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.     

Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.     

Kinetically controlled cellular interactions of polymer-polymer and polymer-liposome nanohybrid systems

Although bioactive polymers such as cationic polymers have demonstrated potential as drug carriers and nonviral gene delivery vectors, high toxicity and uncontrolled, instantaneous cellular interactions of those vectors have hindered the successful implementation In Vivo. Fine control over the cellular interactions of a potential drug/gene delivery vector would be thus desirable. Herein, we have designed nanohybrid systems (100-150 nm in diameter) that combine the polycations with protective outer layers consisting of biodegradable polymeric nanoparticles (NPs) or liposomes. A commonly used polycation polyethylenimine (PEI) was employed after conjugation with rhodamine (RITC). The PEI-RITC conjugates were then encapsulated into (i) polymeric NPs made of either poly(lactide-co-glycolide) (PLGA) or poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA); or (ii) PEGylated liposomes, resulting in three nanohybrid systems. Through the nanohybridization, both cellular uptake and cytotoxicity of the nanohybrids were kinetically controlled. The cytotoxicity assay using MCF-7 cells revealed that liposome-based nanohybrids exhibited the least toxicity, followed by PEG-PLGA- and PLGA-based NPs after 24 h incubation. The different kinetics of cellular uptake was also observed, the liposome-based systems being the fastest and PLGA-based systems being the slowest. The results present a potential delivery platform with enhanced control over its biological interaction kinetics and passive targeting capability through size control.     

Cyclodextrin-modified polyethylenimine polymers for gene delivery

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.     

Handling of cationic antigens in the joint and induction of chronic allergic arthritis. In vivo studies in the rat.

The aims of the present study were to define, under in vivo conditions, factors governing antigen binding and persistence in the rat joint and to establish a chronic arthritis model by means of a natural polycation. The influence of size as well as charge on antigen handling was examined using a range of chemically cationized proteins and natural polycations. Arthritis was induced by intraarticular challenge in preimmunized rats. Immunofluorescence studies revealed that not only pI, which must exceed pH 8-9, but also molecular size was a decisive parameter: only antigens of more than 40 kD were able to persist for significant periods in joint structures. All existing models of antigen induced chronic arthritis in rodents utilize chemically cationized proteins. We extended this system to natural polycations by showing that lysozyme (pI 11.3; MW 14 kD) in tetrameric, charge conserved form (MW 56 kD) as a model-antigen was able to induce chronic arthritis in the rat. After intraarticular challenge of preimmunized animals the course of inflammation was assessed both by 99mTechnetium-pertechnetate (99mTc) scintigram and from the histology. In contrast to monomeric lysozyme, which evoked only a transient inflammatory response (less than two weeks), tetrameric lysozyme induced a chronic arthritis, which still persisted at day 90. Our results show that the ability of cationic antigens to trigger chronic arthritis is vitally size dependent. This is also the first report of a natural polycation acting as an arthritogen, thus providing an experimental basis justifying the search for cationic microbial antigens in human post infectious reactive arthritis.     

Zinc-chelated poly(1-vinylimidazole) and a carbohydrate ligand polycation form DNA ternary complexes for gene delivery

Zinc-chelated poly(1-vinylimidazole) (PVIm-Zn) and a carbohydrate ligand polycation, a poly(l-lysine) conjugated with lactose molecules (PLL-Lac), have formed DNA ternary complexes for gene delivery. The particle size of the PVIm-Zn/DNA complexes with negative zeta potential was decreased by the addition of the PLL-Lac. The resulting PLL-Lac/PVIm-Zn/DNA ternary complexes, which exhibited the pH-dependent dissociation of the PLL-Lac, mediated more gene expression than the PVIm/DNA binary complexes. The PLL-Lac/PVIm-Zn/DNA complexes with the specific recognition of cell surface receptors mediated the highest gene expression without cytotoxicity at a relatively lower charge ratio (positive/negative = 2.5). These results suggest that the pH-dependent dissociation of the carbohydrate ligands after the recognition of cell surface receptors, including the physicochemical and biochemical function of PVIm-Zn, played an important role in gene expression.     

