required for the same ELISA carried out in 96-well plates. The detection in the microchannel ELISA only required 5-8 cells per sample, which is also better than 15-30 cells required in multi-well plates. With the high sensitivity, short assay time, and small reagent consumption, the microchannel ELISA can be economically used for fast detection of E. coli O157:H7.     

Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery

Several families of synthetic polymers, including degradable poly(beta-amino ester)s, have been previously shown to effectively mediate gene transfer. However, the combined impact of potentially significant factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on different gene transfer properties has yet to be systematically investigated. The elucidation of these relationships may aid in the design of nonviral vectors with greatly enhanced transfection properties. To examine these factors, two distinct poly(beta-amino ester) structures, Poly-1 and Poly-2, were generated by adding 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively, to 1-aminobutanol. Twelve unique versions of each structure were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in polymers with either amine or acrylate end-groups and with molecular weights ranging from 3350 to 18000. Using high throughput methods, all polymers were tested in quadruplicate at nine different polymer/DNA ratios ranging from 10:1 w/w to 150:1 w/w. Through the optimization of molecular weight, polymer chain end-group, and polymer/DNA ratio, these polymers successfully mediated gene transfer at levels that surpassed both PEI and Lipofectamine 2000 in vitro.     

Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives

The continually increasing wealth of knowledge about the role of genes involved in acquired or hereditary diseases renders the delivery of regulatory genes or nucleic acids into affected cells a potentially promising strategy. Apart from viral vectors, non-viral gene delivery systems have recently received increasing interest, due to safety concerns associated with insertional mutagenesis of retro-viral vectors. Especially cationic polymers may be particularly attractive for the delivery of nucleic acids, since they allow a vast synthetic modification of their structure enabling the investigation of structure-function relationships. Successful clinical application of synthetic polycations for gene delivery will depend primarily on three factors, namely (1) an enhancement of the transfection efficiency, (2) a reduction in toxicity and (3) an ability of the vectors to overcome numerous biological barriers after systemic or local administration. Among the polycations presently used for gene delivery, poly(ethylene imine), PEI, takes a prominent position, due to its potential for endosomal escape. PEI as well as derivatives of PEI currently under investigation for DNA and RNA delivery will be discussed.This review focuses on structure-function relationships and the physicochemical aspects of polyplexes which influence basic characteristics, such as complex formation, stability or in vitro cytotoxicity, to provide a basis for their application under in vivo conditions. Rational design of optimized polycations is an objective for further research and may provide the basis for a successful cationic polymer-based gene delivery system in the future.     

Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked low-molecular-weight polyethylenimine as gene delivery carriers in cell culture and dystrophic mdx mice

Hyperbranched poly(ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) and low-molecular-weight polyethylenimine (LPEI, M(w) 0.8k, 1.2k, and 2.0k) and evaluated in vitro and in vivo as gene carriers. PEAs effectively condensed plasmid DNA with particle sizes below 200 nm and surface charges between 11.5 and 33.5 mV under tested doses [at the ratios 2-10:1 of polymer/pDNA(w/w)]. The PEAs showed significantly lower cytotoxicities when compared with PEI 25k in two different cell lines. The PEAs (C series) composed of PEI 2k showed higher transgene expression compared to PEAs of PEI 0.8k (A series) or 1.2k (B series). Highest gene transfection efficiency in CHO, C2C12 myoblast, and human skeletal muscle (HSK) cell lines was obtained with TAEI/PEI-2K (C12) at a ratio of 1:2. Both C12, C14(TAEI/PEI-2K at a ratio of 1:4) demonstrated 5-8-fold higher gene expression as compared with PEI 25k in mdx mice in vivo through intramuscular administration. No obvious muscle damage was observed with these new polymers. Higher transfection efficiency and lower toxicity indicate the potential of the biodegradable PEAs as safe and efficient transgene delivery vectors.     

Degradable poly(amino alcohol esters) as potential DNA vectors with low cytotoxicity

The synthesis of a new degradable polymer system, poly(amino alcohol esters) and the resulting polymers' potential for use in gene transfection vectors are reported. The polymerization proceeded in a one step reaction from commercially available bis(secondary amines) monomers (N,N'-dimethyl-1,3-propanediamine and N,N'-dimethyl-1,6-hexanediamine, respectively) through nucleophilic addition to the diglycidyl ester of dicarboxylic acid (diglycidyl adipate). Poly(amino alcohol ester) 1 and 2 were synthesized with a yield of 89% and 91% with Mn = 24,800 and Mn = 36,400, respectively. Poly(amino alcohol ester) 1 degraded hydrolytically in phosphate buffer at pH 7.4 with a half-life of approximately 5 days. Both polymers readily self-assembled with plasmid DNA into nanometer-sized DNA/polymer complexes less than 180 nm diameter and are significantly less cytotoxic than the commonly used DNA delivery polymer, poly(ethylene imine) (PEI).     

