Recognition and selective binding of DNA by ionenes of different charge density

The ability of aliphatic ionenes to recognize and bind DNA or poly(methacrylic acid) (PMA) in the equimolar mixture of these polyanions was studied by fluorescence quenching technique. Within a particular system, the selectivity of competitive interactions was shown to be determined by a component with the lowest degree of polymerization (DP). Ionene polycations with lowest DP values did not exhibit pronounced selectivity in binding DNA or PMA with higher values of DP. Increase in ionene DP resulted in a steady increase in selectivity of interaction and ultimately in almost exclusive binding of one of the two polyanions. The ability of the ionene to recognize and bind DNA in the mixture of polyanions was shown not to correlate with the affinity of the ionene to DNA in their binary mixture. Although ionenes with a higher charge density exhibited preferential binding to PMA, the ionenes with the lowest charge density selectively bound DNA.     

Development of improved nanoparticulate polyelectrolyte complex physicochemistry by nonstoichiometric mixing of polyions with similar molecular weights

Water-based, biodegradable polyelectrolyte complex dispersions (PECs) prepared by mixing oppositely charged polyions are advantageous drug delivery systems due to constituent biocompatibility and nanoparticulate architectures. Reaction phase environmental parameters dictate PEC physicochemical properties, and specifically, complexation between polyelectrolytes having significantly different molecular weights leads to formation of water-insoluble aggregates. Starting with this fact, four-component similar and dissimilar molecular weight PEC chemistries were applied and compared with and without frequency-induced dispergation. The goal was to define nanoparticulate PEC systems with desirable characteristics for use in biological systems. Results show PEC formulations from precursors with similar low molecular weights yielded dispersions with suitable physicochemical characteristics, as verified by photon correlation spectroscopy and TEM, presumably due to efficient ion pairing. Similar low molecular weight PECs fabricated with dispergation exhibited pH-independent stability, as validated by charge and size measurements. These physicochemical advantages lead to an ideal delivery platform.     

Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.     

Structural analysis of chitosan mediated DNA condensation by AFM: influence of chitosan molecular parameters

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (F(A)) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing F(A) of the chitosan, indicating that the charge density of chitosan, proportional to (1 - F(A)), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles.     

Charge density and molecular weight of polyphosphoramidate gene carrier are key parameters influencing its DNA compaction ability and transfection efficiency

A series of polyphosphoramidates (PPAs) with different molecular weights (MWs) and charge densities were synthesized and examined for their DNA compaction ability and transfection efficiency. A strong correlation was observed between the transfection efficiency of PPA/DNA nanoparticles and the MW and net positive charge density of the PPA gene carriers in three different cell lines (HeLa, HEK293, and HepG2 cells). An increase in MW and net positive charge density of PPA carrier yielded higher DNA compaction capacity, smaller nanoparticles with higher surface charges, and higher complex stability against challenges by salt and polyanions. These favorable physicochemical properties of nanoparticles led to enhanced transfection efficiency. PPA/DNA nanoparticles with the highest complex stability showed comparable transfection efficiency as PEI/DNA nanoparticles likely by compensating the low buffering capacity with higher cellular uptake and affording higher level of protection to DNA in endolysosomal compartment. The differences in transfection efficiency were not attributed by any difference in cytotoxicity among the carriers, as all nanoparticles showed a minimal level of cytotoxicity under the transfection conditions. Using PPA as a model system, we demonstrated the structural dependence of transfection efficiency of polymer gene carrier. These results offer more insights into nanoparticle engineering for nonviral gene delivery.     

Quaternized poly(4-vinylpyridine)s as model gene delivery polycations: structure-function study by modification of side chain hydrophobicity and degree of alkylation

To optimize polycation-based gene delivery agents, the influence of molecular characteristics of the polycations on physicochemical properties of polycation/DNA complexes and their relationships to cellular gene transfer need to be understood. With this aim, we have synthesized a series of model polymers based on quaternized poly(4-vinylpyridine)s (CnPVP-beta) with the same DP of 1600 but differing by the number n of methylene groups in N-alkyl ester substituents from 1 to 6 and/or by degree of alkylation beta from 25% to 95%. For polycations CnPVP-95, the efficiency of transfection of a plasmid vector expressing a secreted form of alkaline phosphatase started to be detectable at n = 4, noticeable at n = 5, and again undetectable at n = 6. A decrease in beta of active C5PVP-95 from 95% to 65% resulted in a further noticeable increase of activity with a 9-fold increase for C5PVP-65. This finding was attributed to the proton sponge mechanism due to protonation of non-alkylated pyridine moieties of CnPVP-beta/DNA complexes in slightly acidic media that was supported by the fluorescence quenching assay. The data demonstrate the advantages of partial alkylation of tertiary polyamines with medium-length alkyl agents for preparation of efficient nonviral gene delivery vectors.     

