Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency

Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.     

Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.     

Polycationic block copolymers of poly(ethylene oxide) and poly(propylene oxide) for cell transfection

A facile, one-step synthesis of cationic block copolymers of poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and copolymers of poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO) has been developed. The PEO-PPO-PEO-pDMAEMA (L92-pDMAEMA) and PEO-pDMAEMA copolymers were obtained via free radical polymerization of DMAEMA initiated by polyether radicals generated by cerium(IV). Over 95% of the copolymer fraction was of molecular mass ranging from 6.9 to 7.1 kDa in size, indicating the prevalence of the polyether-monoradical initiation mechanism. The L92-pDMAEMA copolymers possess parent surfactant-like surface activity. In contrast, the PEO-pDMAEMA copolymers lack significant surface activity. Both copolymers can complex with DNA. Hydrodynamic radii of the complexes of the L92-pDMAEMA and PEO-pDMAEMA with plasmid DNA ranged in size from 60 to 400 nm, depending on the copolymer/DNA ratio. Addition of Pluronic P123 to the L92-pDMAEMA complexes with DNA masked charges and decreased the tendency of the complex to aggregate, even at stoichiometric polycation/DNA ratios. The transfection efficiency of the L92-pDMAEMA copolymer was by far greater than that of the PEO-pDMAEMA copolymer. An extra added Pluronic P123 further increased the transfecton efficacy of L92-pDMAEMA, but did not affect that of PEO-pDMAEMA.     

Phosphonium-containing diblock copolymers for enhanced colloidal stability and efficient nucleic Acid delivery

RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.     

The inhibition of a plant proteinase by lysine copolymers is modulated by the hydrophobicity of the interposed amino acid side chains

Summary One Lys/Phe copolymer and two series of copolymers of lysine with either alanine or tyrosine have been used as inhibitors of a plant proteinase that is known to be inhibited by polycationic inhibitors. The copolymers differ in the hydrophobicity of the non-lysine amino acid residue and the single members of each series differ from each other in their degree of polymerization and in their charge density, i.e., the frequency of occurrence of the lysine residue in the synthetic polyamino acid chain. All the tested copolymers show cooperative inhibition, with a Hill coefficient higher than 1. CD measurements indicate that the inhibition is realized through a conformational change of the enzyme molecule. Both the enzyme inhibition and the conformational change are supported by aspecific electrostatic binding between the positively charged groups of the lysine moiety and the negatively charged groups of the enzyme surface. In each series the inhibitory power increases with the charge density, while at the same charge density the inhibitory efficiency depends on the hydrophobicity of the side chain of the non-lysine amino acid in the order Phe>Tyr>Ala.     

Polyethylenimine-graft-poly(ethylene glycol) copolymers: influence of copolymer block structure on DNA complexation and biological activities as gene delivery system

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.     

Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers

As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may provide flexible chemistry for the attachment of targeting moieties. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) were prepared in order to develop hepatocyte targeting gene transfection carriers. The block copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization of N-2-hydroxypropyl methacrylamide (HPMA) and N-3-aminopropyl methacrylamide (APMA), followed by galactosylation and guanidinylation. The molecular weight of GHG copolymers determined by static light scattering method was in the range from 48?600 to 76?240 g/mol. In addition, the galactose content in the GPMA block in the copolymers was determined to be 6.5-8.0 mol % according to the sulfuric acid method. The GHG copolymers complexed completely with plasmid DNA (pDNA) to show positive zeta-potential values with diameter 100-250 nm from charge ratio of 4, which demonstrated the excellent DNA condensing ability of guanidino groups. Furthermore, the MTT assay data of GHG/pDNA complexes on HepG2 cells and HeLa cells indicated that GHG copolymers had significantly lower cytotoxicity than PEI. In addition, the copolymers with GPMA component from 30.23% showed higher transfection efficiency than PEI at charge ratio of 12 in HepG2 cells. The result revealed that the conjugation of galactose groups in the copolymers brought asialoglycoprotein-receptor (ASGP-R) mediated transfection. The employing of HPMA component decreased the aggregation of protein in transfection presence of serum. The GHG copolymers combined the advantages of galactose moieties, guanidino groups, and HPMA component might show potential in safe hepatocyte targeting gene therapy.     