Enhanced gene expression through temperature profile-induced variations in molecular architecture of thermoresponsive polymer vectors

BACKGROUND: Successful non-viral gene targeting requires vectors to meet two conflicting needs-strong binding to protect the genetic material during transit and weak binding at the target site to enable release. Responsive polymers could fulfil such requirements through the switching of states, e.g. the chain-extended coil to chain-collapsed globule phase transition that occurs at a lower critical solution temperature (LCST), in order to transport nucleic acid in one polymer state and release it in another. METHODS: The ability of new synthetic polycations based on poly(ethyleneimine) (PEI) with grafted neutral responsive poly(N-isopropylacrylamide) (PNIPAm) chains to condense DNA into particles with architectures varying according to graft polymer LCST was assessed using a combination of fluorescence spectroscopy, dynamic light scattering (DLS), zeta sizing, gel retardation and atomic force microscopy studies. Transfection assays were conducted under experimental conditions wherein the polymer components were able to cycle across their LCST. RESULTS: Two PEI-PNIPAm conjugate polymers with different LCSTs displayed coil-globule transitions when complexed to plasmid DNA, leading to variations in molecular architecture as shown by changes in emission maxima of an environment-sensitive fluorophore attached to the PNIPAm chains. Gel retardation assays demonstrated differences in electrophoretic mobilities of polymer-DNA complexes with temperatures below and above polymer LCSTs. Atomic force micrographs showed changes in the structures of polymer-DNA complexes for a polymer undergoing a phase transition around body temperature but not for the polymer with LCST outside this range. Transfection experiments in C2C12 and COS-7 cells demonstrated that the highest expression of transgene occurred in an assay that involved a 'cold-shock' below polymer LCST during transfection. CONCLUSIONS: Designed changes in thermoresponsive polycation vector configuration via temperature-induced phase transitions enhanced transgene expression. The results indicate that changes in molecular architecture induced by a carefully chosen stimulus during intracellular trafficking can be used to enhance gene delivery.     

General structure-activity relationship for poly(glycoamidoamine)s: the effect of amine density on cytotoxicity and DNA delivery efficiency

Herein, two new series of poly(glycoamidoamine)s (branched and linear) have been synthesized by polycondensation. The polymer repeat units have been designed to contain D-glucaramide, meso-galactaramide, D-mannaramide, or L-tartaramide structures and five or six ethyleneamine units to investigate the amine density effects on the bioactivity as compared to a similar series of poly(glycoamidoamine)s previously described that contain four ethyleneamines. These delivery vehicles were created to examine the effects that the number of secondary amines in the polymer repeat unit and the polymer structure (branched and linear) have on plasmid DNA (pDNA) binding affinity, polyplex formation, cell viability, and gene expression in the absence and presence of serum in the culture medium. The results reveal that the new polymers with higher amine density in the repeat unit do not significantly enhance the transfection efficiency compared to that of previous models containing four ethyleneamines, but an increase in cytotoxicity is noticed. Linear polymers reveal higher pDNA neutralization efficacy, gene expression, and toxicity than the branched versions containing a similar chemical structure, which may be caused by a higher protonation of the amine groups. With these new vectors, some interesting trends emerged. The galactaramide and tartaramide analogues revealed higher delivery efficiency than the glucaramide and mannaramide structures. In addition, the branched and linear structures containing five ethyleneamines in the repeat unit formed polyplexes at higher N/P ratios, which had lower zeta potential and lower delivery efficacy than the analogues with six ethyleneamines, and also the linear structures generally revealed higher delivery efficiency and toxicity when compared to those of their branched analogues.     

Synthesis and characterization of the novel transfection reagent poly(amino ester glycol urethane)

Poly(ester urethane) (PEU) is a class of biodegradable polymer that has been applied as tissue-engineering scaffolds with minimum toxicity. Despite its unique biocompatibility, there have been no reports in modifying the PEU backbone to design a soluble, PEU-based DNA carrier. We have developed a method of incorporating tertiary amines and poly(ethylene glycol) (PEG) into PEU to synthesize a soluble poly(amino ester glycol urethane) (PaEGU) as a novel transfection reagent. Parallel to this, we have synthesized poly(amino ester) (PaE) and poly(amino ester urethane) (PaEU) as the control polymers. The test transfection reagent PaEGU and the control PaE were similar in their properties of being soluble and buffering pH in water and their capabilities of self-assembling with DNA and transfecting the target cells. Significantly, PaEGU exhibited faster hydrolysis kinetics than PaE, half-lives of 19 and 36 h for PaEGU and PaE, respectively, underlying PaEGU's unique property of low cytotoxicity. However, in comparison to PaEGU, the other control polymer, PaEU, was not readily dissolved in water, indicating the importance of PEG units in PaEGU in increasing polymer hydrophilicity. This study demonstrated a useful synthesis scheme for the PEU-based transfection reagent PaEGU. The combination of tertiary amine, ester, PEG, and urethane units in the polymer backbone constitutes a feasible approach for the future design of low-toxicity gene transfer vectors.     

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N′-cystamine-bis-acrylamide)/pDNA complexes

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)—25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N′-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.     

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.     

Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B'-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.