Transfection Efficiency Of Polyplexes Research Articles

Linear Poly(ethylenimine-propylenimine) Random Copolymers for Gene Delivery: From Polymer Synthesis to Efficient Transfection with High Serum Tolerance.

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

Influence of the media ionic strength on the formation and in vitro biological performance of polycation-DNA complexes

Cationic polymer-DNA complexes, or polyplexes, have been subject to intensive investigation as potentially efficient non-viral systems for gene therapy. Yet the effects of ionic strength, a physiologically relevant parameter, on the formation, physicochemical properties (e.g. size and colloidal stability) and transfection efficiency of polyplexes are still poorly investigated and understood. In this work, we analyze the effect of ionic strength on the formation and transfection efficiency of poly-L-lysine (PLL), branched polyethylenimine (PEI) and bioreducible poly-L-Lysine (bPLL) polycations complexed with plasmid DNA, using different preparation paths. In path I, the polycation and DNA are mixed in water and transferred to saline media afterwards. In path II, the polycation and DNA are mixed already in the presence of salt. Despite that the final compositions are identical, for monovalent salt (NaCl) concentrations ≥ 70 mM, the two pathways give rise to polyplexes with different sizes and stability. Path I polyplexes are smaller and colloidally more stable than their path II analogues, irrespective of polycation. Regarding the different polycations used, PLL-DNA polyplexes are smaller and more stable than PEI-polyplexes, regardless of path, while bPLL-DNA particles aggregate very easily in saline media. Conversely, when applied to 2D A549 cell cultures, the two assembly pathways do not show significant differences in transfection efficiency, but regarding cellular-uptake, PEI and path I offer better results. Overall, we show that slight differences in ionic strength at the time of polyplex formation strongly influence the size and stability of polycation-DNA complexes, but they do not translate into significant differences in the transfection of 2D in vitro A549 cell-cultures. Notwithstanding, caution should be exercised as the size differences observed could impact transfection in more complex in vivo models.

Charge and Assembly Reversible Micelles Fueled by Intracellular ATP for Improved siRNA Transfection.

Hydrophobic modification on polycations were commonly used to improve the stability and transfection efficiency of polyplexes. However, the improved stability often means undesired release of the encapsulated siRNA, limiting the application of cationic micelles for siRNA delivery. The current strategy of preparing bioresponsive micelles based on the cleavage of sensitive linkages between polycation and hydrophobic part was far from sufficient, owing to the siRNA binding of the separated polycations from micelles leading to the incomplete release of siRNA. In this study, we propose a new strategy by the combination of micelles disassembly and separated polycations charge reversal. FPBA (3-fluoro-4-carboxyphenylboronic acid) grafted PEI 1.8 k (polyethylenimine) as the polycations of PEI-FPBA and dopamine (with diol-containing moiety) conjugated with cholesterol as the hydrophobic part (Chol-Dopa). The PFCDM micelles was assembled by PEI-FPBA and Chol-Dopa, based on the FPBA-Dopa conjugation. The prepared PFCDM showed strong siRNA loading ability and superior stability in the presence of PBS or serum. Besides, the PFCDM exhibited excellent ATP sensibility. The intracellular ATP could effectively trigger the disassembly of micelles and charge reversal of PEI-FPBA, resulting in the burst release of siRNA in the cytosol. With the property of extracellular stability and intracellular instability, PFCDM displayed good performance on in vitro and in vivo luciferase silencing on 4T1 cells. It should also be noted that the assembly of low molecular weight PEI was relatively safe to cells compared with 25 k PEI. To sum up, the ATP-fueled assembly and charge reversible micelles gave examples for polyplexes to achieve better stability and on demand cargo release at the same time and shows potential to be used for in vitro and in vivo siRNA transfection.

Peptides mediating DNA transport on microtubules and their impact on non-viral gene transfer efficiency.

