Development of a nonviral gene delivery vehicle for systemic application

Polycation vehicles used for in vitro gene delivery require alteration for successful application in vivo. Modification of polycations by direct grafting of additional components, e.g., poly(ethylene glycol) (PEG), either before or after DNA complexation, tend to interfere with polymer/DNA binding interactions; this is a particular problem for short polycations such as linear, beta-cyclodextrin-containing polycations (betaCDPs). Here, a new method of betaCDP polyplex (polycation/DNA composite structures) modification is presented that exploits the ability to form inclusion complexes between cyclodextrins and adamantane. Surface-PEGylated betaCDP polyplexes are formed by self-assembly of the polyplexes with adamantane-PEG conjugates. While unmodified polyplexes rapidly aggregate and precipitate in salt solutions, the PEGylated betaCDP polyplexes are stable at conditions of physiological salt concentration. Addition of targeting ligands to the adamantane-PEG conjugates allows for receptor-mediated delivery; galactosylated betaCDP-based particles reveal selective targeting to hepatocytes via the asialoglycoprotein receptor. Galactosylated particles transfect hepatoma cells with 10-fold higher efficiency than glucosylated particles (control), but show no preferential transfection in a cell line lacking the asialoglycoprotein receptor. Thus, surface modification of betaCDP-based polyplexes through the use of cyclodextrin/adamantane host/guest interactions endows the particles with properties appropriate for systemic application.     

Linear cationic click polymers/DNA nanoparticles: in vitro structure-activity relationship and in vivo evaluation for gene delivery

The aim of this work was to explore the structure--activity relationships (SAR) of a series of novel linear cationic click polymers with various structures for in vitro gene delivery and in vivo gene transfer. The experimental results revealed that the minimal structure variation could result in a crucial effect on DNA-binding ability, buffering capacity, and the cellular delivery capacity of polymer, all of which brought about the obvious effects on their transfection efficiencies. The polymer synthesized from diazide monomer containing bis-ethylenediamine unit and dialykene monomer containing bis-ethylene glycol unit (B(2)) could effectively condense DNA into complex nanoparticles (B(2)Ns), which showed the highest in vitro transfection efficiency. The biodistribution and transfection efficiency of B(2)Ns in nude mice bearing tumor demonstrated the ability of effectively delivering DNA into tumor tissue. These results implied that this gene vector based on linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.     

Synthesis and characterization of poly (amino ester) for slow biodegradable gene delivery vector

Many therapeutic carrier materials were exploited for human gene therapy from viral to polymeric vectors. This research describes the evaluation of two biodegradable ester-bonded polymers synthesized by double-monomer polycondensation for a non-viral cationic polymer-based gene delivery system. The backbone was constructed to include inner tertiary amines and outer primary amines. Self-assembly with DNA resulted in the production of regularly nano-sized spherical polyplexes with good transfection efficiency, especially in the presence of serum. The polymers showed a relatively slow degradability for an amine-containing ester polymer, as they maintained DNA/polymer complex for 7 days in physiological buffer conditions. Finally, the low toxicity and slow degradation concluded these polymers reliable for long-term therapeutic applications.     

Synthesis of linear polyethylenimine derivatives for DNA transfection

A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.     

Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes ("polyplexes") from the endosome. A possible way to enhance endosomal escape is the use of cationic polymers with a pKa around or slightly below physiological pH ("proton sponge"). We synthesized a new polymer with two tertiary amine groups in each monomeric unit [poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated as pDAMA]. One pKa of the monomer is approximately 9, providing cationic charge at physiological pH, and thus DNA binding properties, the other is approximately 5 and provides endosomal buffering capacity. Using dynamic light scattering and zeta potential measurements, it was shown that pDAMA is able to condense DNA in small particles with a surface charge depending on the polymer/DNA ratio. pDAMA has a substantial lower toxicity than other polymeric transfectants, but in vitro, the transfection activity of the pDAMA-based polyplexes was very low. The addition of a membrane disruptive peptide to pDAMA-based polyplexes considerably increased the transfection efficiency without adversely affecting the cytotoxicity of the system. This indicates that the pDAMA-based polyplexes alone are not able to mediate escape from the endosomes via the proton sponge mechanism. Our observations imply that the proton sponge hypothesis is not generally applicable for polymers with buffering capacity at low pH and gives rise to a reconsideration of this hypothesis.     

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.     

Cationic amphiphilic star and linear block copolymers: synthesis, self-assembly, and in vitro gene transfection

A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.     