Microscopic insight into the DNA condensation process of a zwitterion‐functionalized polycation

Zwitterion‐functionalized polycations are ideal gene carriers with long circulation, high cellular uptaking and low cell viability. However, the trade‐off between the DNA condensation efficiency and the cell viability must be addressed. The purpose of this study is to provide a microscopic insight into the DNA condensation process and to explore the effect of a zwitterionic block of zwitterion‐functionalized polycation, which is of great significance in designing novel gene delivery systems. Poly[2‐(dimethylamino)ethyl methacrylate‐b‐(sulfobetaine methacrylate)] (PDMAEMA‐b‐PSBMA) copolymers were synthesized and used as the model systems. Different from the conventional concept that the PSBMA zwitterionic block act only as the “stealthy” groups, the subtle differences in physical and colloidal characteristics between the polycation/DNA polyplexes show that the PSBMA segment is capable of wrapping DNA attributed to the quaternary ammonium cations, without compromising the DNA condensation capability. On the other hand, the incorporation of PSBMA block reduces the surface charge of the polyplexes, which substantially result in the inefficient transfection and the reduced cytotoxicity.     

Molecular Dynamics Simulations of DNA-Polycation Complex Formation

Complexes formed from DNA and polycations are of interest because of their potential use in gene therapy; however, there remains a lack of understanding of the structure and formation of DNA-polycation complexes at atomic scale. In this work, molecular dynamics simulations of the DNA duplex d(CGCGAATTCGCG) in the presence of polycation chains are carried out to shed light on the specific atomic interaction that result in complex formation. The structures of complexes formed from DNA with polyethylenimine, which is considered one of the most promising DNA vector candidates, and a second polycation, poly-L-lysine, are compared. After an initial separation of ∼50 Å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes on complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, with polycation intrusion into the major and minor grooves dependent on the identity and charge state of the polycation. The ability of the polycation to effectively neutralize the charge of the DNA phosphate groups and the resulting influence on the DNA helix interaction are discussed.     

Polycation-mediated enhancement of retroviral transduction efficiency depends on target cell types and pseudotyped Env proteins: implication for gene transfer into neural stem cells

Polycations such as polybrene (PB) are routinely used for most retroviral vector-mediated gene transfer studies because they can increase the infectivity of retroviruses. However, it was not systematically determined if addition of the polycation is an essential prerequisite for all retroviral transductions. To test this, we measured the effects of the polycation on transduction efficiency using various combinations of target cells and pseudotyped viral envelope (Env) proteins. Here, we show polycations do not always increase retroviral transduction efficiency and that their enhancing effect depends on both the type of target cells and Env proteins. The findings presented here also suggest that high transduction rates can be achieved in primary neural stem cells in vitro and in vivo by choosing an appropriate Env protein for pseudotyping without using polycations which are potentially toxic to primary cells and may change the intrinsic characteristics of cells.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

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.     

Handling of cationic antigens in the joint and induction of chronic allergic arthritis

The aims of the present study were to define, under in vivo conditions, factors governing antigen binding and persistence in the rat joint and to establish a chronic arthritis model by means of a natural polycation. The influence of size as well as charge on antigen handling was examined using a range of chemically cationized proteins and natural polycations. Arthritis was induced by intraarticular challenge in preimmunized rats. Immunofluorescence studies revealed that not only pI, which must exceed pH 8–9, but also molecular size was a decisive parameter: only antigens of more than 40 kD were able to persist for significant periods in joint structures. All existing models of antigen induced chronic arthritis in rodents utilize chemically cationized proteins. We extended this system to natural polycations by showing that lysozyme (pI 11.3; MW 14 kD) in tetrameric, charge conserved form (MW 56 kD) as a modelantigen was able to induce chronic arthritis in the rat. After intraarticular challenge of preimmunized animals the course of inflammation was assessed both by^99mTechnetium-pertechnetate (^99mTc) scintigram and from the histology. In contrast to monomeric lysozyme, which evoked only a transient inflammatory response (less than two weeks), tetrameric lysozyme induced a chronic arthritis, which still persisted at day 90. Our results show that the ability of cationic antigens to trigger chronic arthritis is vitally size dependant. This is also the first report of a natural polycation acting as an arthritogen, thus providing an experimental basis justifying the search for cationic microbial antigens in human post infectious reactive arthritis.     