Controllable stability of DNA-containing polyelectrolyte complexes in water-salt solutions

Destruction of polyelectrolyte complexes (PECs) formed by DNA and synthetic polyamines of different structures was carried out by addition of low molecular weight electrolyte to PEC solution at different pHs. The dissociation was studied by the fluorescence quenching technique using the ability of cationic dye ethidium bromide to intercalate into free sites of DNA double helix followed by ignition of ethidium fluorescence. Structure of amine groups of the polycation was shown to be a decisive factor of PEC stability. PECs formed by polycations with quaternary amine groups, i.e., poly(N-alkyl-4-vinylpyridinium) bromides, poly(N, N-dimethyldiallylammonium) chloride, and ionene bromide, were pH independent and the least tolerant to destruction by the added salt. Primary amine groups of basic polypeptides poly-L-lysine hydrobromide and poly-L-arginine hydrochloride as well as synthetic polycation poly(vinyl-2-aminoethyl ether) provided the best stability of PECs in water-salt solutions under wide pH range. Moderate and pH-dependent stability was revealed for PECs included poly(N,N-dimethylaminoethylmethacrylate) with tertiary amine groups in the chain or branched poly(ethylenimine) with primary, secondary, and tertiary amine groups in the molecule. The data obtained appear to be the basis for design of DNA-containing PECs with given and controllable stability. The design may be accomplished not only by proper choice of polyamine of one or another type, but by using of tailor-made polycations with given composition of amine groups of different structure in the chain as well. Thus, quaternization of a part of tertiary amine groups of poly(N, N-dimethylaminoethylmethacrylate) resulted in expected decrease of stability of DNA-containing PECs in water-salt solutions. The destruction of PEC formed by random copolymer of 4-vinylpyridine and N-ethyl-4-vinylpyridinium bromide was pH sensitive and could be performed under pH and ionic strength closed to the physiological conditions. This result appears to be particularly promising for addressing DNA packed in PEC species to the target cell.     

Structural effects of carbohydrate-containing polycations on gene delivery. 2. Charge center type

Recent polycation structure-gene delivery studies reveal that subtle changes in the molecular structure of polycations have substantial influences on DNA-binding and condensation and on in vitro toxicity and gene delivery efficiency. In Part 1 of this structure-property study using carbohydrate-containing polycations (1), it is demonstrated that as the amidine charge center is removed further from the carbohydrate unit within the polycation structure, the toxicity increases. Inclusion of larger carbohydrate species within the polycation backbone also reduces the toxicity. Here, the effect that polycation charge center type has on toxicity and gene delivery efficiency is investigated. A series of quaternary ammonium polycations containing N,N,N',N'-tetramethyl-1,6-hexanediamine, d-trehalose, and beta-cyclodextrin are synthesized in order to elucidate the effects of charge center type (by comparison to the data given in Part 1) on gene delivery. In all cases, it is found that the quaternary ammonium analogues exhibit lower gene expression values and similar toxicities to their amidine analogues. Additionally, transfection experiments conducted in the presence of chloroquine reveal increased gene expression from quaternary ammonium containing polycations and not from their amidine analogues.     

Structural effects of carbohydrate-containing polycations on gene delivery. 3. Cyclodextrin type and functionalization

Linear cationic beta-cyclodextrin (beta-CD)-based polymers can form polyplexes with plasmid DNA and transfect cultured cells. The effectiveness of the gene delivery and the cellular toxicity has been related to structural features in these polycations. Previous beta-CD polycations were prepared from the cocondensation of 6(A),6(D)-dideoxy-6(A),6(D)-diamino-beta-CD monomers with other difunctionalized monomers such as dimethyl suberimidate (DMS). Here, the type of CD and its functionalization are varied by synthesizing numerous 3(A),3(B)-dideoxy-3(A),3(B)-diamino-beta- and gamma-CD monomers. Both alkyl- and alkoxydiamines are prepared in order to vary the nature of the spacing between the CD and the primary amines in the monomers. These diamino-CD-monomers are polymerized with DMS to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When these polycations are used to form polyplexes with plasmid DNA, longer alkyl regions between the CD and the charge centers in the polycation backbone increase transfection efficiency and toxicity in BHK-21 cells, while increasing hydrophilicity of the spacer (alkoxy versus alkyl) provides for lower toxicity. Further, gamma-CD-based polycations are shown to be less toxic than otherwise identical beta-CD-based polycations.     

Polycations as prostaglandin sythesis inducers. II. structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (>80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. Full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Polycations as prostaglandin synthesis inducers. II. Structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (greater than 80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Gene carriers based on hexanediol diacrylate linked oligoethylenimine: effect of chemical structure of polymer on biological properties

Two biodegradable polycations based on hexanediol diacrylate linked oligoethylenimine (OEI) were synthesized by applying different reaction temperatures, 20 degrees C (LT-OEI-HD) and 60 degrees C (HT-OEI-HD). Their structural properties were analyzed by NMR, FTIR, and SEC/MALLS (size exclusion chromatography coupled with multiangle laser light scattering detection). Reaction temperature strongly influenced molecular weight and ester/amide ratio and thus resulted in polycations with different biological activities and degradation profiles. LT-OEI-HD was an ester-based polycation of 8.7 kDa which degraded rapidly at pH 7 and pH 9 respectively. HT-OEI-HD had a molecular weight of 26.6 kDa, was mainly based on amides, and degraded more slowly than LT-OEI-HD. Both polymers mediated gene transfer as efficiently as linear polyethylenimine of 22 kDa in two cell lines while being less toxic at their optimal conjugate/plasmid (C/P) ratios. LT-OEI-HD needed higher C/P ratios for gene delivery; however, it was significantly less toxic than HT-OEI-HD.     

Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.     

A nanoparticle-based model delivery system to guide the rational design of gene delivery to the liver. 1. Synthesis and characterization

Nonviral gene delivery systems are amenable to forming colloidal particles with a wide range of physicochemical properties that include size, surface charge, and density and type of ligand presented. However, it is not known how to best design these particles without having a set of physicochemical design constraints that have been optimized for the intended gene delivery application. Here, a nanoparticle-based model delivery system is developed that can mimic the surface properties of nonviral gene delivery particles, and this model system is used to define design constraints that should be applied to next generation gene delivery particles. As a test case, a well-defined nanoparticle-based system is developed to guide the rational design of gene delivery to hepatocytes in the liver. The synthetic scheme utilizes monodisperse polystyrene particles and provides for variation of mean particle size and particle size distribution through variation in reaction conditions. The nanoparticles are PEGylated to provide stability in serum and also incorporate targeting ligands, e.g., galactose, at tunable densities. Four nanoparticles are synthesized from uniformly sized polystyrene beads specifically for the purpose of identifying design constraints to guide next generation gene delivery to the liver. These four nanoparticles are Gal-50 and Gal-140, that are galactosylated 50 and 140 nm nanoparticles, and MeO-50 and MeO-140, that are methoxy-terminated 50 and 140 nm nanoparticles. All four particles have the same surface charge, and Gal-50 and Gal-140 have the same surface galactose density. The availability of galactose ligands to receptor binding is demonstrated here by agglutination with RCA120.     

Powder and mechanical properties of microcrystalline cellulose with different degrees of polymerization

This study investigated the influence of the degree of polymerization (DP) of cellulose materials (microcrystalline cellulose [MCC]) on some powder properties and the compression behavior of these materials. The DP was determined by measurements of viscosity (H). The weight average of molecular weight and the weight average of the different DPs were investigated after MCC was modified to cellulose tricarbanilate by light scattering measurements. The DP showed a remarkable influence on the physicochemical properties of the cellulose materials and, consequently, on the behavior of these materials during compression. MCC types with a high DP value showed greater water absorption than the types with a low DP value. No relevant relationship between the crystallinity index and the DP could be observed. DP 190 showed lower compactibility and compressibility parameters than DP 244 and 299. No significant differences could be observed between DP 244 and 299 when the same particle size fraction was compressed. Furthermore, the compressibility was increased by increasing the DP.     

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.     

DNA-polycation complexes: effect of polycation structure on physico-chemical and biological properties

The purpose of the study was to investigate the influence of cationic polymer structure on the formation of DNA-polycation complexes and their transfection activity. Primary, tertiary, and quaternary polyamines with molecular masses ranging from 8000 to 200,000 were investigated. DNA-cationic polymer interaction was characterized by low gradient viscometry, dynamic light scattering, circular dichroism, UV spectrometry, flow birefringence, DNA electrophoresis, and electron microscopy. Transfection activity of the complexes was evaluated by the expression of reporter gene (beta-galactosidase) and using synthetic FITC-labelled oligonucleotides. Complex formation was found to be dependent on the structure and molecular weight of the polymer and the ionic strength of the solution. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. Cell lines of different origin were used for testing of transfection activity of the complexes. The sensitivity of the cells to transfection was established to be highly dependent on the cell line. DNA-polycation complexes are non-toxic according to MTT. Polyallylamine, and polydimethylaminoethylmethacrylate were found to be the most promising polycations for gene delivery. Transfection efficacy of their complexes with DNA to T-98G cells reaches up to 90-100%. It was found that optimal molecular mass of polydimethylaminoethylmethacrylate is in the range of 8000-50,000 Da.     

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.     

Poly (beta-amino ester) as an in vivo nanocarrier for therapeutic nucleic acids

Therapeutic nucleic acids are an emerging class of therapy for treating various diseases through immunomodulation, protein replacement, gene editing, and genetic engineering. However, they need a vector to effectively and safely reach the target cells. Most gene and cell therapies rely on ex vivo gene delivery, which is laborious, time-consuming, and costly; therefore, devising a systematic vector for effective and safe in vivo delivery of therapeutic nucleic acids is required to target the cells of interest in an efficient manner. Synthetic nanoparticle vector poly beta amino ester (PBAE), a class of degradable polymer, is a promising candidate for in vivo gene delivery. PBAE is considered the most potent in vivo vector due to its excellent transfection performance and biodegradability. PBAE nanoparticles showed tunable charge density, diverse structural characteristics, excellent encapsulation capacity, high stability, stimuli-responsive release, site-specific delivery, potent binding to nucleic acids, flexible binding ability to various conjugates, and effective endosomal escape. These unique properties of PBAE are an essential contribution to in vivo gene delivery. The current review discusses each of the components used for PBAE synthesis and the impact of various environmental and physicochemical factors of the body on PBAE nanocarrier.