Chiral cationic polyamines for chiral microcapsules and siRNA delivery

Reported herein is the use of chiral cationic polyamines for two intriguing applications: fabrication of chiral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. The microcapsules are easily fabricated from homochiral polymers, and the resulting architectures can be used for supramolecular chiral catalysis and many other potential applications. Enantiospecific delivery of siRNA to Huh 7 cells is seen by one ‘enantiomer’ of the polymers delivering siRNA with significantly improved transfection efficiency and reduced toxicity compared to the ‘enantiomeric’ polymer and commercially available transfection reagents. Taken together, the use of these easily accessible polyamine structures for diverse applications is highlighted in this Letter herein and can lead to numerous future research efforts.     

Membrane activity and transfection ability of amphipathic polycations as a function of alkyl group size

Cationic membrane disruptive peptides such as melittin would appear to have attributes necessary for DNA delivery: DNA binding via electrostatic interactions and membrane lysis to enable cytoplasmic delivery. However, the relatively small overall charge of membrane disruptive peptides results in weak interactions with DNA. As a model of cationic membrane disruptive peptides, amphiphilic polyvinyl ethers were synthesized. The number of positively charged groups incorporated into these polymers is substantially greater than membrane-active peptides, which enables these polymers to form stable complexes with DNA. By varying the length of the hydrophobic groups incorporated into the polymer from one to four carbons, the dependence of membrane activity on side chain length was established. The ability of these polymers to transfect DNA in tissue culture was tested, and it was found that transfection efficiency is dependent upon the membrane disruptive activity of the polymer. Comparison of melittin and synthetic polymers suggests that transfection and toxicity appear to be dependent upon their affinity for DNA. This demonstration of relationships among membrane lysis, transfection, DNA binding, and polymer side-chain composition establishes a new class of transfection reagents and may guide in the design of polymers and formulations that will enable efficient in vivo transfection.     

Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection

Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer‐based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra‐ and extracellular barriers. The recent progress has shown that stimuli‐responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA‐polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 363–375, 2015.     

Biodegradable poly(vinyl alcohol)-polyethylenimine nanocomposites for enhanced gene expression in vitro and in vivo

Use of cationic polymers as nonviral gene vectors has several limitations such as low transfection efficiency, high toxicity, and inactivation by serum. In this study, varying amounts of low molecular weight branched polyethylenimine 1.8 kDa (bPEI 1.8) were introduced on to a neutral polymer, poly(vinyl alcohol) (PVA), to bring in cationic charge on the resulting PVA-PEI (PP) nanocomposites. We rationalized that by introducing bPEI 1.8, buffering and condensation properties of the proposed nanocomposites would result in improved gene transfer capability. A series of PVA-PEI (PP) nanocomposites was synthesized using well-established epoxide chemistry and characterized by IR and NMR. Particle size of the PP/DNA complexes ranged between 120 to 135 nm, as determined by dynamic light scattering (DLS), and DNA retardation assay revealed efficient binding capability of PP nanocomposites to negatively charged nucleic acids. In vitro transfection of PP/DNA complexes in HEK293, HeLa, and CHO cells revealed that the best working formulation in the synthesized series, PP-3/DNA complex, displayed ~2-50-fold higher transfection efficiency than bPEIs (1.8 and 25 kDa) and commercial transfection reagents. More importantly, the PP/DNA complexes were stable over a period of time, along with their superior transfection efficiency in the presence of serum compared to serum-free conditions, retaining the nontoxic property of low molecular weight bPEI. The in vivo administration of PP-3/DNA complex in Balb/c mice showed maximum gene expression in their spleen. The study demonstrates the potential of PP nanocomposites as promising nonviral gene vectors for in vivo applications.     

Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3)-fold and 1.5±0.7×10(3)-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p?=?0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.     

Low charge density cationic polymers for gene delivery: exploring the influence of structural elements on in vitro transfection

A series of end-functionalized poly(trimethylene carbonate) DNA carriers, characterized by low cationic charge density and pronounced hydrophobicity, is used to study structural effects on in vitro gene delivery. As the DNA-binding moieties are identical in all polymer structures, the differences observed between the different polymers are directly related to the functionality and length of the polymer backbone. The transfection efficiency and cytotoxicity of the polymer/DNA complexes are thus found to be dependent on a combination of polymer charge density and functionality, highlighting the importance of such structural considerations in the development of materials for efficient gene delivery.     