Multi‐armed poly(L‐glutamic acid)‐graft‐oligoethylenimine copolymers as efficient nonviral gene delivery vectors

Background

The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low‐toxic, highly efficient gene delivery.

Methods

A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi‐armed poly(L ‐glutamic acid) backbone. The molecular structures of multi‐armed poly(L ‐glutamic acid)‐graft‐OEI (MP‐g‐OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP‐g‐OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP‐g‐OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney‐293 cells for their cytotoxicity and transfection efficiency.

Results

The particle sizes of MP‐g‐OEI/DNA complexes were in a range of 109.6–182.6 nm and the zeta potentials were in a range of 29.2–44.5 mV above the N/P ratio of 5. All the MP‐g‐OEI copolymers exhibited lower cytotoxicity and higher gene transfection efficiency than PEI25k in the absence and presence of serum with different cell lines. Importantly, the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay revealed that the cytotoxicity of MP‐g‐OEI copolymers varied with their molecular weight and charge density, and two of MP‐g‐OEI copolymers (OEI600‐MP and OEI1800‐MP) could achieve optimal transfection efficiency at a similar low N/P ratio as that for PEI25k.

Conclusions

MP‐g‐OEI copolymers demonstrated considerable potential as nonviral vectors for gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Linear polyethyleneimine grafted to a hyperbranched poly(ethylene glycol)-like core: a copolymer for gene delivery

A block copolymer of a hyperbranched poly(ethylene glycol)-like core and linear polyethylenimine (HBP) was synthesized by a facile synthetic route that included (1) a single-step cationic copolymerization of diepoxy and polyhydroxyl monomers, (2) derivatization of hydroxyl groups of the core HBPEG copolymer with either tosyl or chloromethylbenzoyl chlorides resulting in a corresponding macroinitiator, and (3) synthesis of HBPEG-block-poly(alkyl oxazolines). HBPEG-block-linear polyethyleneimine (HBP) was obtained by hydrolysis of HBPEG-block-poly(alkyl oxazolines). Linear PEI-bearing hyperbranched polycations (HBP) had lower inherent toxicity in cell culture than PEG-grafted linear polyethyleneimines (PEGLPEI). PEGLPEI formed a complex with DNA with an average diameter of 250 nm. The complexes were loosely condensed and formed aggregates and precipitates during storage. By contrast, hyperbranched polycations (HBP) formed approximately 50 nm nanocomplexes with DNA that were stable for several weeks and showed resistance to DNAse I-mediated degradation. The 'inverted' block copolymers showed several orders of magnitude higher transfection efficiency than PEGLPEI in vitro. Because of the biocompatibility and higher transfection efficiency, the 'inverted' block copolymer merits further investigation as a gene carrier.     

Synthesis and in vitro evaluation of novel star-shaped block copolymers (blocked star vectors) for efficient gene delivery

Novel 4-branched diblock copolymers consisting of cationic chains as an inner domain and nonionic chains as an outer domain were prepared by iniferter-based living radial polymerization and evaluated as a polymeric transfectant. The cationic polymerization of 3-(N,N-dimethylamino)propyl acrylamide (DMAPAAm) using 1,2,4,5-tetrakis( N,N-diethyldithiocarbamylmethyl)benzene as a 4-functional iniferter followed by the nonionic block polymerization of N,N-dimethylacrylamide (DMAAm) afforded 4-branched diblock copolymers with controlled compositions. By changing the solution or irradiation conditions, 4-branched PDMAPAAms with molecular weights of 10,000, 20,000, and 50,000 were synthesized. In addition, by graft polymerization, PDMAPAAm-PDMAAm blocked copolymers with copolymer composition (unit ratio of DMAAm/DMAPAAm) ranging from 0.18 to 1.0 for each cationic polymer were synthesized. All polymers were shown to interact with and condense plasmid DNA to yield polymer/DNA complexes (polyplexes). A transfection study on COS-1 cells showed that the polyplexes from block copolymers with cationic chain length of approximately 50,000 and a nonionic chain length of 30,000, which were approximately 200 nm in diameter and very stable in aqueous media, had the most efficient luciferase activity with minimal cellular cytotoxicity under a charge ratio of 20 (vector/pDNA). The PDMAPAAm-PDMAAm-blocked, star-shaped polymers are an attractive novel class of nonviral gene delivery systems.     

