Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine

A new polyethylenimine (PEI)-derived biodegradable polymer was synthesized as a nonviral gene carrier. Branches of PEI were ketalized, and capabilities of nucleic acid condensation and delivery efficiency of the modified polymers were compared with ones of unketalized PEI. Ketalized PEI was able to efficiently compact both plasmid DNA and siRNA into nucleic acids/ketalized PEI polyplexes with a range of 80-200 nm in diameter. Nucleic acids were efficiently dissociated from the polyplexes made of ketalized PEI upon hydrolysis. In vitro study also demonstrated that ketalization enhanced transfection efficiency of the polyplexes while reducing cytotoxicity, even at high N/ P ratios. Interestingly, transfection efficiency was found to be inversely proportional to molecular weights of ketalized PEI, while RNA interference was observed in the opposite way. This study implies that selective delivery of plasmid DNA and siRNA to the nucleus and the cytoplasm can be achieved by tailoring the structures of polymeric gene carriers.     

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.     

The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells

The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9 kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06 nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5 nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1 h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.     

Physicochemical and biological study of selected hydrophobic polyethylenimine-based polycationic liposomes and their complexes with DNA

Non-viral gene therapy is based on the development of efficient and safe gene carrier systems able to transfer DNA into cells. Polyethylenimine (PEI), the most promising non-viral vector, with its high cationic-charge-density potential is able (1) to compact DNA in complexes (polyplexes) smaller than those formed by liposomes (lipoplexes) and (2) to destabilize the endosomal membrane by a 'proton sponge' effect. Several PEI's hydrophobic modifications were reported in the last several years but in some cases a reduced transfection efficiency was observed. The mechanism underlying this phenomenon is not well understood so far. In order to extensively investigate these mechanisms, we reported a physicochemical and biological study of selected hydrophobic PEI's derivatives grafted with chains of different length and percentages of substitution able to form vesicles (polycationic liposomes) and to bind DNA. Their properties were studied by means of dynamic light scattering, freeze-fracture microscopy, potentiometric titrations, gel retardation assays, polyanion exchange reactions, toxicity assays, in vitro transfections, and fluorescence microscopy. Our results indicate that even if polyplexes are able to pass through the cellular membrane, the stability of PEI's hydrophobic polyplexes likely explain their different transfection efficiency in vitro.     

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2-6 months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This "direct" transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.     

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.     

Extracellular barriers to in Vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver

Polyplex-mediated gene therapy is a promising alternative to viral gene therapy. One challenge to these synthetic carriers is reduced transfection efficiencies in vivo compared to those achieved in vitro. Many of the intracellular barriers to gene delivery have been elucidated, but similar quantification of extracellular barriers to gene delivery remains a need. In this study, the unpackaging of polyplexes by serum proteins, soluble glycosaminoglycans, and an extracellular matrix extract was demonstrated by a YOYO-1 fluorescence quenching assay. Additionally, exposing polyplexes to serum or proteoglycans before in vitro transfection caused decreased cellular uptake of DNA. Lastly, PEI polyplexes and PEGylated PEI polyplexes were injected into the portal vein of mice, and the intrahepatic distributions of labeled DNA and polymer were assessed by confocal microscopy. PEI polyplexes delivered DNA to the liver, but extensive vector unpackaging was observed, with PEI primarily colocalized with the extracellular matrix. PEGylated polyplexes mediated less DNA delivery to the liver, possibly due to premature vector unpackaging in the blood. Through this work, both the blood and the extracellular matrix have been determined to be significant extracellular barriers to polyplex-mediated in vivo gene delivery to the liver.     

Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.     

Structure-activity relationships of water-soluble cationic methacrylate/methacrylamide polymers for nonviral gene delivery.