Synthetic vectors such as cationic polymers and cationic lipids remain attractive tools for non-viral gene transfer which is a complex process whose effectiveness relies on the ability to deliver a plasmid DNA (pDNA) into the nucleus of non-dividing cells. Once in the cytosol, the transport of pDNAs towards the nuclear envelope is strongly impaired by their very low cytosolic mobility due to their large size. To promote their movement towards the cell nucleus, few strategies have been implemented to exploit dynein, the microtubule’s (MT’s) motor protein, for propagation of cytosolic pDNA along the MTs towards the cell nucleus. In the first part of this review, an overview on MTs, dynein, dynein/virus interaction feature is presented followed by a summary of the results obtained by exploitation of LC8 and TCTEL1 dynein light chain association sequence (DLC-AS) for non-viral transfection. The second part dedicated to the adenoviral protein E3-14.7K, reports the transfection efficiency of polyplexes and lipoplexes containing the E3-14.7K-derived P79-98 peptide linked to pDNA. Here, several lines of evidence are given showing that dynein can be targeted to improve cytosolic pDNA mobility and accumulate pDNA near nuclear envelope in order to facilitate its transport through the nuclear pores. The linkage of various DLC-AS to pDNA carriers led to modest transfection improvements and their direct interaction with MTs was not demonstrated. In contrast, pDNA linked to the P79-98 peptide interacting with TCTEL1 via a cytosolic protein (fourteen seven K-interacting protein-1 (FIP-1)), interaction with MTs is evidenced in cellulo and transfection efficiency is improved.

Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes

Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI25 (25kDa PEI) or carboxyalkylated-PEI10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143–173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity.

A Zwitterionic-Shielded Carrier with pH-Modulated Reversible Self-Assembly for Gene Transfection.

Cationic vectors are ideal candidates for gene delivery thanks to their capability to carry large gene inserts and their scalable production. However, their cationic density gives rise to high cytotoxicity. We present the proper designed core-shell polyplexes made of either poly(ethylene imine) (PEI) or poly(2-dimethylamino ethyl methacrylate) (PDMAEMA) as the core and zwitterionic poly(acrylic acid)-block-poly(sulfobetaine methacrylate) (PAA-b-PSBMA) diblock copolymer as the shell. Gel retardation and ethidium bromide displacement assays were used to determine the PEI/DNA or PDMAEMA/DNA complexation. At neutral pH, the copolymer serves as a protective shell of the complex. As PSBMA is a nonfouling block, the shell reduced the cytotoxicity and enhanced the hemocompatibility (lower hemolysis activity, longer plasma clotting time) of the gene carriers. PAA segments in the copolymer impart pH sensitivity by allowing deshielding of the core in acidic solution. Therefore, the transfection efficiency of polyplexes at pH 6.5 was better than at pH 7.0, from β-galactosidase assay, and for all PAA-b-PSBMA tested. These results were supported by more favorable physicochemical properties in acidic solution (zeta potential, particle size, and interactions between the polymer and DNA). Thus, the results of this study offer a potential route to the development of efficient and nontoxic pH-sensitive gene carriers.

Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery.

Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

Synthesis and evaluation of novel chitosan derivatives for gene delivery

A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efficiencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors

Poloxamine/fibrin hybrid hydrogels for non-viral gene delivery.

Hydrogels have been widely investigated for localized, sustained gene delivery because of the similarity of their physical properties to native extracellular matrix and their ability to be formed under mild conditions amenable to the incorporation of bioactive molecules. The objective of this study was to develop bioactive hydrogels composed of macromolecules capable of enhancing the efficiency of non-viral vectors. Hybrid hydrogels were prepared by simultaneous enzymatic and Michael-type addition crosslinking of reduced fibrinogen and an acrylated amphiphilic block copolymer, Tetronic T904, in the presence of dithiothreitol (DTT) and thrombin. T904/fibrin hydrogels degraded by surface erosion in the presence of plasmin and provided sustained release of polyplex vectors up to an order of magnitude longer than pure fibrin gel control. In addition, the rate of gel degradation and time-course of polyplex vector release were readily controlled by varying the T904/fibrinogen ratio in the gel composition. When added to transfected neuroblastoma (N2A) cells, both native T904 itself and hydrogel degradation products significantly increased polyplex transfection efficiency with minimal effect on cell viability. To evaluate gel-based transfection, N2A cells encapsulated in small fibrin clusters were covered by or suspended within polyplex-loaded hydrogels. Cells progressively degraded and invaded the hybrid hydrogels, exhibiting increasing gene expression over 2 weeks and then diminishing but persistent gene expression for over 1 month. In conclusion, these results demonstrate that T904/fibrin hybrid hydrogels can be promising tissue engineering scaffolds that provide local, controlled release of non-viral vectors in combination with the generation of bioactive gel degradation products that actively enhance vector efficiency. Copyright © 2014 John Wiley & Sons, Ltd.