Phosphonium-containing polyelectrolytes for nonviral gene delivery

Nonviral gene therapy focuses intensely on nitrogen-containing macromolecules and lipids to condense and deliver DNA as a therapeutic for genetic human diseases. For the first time, DNA binding and gene transfection experiments compared phosphonium-containing macromolecules with their respective ammonium analogs. Conventional free radical polymerization of quaternized 4-vinylbenzyl chloride monomers afforded phosphonium- and ammonium-containing homopolymers for gene transfection experiments of HeLa cells. Aqueous size exclusion chromatography confirmed similar absolute molecular weights for all polyelectrolytes. DNA gel shift assays and luciferase expression assays revealed phosphonium-containing polymers bound DNA at lower charge ratios and displayed improved luciferase expression relative to the ammonium analogs. The triethyl-based vectors for both cations failed to transfect HeLa cells, whereas tributyl-based vectors successfully transfected HeLa cells similar to Superfect demonstrating the influence of the alkyl substituent lengths on the efficacy of the gene delivery vehicle. Cellular uptake of Cy5-labeled DNA highlighted successful cellular uptake of triethyl-based polyplexes, showing that intracellular mechanisms presumably prevented luciferase expression. Endocytic inhibition studies using genistein, methyl β-cyclodextrin, or amantadine demonstrated the caveolae-mediated pathway as the preferred cellular uptake mechanism for the delivery vehicles examined. Our studies demonstrated that changing the polymeric cation from ammonium to phosphonium enables an unexplored array of synthetic vectors for enhanced DNA binding and transfection that may transform the field of nonviral gene delivery.     

Preparation of low molecular weight N-maleated chitosan-graft-PAMAM copolymer for enhanced DNA complexation

Low molecular weight N-maleated chitosan-graft-PAMAM (polyamidoamine) copolymer was prepared through N-maleated chitosan (NMC) by Michael type addition reaction to enhance its solubility in water as well as its cationic character for enhancement of DNA complexation. FTIR, (1)H NMR, XRD and GPC were used to characterize the graft copolymers. The copolymer showed better DNA complexation ability at low N/P ratio than that of chitosan due to increased surface charge density by the incorporation of PAMAM molecule on to chitosan backbone. The copolymer can effectively protect the DNA toward anionic surfactant. In vitro release study showed efficient DNA release occurred at physiological pH (pH 7.4). In vitro cell cytotoxicity test indicated toward less cytotoxicity of NMC-graft-PAMAM copolymers compared to that of 25kDa PEI. Thus, the synthesized NMC-graft-PAMAM copolymers have great potential of finding application in drug and gene delivery.     

Biodegradable amphiphilic block copolymers bearing protected hydroxyl groups: synthesis and characterization

A new biodegradable amphiphilic block copolymer, poly(ethylene glycol)-b-poly(L-lactide-co-9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one) [PEG-b-P(LA-co-PTO)], was successfully prepared by ring-opening polymerization (ROP) of L-lactide (LA) and functionalized carbonate monomer 9-phenyl-2,4,8,10-tetraozaspiro[5,5]undecan-3-one (PTO) in the presence of monohydroxyl poly(ethylene glycol) as macroinitiator using Sn(Oct)2 as catalyst. NMR, FT-IR, and GPC studies confirmed the copolymer structure. It could self-assemble into micelles in aqueous solution with critical micelle concentration (CMC) in the magnitude of mg/L, which changed with the composition of the copolymer. After catalytic hydrogenation, copolymers with active hydroxyl groups were obtained. Adhesion and proliferation of Vero cells on the copolymer films showed that the synthesized copolymers were good biocompatible materials. In vitro degradation of the copolymer before and after deprotection was investigated in the presence of proteinase K. The free hydroxyl groups on the copolymers were capable of further modification with biotin. This new amphiphilic block copolymer has great potential for both drug encapsulation and conjugate because of its low CMC and the presence of active hydroxyl groups.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