Poly(2-aminoethyl methacrylate) with well-defined chain length for DNA vaccine delivery to dendritic cells

Poly(2-aminoethyl methacrylate) (PAEM) homopolymers with defined chain length and narrow molecular weight distribution were synthesized using atom transfer radical polymerization (ATRP), and a comprehensive study was conducted to evaluate the colloidal properties of PAEM/plasmid DNA polyplexes, the uptake and subcellular trafficking of polyplexes in antigen-presenting dendritic cells (DCs), and the biological performance of PAEM as a potential DNA vaccine carrier. PAEM of different chain length (45, 75, and 150 repeating units) showed varying strength in condensing plasmid DNA into narrowly dispersed nanoparticles with very low cytotoxicity. Longer polymer chain length resulted in higher levels of overall cellular uptake and nuclear uptake of plasmid DNA, but shorter polymer chains favored intracellular and intranuclear release of free plasmid from the polyplexes. Despite its simple chemical structure, PAEM transfected DCs very efficiently in vitro in media with or without serum and led to phenotypic maturation of DCs. When a model antigen-encoding ovalbumin plasmid was used, transfected DCs stimulated the activation of na?ve CD8(+) T cells to produce high levels of interferon-γ. The efficiency of transfection, DC maturation, and CD8(+) T cell activation showed varying degrees of polymer chain-length dependence. These structurally defined cationic polymers may have much potential as efficient DNA vaccine carriers and immunostimulatory adjuvants. They may also serve as a model material system for elucidating structural and intracellular mechanisms of polymer-mediated DNA vaccine delivery.     

Synthesis, characterization, and evaluation as transfection reagents of ampholytic star copolymers: effect of star architecture

Five star polymers based on the positively ionizable hydrophilic 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the hydrophobic but hydrolyzable tetrahydropyranyl methacrylate (THPMA) were prepared by group-transfer polymerization (GTP) using ethylene glycol dimethacrylate (EGDMA) as the coupling agent. In particular, four isomeric star copolymers (one heteroarm, two star block, and the statistical star), all with a 3:1 DMAEMA:THPMA molar ratio, plus one star homopolymer of DMAEMA, with degrees of polymerization of the arms equal to 15, were synthesized. After star polymer preparation and preliminary characterization, the THPMA units were hydrolyzed to negatively ionizable hydrophilic methacrylic acid (MAA) untis, thus yielding star polyampholytes. All the star polyampholytes as well as the commercially available transfection reagent SuperFect were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 bearing the enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was affected by star architecture. The DMAEMA15-star-MAA5 polyampholyte presented the highest transfection efficiency of all the star polymers tested but lower than that of SuperFect at its optimum conditions. All four star copolymers showed decreased toxicity compared to the DMAEMA star homopolymer for the same amounts of star polymer tested and also compared to the SuperFect at its optimum conditions.     

Synthesis and in vitro evaluation of glutamide-containing cationic lipids for gene delivery

A novel series of cationic amphiphiles based on dialkyl glutamides with cationic pyridinium head group were synthesized as potential gene delivery agents. Four cationic lipids with glutamide as linker and varying chain lengths were tested for their transfection efficiency in three cell lines. The DNA-lipid complexes were characterized for their ability to bind to DNA, protection from nuclease digestion, size, zeta-potential, and toxicity. All four lipids demonstrated efficient transfection in MCF-7, COS, and HeLa cells, and the reporter gene expression was much higher with DOPE as the helper lipid in the formulation when compared to cholesterol. Among these 14-carbon lipids, lipid 2 has shown the highest transfection efficiency, complete protection of DNA from nuclease digestion, and low toxicity. Interestingly, lipid 2 has also shown remarkable enhancement in transfection in the presence of serum.     

A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine

This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.     

New class of polymers for the delivery of macromolecular therapeutics

Cationic polymers show promise for the in vitro and in vivo delivery of macromolecular therapeutics. Known cationic polymers, e.g., poly(L)lysine (PLL) and polyethylenimine (PEI), have been employed in native and modified forms for the delivery of plasmid DNA (pDNA) and reveal varying levels of toxicity. Here, we report the preparation of a new class of cationic polymers that are specifically designed to deliver macromolecular therapeutics. Linear, cationic, beta-cyclodextrin (beta-CD)-containing polymers (CD-polymers) are synthesized by copolymerizing difunctionalized beta-CD monomers (AA) with other difunctionalized comonomers (BB) such that an AABBAABB product is formed. The beta-CD polymers are able to bind approximately 5 kbp pDNA above polymer to DNA (+/-) charge ratios of 1.5, compact the bound pDNA into particles of approximately 100-150 nm in size at charge ratios above 5+/-, and transfect cultured cells at charge ratios above 10+/-. In vitro transfections with the new beta-CD-polymers are comparable to the best results obtained in our hands with PEI and Lipofectamine. Some cell line-dependent toxicities are observed for serum-free transfections; however, no toxicity is revealed at charge ratios as high as 70+/- in transfections conducted in 10% serum. Single IV and IP doses as high as 200 mg/kg in mice showed no mortalities.     

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.