Influence of pDNA availability on transfection efficiency of polyplexes in non-proliferative cells

We succeeded in visualizing plasmid DNA (pDNA) in the nucleus and cytosol of non-proliferative cells after transfection with linear polyethylenemine (lPEI) and histidinylated lPEI (His16-lPEI). This was possible with confocal microscope by using pDNA labelled with quantum dots. Indeed pDNA labelled with Cy3 leads to false positive nuclear localization because the saturation of the fluorescence signal overestimated the volume occupied by Cy3-pDNA. Moreover, Cy3 brightness was too weak to detect low amount of pDNA. About 20 to 40 pDNA copies were detected in the nucleus after the transfection of pDNA labelled with quantum dots. Transfection efficiency and cellular imaging data suggested that the cytosolic availability of pDNA, including endosome escape and/or polyplexes dissociation, is crucial for its nuclear delivery. In vitro transcription assay and transfection of cells allowing cytosolic gene expression concluded to better cytosolic availability of pDNA within His16-lPEI polyplexes. Cryo-TEM analyses revealed that His16-lPEI polyplexes exhibited a spherical shape and an amorphous internal structure which differed from the high degree of order of lPEI polyplexes. Altogether, this comparative study indicated that the high transfection efficiency of non-proliferative cells with His16-lPEI polyplexes was related to the amorphous structure and the facilitated dissociation of the assemblies.

Efficacy and mechanism of poloxamine‐assisted polyplex transfection

Amphiphilic block copolymers acting as biological response modifiers provide an attractive approach for improving the transfection efficiency of polycationic polymer/DNA complexes (polyplexes) by altering cellular processes crucial for efficient transgene expression. The present study aimed to investigate the effect of the poloxamine Tetronic T904, a four-arm polyethylene oxide/polypropylene oxide block copolymer, on polyplex transfection and to determine its mechanism of action by analyzing the cellular uptake of polyplex, the nuclear localization of plasmid and RNA transcript production. T904 significantly increased the transfection efficiency of polyplexes based on 25-kDa branched polyethylenimine in a dose-dependent manner in the presence of serum in C6 glioma cells, as well as human fibroblasts and mesenchymal stem cells. The activity of T904 was not promoter-dependent, increasing the expression of reporter genes under both cytomegalovirus and SV40 promoters. Although T904 did not affect the internalization or nuclear uptake of plasmid, mRNA expression levels from both promoters showed dose-dependent increases that closely paralleled increases in gene expression. The present study demonstrates that T904 significantly increases polyplex transfection efficiency and suggests a mechanism of increased transcriptional activity. As a four-arm, hydroxyl-terminated polymer, T904 is amenable to a variety of end group functionalization and covalent cross-linking strategies that have been developed for preparing hydrogels from multi-arm polyethylene glycol, making it particularly attractive for scaffold-mediated gene delivery.

An E3‐14.7K Peptide that Promotes Microtubules‐Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

Gelatin coating to stabilize the transfection ability of nucleic acid polyplexes

Amphiphilic polymers are effective in complexing and delivering therapeutic nucleic acids, such as plasmid DNA (pDNA) and short interfering RNA (siRNA). However, long-term stability of the complexes is not desirable, as it may have an impact on the transfection efficiency in vivo. To develop a method to preserve complex stability we first showed that pDNA complexes formed with the amphiphilic polymer linoleic acid-substituted polyethylenimine (PEI–LA) and incubated at 37°C lost ∼90% of their transfection efficiency after only 24h of complex formation. Polyethyleneglycol modification of complexes to control the increase in complex size and incubation in scaffolds used for implantation did not preserve the transfection ability of the complexes. Among a variety of approaches explored, gelatin coating of complexes was found to be the best at maintaining the original transfection efficiency. Mechanistic studies suggested that improved complex uptake, not size stability, was responsible for retention of the transfection efficiency. Similarly to the results with pDNA, gelatin coating also prevented the decreases in uptake and silencing efficiency of siRNA complexes observed following incubation at 37°C. Gelatin-stabilized complexes were, furthermore, effective in vivo and led to subcutaneous transgene expression with a low pDNA dose that was otherwise ineffective. We conclude that a simple gelatin coating approach offers an efficient means to preserve the transfection efficiency of polyplexes.

Functional Poly(Dimethyl Aminoethyl Methacrylate) by Combination of Radical Ring‐Opening Polymerization and Click Chemistry for Biomedical Applications

AbstractIn this work, the macromolecular design and modular synthesis of degradable and biocompatible copolymers via radical polymerization and click chemistry is highlighted and the resulting systems are evaluated as gene delivery carriers. Poly(ethylene glycol) (PEG) grafted poly[2‐methylene‐1,3‐dioxepane (MDO)‐co‐propargyl acrylate (PA)‐co‐2‐(dimethyl aminoethyl methacrylate (DMAEMA)] (MPD) is synthesized using radical polymerization and azide‐alkyne click chemistry. The polymers are less cytotoxic and are able to condense plasmid DNA into nanosized particles. The low transfection efficiency of polyplexes in HepG2 cells is significantly improved by mixing Tat peptide with polyplexes.