Specificity of DNA basic polypeptide interactions. II+ Influence of aromatic amino acid residues investigated with agarose bound lysine copolypeptides.

Binding affinities towards DNA and base pair specificities of lysine copolymers, containing different amounts of Phe, Tyr or Trp residues, were estimated using a previously described chromatographic method. Incorporation of few aromatic residues into polylysine causes a decrease in the binding affinity, however, further raising the aromatic residue - lysine ratio results in a continous increase of affinity, which is most pronounced with the Tyr copolymers and not observed with polymers containing neutral aliphatic amino acid residues. AT-specificity increases concomitant with binding affinity in the case of the Tyr copolymers but not with the Phe copolymers. The interaction of DNA with the alternating Phe-Lys polymer is significantly stronger than with the random copolymer of equal residue composition. The molecular and conformational reasons determining specificity are discussed.     

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.     

Poly(L-lysine)-graft-chitosan copolymers: synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by 1H NMR, 13C NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 approximately 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.     

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.     

The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

To evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid-DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP(+)/DNA(-)) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP(+)/DNA(-) ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid-DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study.     

Reversibly shielded DNA polyplexes based on bioreducible PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers mediate markedly enhanced nonviral gene transfection

Reversibly shielded DNA polyplexes based on bioreducible poly(dimethylaminoethyl methacrylate)-SS-poly(ethylene glycol)-SS-poly(dimethylaminoethyl methacrylate) (PDMAEMA-SS-PEG-SS-PDMAEMA) triblock copolymers were designed, prepared and investigated for in vitro gene transfection. Two PDMAEMA-SS-PEG-SS-PDMAEMA copolymers with controlled compositions, 6.6-6-6.6 and 13-6-13 kDa, were obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization of dimethylaminoethyl methacrylate (DMAEMA) using CPADN-SS-PEG-SS-CPADN (CPADN: 4-cyanopentanoic acid dithionaphthalenoate; PEG: 6 kDa) as a macro-RAFT agent. Like their nonreducible PDMAEMA-PEG-PDMAEMA analogues, PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers could effectively condense DNA into small particles with average diameters less than 120 nm and close to neutral zeta potentials (0 ～ +6 mV) at and above an N/P ratio of 3/1. The resulting polyplexes showed excellent colloidal stability against 150 mM NaCl, which contrasts with polyplexes of 20 kDa PDMAEMA homopolymer. In the presence of 10 mM dithiothreitol (DTT), however, polyplexes of PDMAEMA-SS-PEG-SS-PDMAEMA were rapidly deshielded and unpacked, as revealed by significant increase of positive surface charges as well as increase of particle sizes to over 1000 nm. Release of DNA in response to 10 mM DTT was further confirmed by gel retardation assays. These polyplexes, either stably or reversibly shielded, revealed a low cytotoxicity (over 80% cell viability) at and below an N/P ratio of 12/1. Notably, in vitro transfection studies showed that reversibly shielded polyplexes afforded up to 28 times higher transfection efficacy as compared to stably shielded control under otherwise the same conditions. Confocal laser scanning microscope (CLSM) studies revealed that reversibly shielded polyplexes efficiently delivered and released pDNA into the perinuclei region as well as nuclei of COS-7 cells. Hence, reduction-sensitive reversibly shielded DNA polyplexes based on PDMAEMA-SS-PEG-SS-PDMAEMA are highly promising for nonviral gene transfection.