A number of water-soluble cationic carriers was evaluated as transfectant. Almost all studied cationic methacrylate/methacrylamide polymers were able to condense the structure of plasmid DNA, yielding polymer/plasmid complexes (polyplexes) with a size of 0.1-0.3 micron and a slightly positive zeta-potential, which can be taken up by cells, e.g., via endocytosis. However, the transfection efficiency and the cytotoxicity of the polymers differed widely: the highest transfection efficiency and cytotoxicity were observed for poly[2-(dimethylamino)ethyl methacrylate], p(DMAEMA). Assuming that polyplexes enter cells via endocytosis, p(DMAEMA) apparently has advantageous properties to escape the endosome. A possible explanation is that, due to its average pK(a) value of 7.5, p(DMAEMA) is partially protonated at physiological pH and might behave as a proton sponge. This might cause a disruption of the endosome, which results in the release of both the polyplexes and cytotoxic endosomal/lysosomal enzymes into the cytosol. On the other hand, the analogues of p(DMAEMA) studied here have a higher average pKa value and have, consequently, a higher degree of protonation and a lower buffering capacity. This might be associated with a lower tendency to destabilize the endosome, resulting in both a lower transfection efficiency and a lower cytotoxicity. Furthermore, molecular modeling showed that, of all studied polymers, p(DMAEMA) has the lowest number of interactions with DNA. We therefore hypothesized that the superior transfection efficiency of p(DMAEMA) containing polyplexes can be ascribed to an intrinsic property of p(DMAEMA) to destabilize endosomes combined with an easy dissociation of the polyplex once present in the cytosol and/or the nucleus.     

DNA compaction into new DNA vectors based on cyclodextrin polymer: surface enhanced Raman spectroscopy characterization

The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.     

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.     

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells

BACKGROUND: Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. METHODS: After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. RESULTS: PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. CONCLUSIONS: We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.     

Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery

Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More specifically, three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers, i.e., ethylenediamine (EDA), diethylenetriamine (DETA), or triethylenetetramine (TETA). Complete addition reactions were confirmed by (1)H NMR. The molecular weight, buffer capacity, and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC), acid-base titration, and liquid chromatography-mass spectroscopy (LC-MS), respectively. Physicochemical characteristics of polymer/pDNA complexes (polyplexes) were analyzed by gel electrophoresis, particle size, and zeta-potential measurements. All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of approximately 32 mV. The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3), primary bovine aortic endothelial cell (BAEC), and rat aortic smooth muscle cell (A7R5) lines. Interestingly, polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k. The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium. Furthermore, confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI, most likely due to environmentally triggered release. Therefore, SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.     

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.     

Methacrylamide polymers with hydrolysis-sensitive cationic side groups as degradable gene carriers

Water-soluble polymers with hydrolyzable cationic side groups (structure of the monomers are shown in Figure 1) were synthesized and evaluated as DNA delivery systems. The polymers, except for pHPMA-NHEM, were able to condense plasmid DNA into positively charged nanosized particles. The rate of hydrolysis at 37 degrees C and pH 7.4 of the side groups differed widely; the fastest rate of hydrolysis was observed for HPMA-DEAE (half-life of 2 h), while HPMA-DMAPr had the lowest rate of hydrolysis (half-life of 70 h). In line with this, pHPMA-DEAE-based polyplexes showed the fastest destabilization of the polyplexes at 37 degrees C and pH 7.4. Polyplexes based on pHPMA-DEAE, pHPMA-DMAE, and pHPMA-MPPM showed release of intact DNA within 24, 48, and 48 h, respectively, after incubation at 37 degrees C and pH 7.4. PHPMA-DEAE and pHPMA-MPPM based polyplexes showed the highest transfection activity (almost twice as active as pEI). Importantly, the pHPMA-DEAE, pHPMA-MPPM, and pHPMA-BDMPAP polyplexes preserved their transfection activity in the presence of serum proteins. All polymers investigated showed a substantial lower in vitro cytotoxicity than pEI. In conclusion, pHPMA-based polyplexes are an attractive class of biodegradable vectors for nonviral gene delivery.     

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.     

Correlating transfection barriers and biophysical properties of cationic polymethacrylates

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real-time polymerase chain reaction (real-time PCR). Three polycations polymers with similar chemical structure were used in this study: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer, and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, the transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier as compared to both PDMA and PEO-b-PDMA. Correlations between cytotoxicity, cellular uptake of plasmid DNA, expression levels of transgene and protein, and the physical properties of the polymers were observed. With the PEO-b-PDEA studies, cytotoxicity was due primarily to the excess of polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA polyplexes to interact with cell effectively was identified as a critical barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with physical characterization techniques could provide useful insights into the transfection barrier at different cellular levels.