Role of boronic acid moieties in poly(amido amine)s for gene delivery

The effects of the presence of two different types of phenylboronic acids as side groups in disulfide-containing poly(amido amine)s (SS-PAA) were investigated in the application of these polymers as gene delivery vectors. To this purpose, a para-carboxyphenylboronic acid was grafted on a SS-PAA with pending aminobutyl side chains, resulting in p(DAB–4CPBA) and an ortho-aminomethylphenylboronic acid was incorporated through copolymerization, resulting in p(DAB–2AMPBA). Both polymers have 30% of phenylboronic acid side chains and 70% of residual aminobutyl side chains and were compared with the non-boronated benzoylated analogue p(DAB–Bz) of similar M w. It was found that the presence of phenylboronic acid moieties improved polyplex formation with plasmid DNA since smaller and more monodisperse polyplexes were formed as compared to their non-boronated counterparts. The transfection efficiency of polyplexes of p(DAB–4CPBA) was approximately similar to that of p(DAB–Bz) and commercial PEI (Exgen), both in the absence and the presence of serum, indicating that p(DAB–4CPBA) and p(DAB–Bz) are potent gene delivery vectors. However, the polymers with phenylboronic acid functionalities showed increased cytotoxicity, which is stronger for the ortho-aminophenylboronic acid containing polyplexes of p(DAB–2AMPBA) than for the p(DAB–4CPBA) analog. The cytotoxic effect may be caused by increased membrane disruptive interaction as was indicated by the increased hemolytic activity observed for these polymers.

Pluronic-based cationic block copolymer for forming pDNA polyplexes with enhanced cellular uptake and improved transfection efficiency

PEGylated cationic polymers have been extensively studied for substituting virus as gene delivery vehicles. These polymers can produce water-soluble polyionic complexes (polyplexes) with plasmid DNA (pDNA) and show enhanced stability compared to non-PEGylated polyplexes. However, PEGylation always diminishes the transfection efficiency of polyplexes probably due to poor cellular internalization of the particles and difficulty in releasing the pDNA cargo from the complexes intracellularly for gene expression. As non-ionic surfactants, Pluronic block copolymers have been shown to interact with plasma membrane and promote cellular uptake of various small molecules and biomacromolecules. To evaluate whether Pluronic could improve the transfection efficiency of polyplexes, Pluronic P85- and PEG-based cationomers comprising poly{ N-[ N-(2-aminoethyl)-2-aminoethyl] aspartamide (P[Asp(DET)]) cationic blocks were synthesized and tested for their transfection ability. In this study, it was demonstrated that although the stability of the PEG-based polyplexes was better than that of the P85-based polyplexes based cationic polymers, the P85-based polyplex could achieve significantly higher transfection than the PEG counterparts. The improvement of gene delivering ability was shown to be correlated with the enhanced cellular internalization of the P85-based polyplexes.

The influence of cyclodextrin modification on cellular uptake and transfection efficiency of polyplexes

Cyclodextrin-modified polycations have been studied widely due to their low cytotoxicity, low immunogenicity and the ability to form inclusion complexes. However, the influence of CD modification on cellular uptake and transfection efficiency of polyplexes is still unclear. In this research, cyclodextrin-modified polyethylenimines (PEI-CD) with different CD-grafting levels were synthesized, which were named PEI-CD(15) and PEI-CD(41), respectively, according to the CD number per PEI chain. CD modification showed great influence on the DNA condensation ability of the polycation. PEI-CD(15) could protect DNA completely above N/P ratio of 2. The particle sizes of these polyplexes were about 120 nm. However, PEI-CD(41) could not protect DNA below N/P of 6, and PEI-CD(41)/DNA polyplexes were larger than 1 μm, even at N/P ratio of 10. Therefore, this research was mainly focused on PEI-CD(15). It was interesting that the PEI-CD(15)/DNA polyplexes at N/P ratio of 8 and 10 displayed excellent stability in physiological salt conditions, probably due to the hydration shell of CDs. The influence of CD modification on the cellular uptake and transfection efficiency of polyplexes depended on the type of the cells. Uptake inhibition experiments indicated that PEI/DNA polyplexes were internalized by HEK293T cells by both clathrin-mediated endocytosis and caveolae-mediated endocytosis. The route of caveolae-mediated endocytosis was significantly promoted after CD modification. So the cell uptake and transfection efficiency of PEI-CD(15)/DNA polyplexes were significantly improved for HEK293T cells. However, the uptake and transfection efficiency of PEI-CD(15)/DNA polyplexes in HepG2 cells was similar to that of PEI/DNA polyplexes, probably due to the lack of endogenous caveolins.