Cationic carriers of genetic material and cell death: A mitochondrial tale

Central to gene therapy technology has been the use of cationic polymers as vectors for DNA and RNA (polyfectins). These have been presumed to be safer than viral systems which, for example, have been found to switch on oncogenes. Two key polycations that have been intensively researched for use as synthetic vectors are poly(ethylenimine) and poly(l-lysine). A frequent stumbling block with these polyfectins is that long-term gene expression in cell lines has not been achieved. Recently it has transpired that both of these polycations can induce mitochondrially mediated apoptosis. It is the aim of this review to discuss the mechanisms behind the observed polycation toxicity including roles for little studied cellular organelles in the process such as the lysosome and endoplasmic reticulum.     

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.     

DNA complexes with polycations used for delivery of DNA into cells

The formation and physicochemical properties of high-molecular thymus and plasmid DNA complexes with synthetic polymers based on (dimethyl-amino)ethyl methacrylate (DMAEM), (diethyl-amino)ethyl methacrylate (DEAEM), and polyvinyl amine (PVA) were investigated in solutions of different ionic strength by low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The toxicity of complexes in T98G cells was studied. It was shown that, when the ratio of polycations to DNA charged groups concentration (N+/P) reaches values > 1, DNA condensation occurs. It is accompanied by increasing optical density of solutions. Changes in DNA size after condensation were estimated. Phase diagrams of systems DNA/polycation in the presence of NaCl were obtained. It was shown by MTT-analysis that DNA complexes with polycations in the range of concentrations used have low toxicity.     

Aliphatic ionenes as gene delivery agents: elucidation of structure-function relationship through modification of charge density and polymer length

Polycation-based gene delivery agents are generally polydisperse populations whose properties are averaged among the different molecular weight species. Therefore, to understand the physicochemical properties of polycations and their relationships to cellular gene transfer, one needs to control the molecular weight of the polymer as well as its cationic charge density. To investigate the structure-function correlation of polycations with respect to the degree of polymerization (DP) and charge density, a series of model materials based on aliphatic ionenes was synthesized and fractionated into distinct molecular weight fractions with DP range from 14 to 32. The aliphatic ionene fractions and their polyelectrolyte complexes (PEC) with DNA were studied using physicochemical and biological methods. Ionene polymers were shown to possess low cytotoxicity (minimal viability of the P388D1 murine macrophage cells 80%). DP and charge density of the ionenes were shown to be the factors of effective control of PEC dissociation in water-salt solutions, with a diminished role of charge density upon lengthening the ionene chain. These polymer characteristics were also important for DNA-ionene PEC resistivity to DNase activity and the ability of ionenes to serve as gene delivery vectors in vitro and exhibited good correlation with the results of salt-induced dissociation of PEC. These data may be useful for developing correlations and mathematical models to predict synthetic gene delivery vector efficiency.     

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.     

Design, synthesis, and biocompatibility of an arginine-based polyester

Polycations are very useful in biotechnology. However, most existing polycations have high toxicity that significantly limits their clinical translation. We designed poly(ethylene argininylaspartate diglyceride) (PEAD) that is based on arginine, aspartic acid, glycerol, and ethylene glycol. A set of in vitro assays demonstrated that PEAD exhibited no cytotoxicity at 1 mg/mL, which is at least 100 times higher than the widely used polycation-polyethylenimine. Subcutaneous injection of 1 mg PEAD in rats did not cause an adverse response acutely or after 4 weeks. Zeta potential measurements revealed that PEAD has high affinity to biological polyanions such as DNA and hyaluronic acid. This polycation represents a new platform of biocompatible polycations that may lead to clinical innovations in gene therapy, controlled release, tissue engineering, biosensors, and medical devices.     

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.