The involvement of microtubules and actin filaments in the intracellular transport of non-viral gene delivery system

It is known that two cytoskeleton components, microtubules and actins filaments, are required for efficient endocytosis. The relative importance of these two components in the cellular uptake of 2-(dimethylamino)ethyl methacrylate (DMAEMA)-DNA polyplexes was investigated in this study by applying microtubule depolymerising agent, colchicine, and actin polymerising inhibitor, cytochalasin D, in a cell transfection study. The effect of colchicine on transfection efficiency of polyplexes was found to be a time-dependent phenomenon, whereby the level of gene expression was inhibited at early stage, presumably to the disruption of a transport of vesicles along microtubules by colchicine. As time progressed, the level of gene expression was significantly enhanced relative to the control, possibly due to the failure in transport of vesicles from endosomes to late lysosomes, or due to the breakdown of nuclear membrane when mitosis was arrested at metaphase by colchicine. On the other hand, transfection efficiency was significantly reduced at all time points by cytochalasin D, which is considered to primarily affects invagination of vesicles at the early stage of endocytosis by inhibiting actin polymerisation. Further investigation to identify the endocytotic route of DMAEMA polyplexes was conducted applying clathrin- and caveolae- pathways inhibitors in cell transfection study. The results indicate that DMAEMA polyplexes were internalized primarily through clathrin-mediated pathway, with a minor fraction possibly entering cells via a caveolae-mediated pathway.

Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro

Chitosan and trimethylchitosan (TMC)-siRNA nanoparticles were produced by simple complexation technique or by ionic gelation using tripolyphosphate (TPP). The obtained complexes were characterized in terms of physicochemical properties such as size, zeta potential, complexation efficiency and stability. Furthermore, cytotoxicity, cell uptake and transfection efficiency of polyplexes were evaluated in vitro. Under pH condition of cell culture medium, a strong decrease in siRNA condensation efficiency was observed with chitosan nanoparticles. This characteristic resulted in low transfection efficiencies in HEK293 cell line. Formulation of chitosan polyplexes with TPP led to improvement of polyplexes stability but no significant increase in transfection efficiency was observed compared to simple chitosan complexes. By contrast, TMC complexes did not have pH dependency on siRNA complexation. TMC-siRNA nanoparticles were stable in physiological condition. Accordingly, cellular uptake was increased compared to chitosan polyplexes. However, improvement of transfection efficiency was low regarding to cellular uptake of these complexes. Chitosan and TMC complexes present some characteristics favourable for siRNA delivery, such as ability to integrate siRNA into small discrete particles or low toxicity of the complexes. This study also highlights the importance of complexes stability in physiological environment for siRNA transfection purposes.

A new synthesis method and degradation of hyper-branched polyethylenimine grafted polycaprolactone block mono-methoxyl poly (ethylene glycol) copolymers (hy-PEI-g-PCL-b-mPEG) as potential DNA delivery vectors

Hyper-branched polyethylenimine grafted polycaprolactone block mono-methoxyl poly (ethylene glycol) copolymer (hy-PEI-g-PCL-b-mPEG) was obtained through the conjugation of mPEG–PCL with hyper-branched PEI (hy-PEI) based on the Michael addition. mPEG–PCL was synthesized by ring-opening polymerization of caprolactone using mPEG as the initiator. Compared earlier syntheses, this method offered a reduced number of reaction steps, milder reaction conditions, and a more efficient purification process. FTIR, 1H NMR and 13C NMR spectra proved the structure of the copolymers and controllability of this new synthesis method. Using 1H NMR spectroscopy the degradation of these copolymers was evaluated. Cytotoxicity of copolymers and gene transfection efficiency of polyplexes displayed prominent composition dependence. Increasing the graft density of mPEG–PCL on hy-PEI and longer lengths of both PCL and mPEG within the copolymers investigated here reduced transfection and cytotoxicity on A549 cells. The hy-PEI-g-PCL-b-mPEG copolymers with very short PCL segments (342 Da and 570 Da) demonstrated 6-fold higher transfection efficiency than hy-PEI25k on A549 cells. The polyplexes of the most promising candidate, hy-PEI25k-g-(PCL570-b-mPEG2k)1, exhibited lower hemolysis compared to those of hy-PEI25k.