Poly(ß-amino ester)s for DNA delivery

Nucleotide-based therapeutics have the potential to treat many inherited and acquired diseases. However, for this form of therapy to become clinically successful, safe and efficient delivery vehicles need to be developed. In this article, we review the synthesis, properties, and use of poly(ß-amino ester)s as vectors for gene delivery. High-throughput synthesis and screening studies have identified poly(ß-amino ester)s that can complex DNA and mediate transfection with efficiencies that are superior to the best commercially available polymer- and lipid-based transfection reagents. Structure-function studies show that high-molecular-weight (>10 kDa) amine-terminated polymers with primary alcohol side chains are the most efficient vectors to date. In vivo, the most effective polymer, C32, delivers plasmid DNA at high levels following intra-tumor injection, with excellent biocompatibility. Interestingly, C32 inhibits transfection to surrounding muscle tissue, making it a good candidate for local gene therapy. In addition to simple polymer/DNA complexes insoluble microparticles can be formed using poly(ß-amino ester)s to physically encapsulate DNA and with sizes appropriate for phagocytosis by antigen-presenting cells. Uptake of these particles by macrophages results in protein expression levels up to 5 orders of magnitude higher than traditional poly(lactic-co-glycolic acid) microparticles containing DNA and can be potent stimulators of antigen presenting cells. Furthermore, in vivo delivery of poly(ß-amino ester) microparticle genetic vaccines leads to an antigen-specific, immune-mediated rejection of a lethal tumor dosage. Taken together, these results show that poly(ß-amino ester)s have broad potential as delivery systems for drug and gene therapies.     

Nanotechnology-Based Strategies to Overcome Current Barriers in Gene Delivery

Nanomaterials are currently being developed for the specific cell/tissue/organ delivery of genetic material. Nanomaterials are considered as non-viral vectors for gene therapy use. However, there are several requirements for developing a device small enough to become an efficient gene-delivery tool. Considering that the non-viral vectors tested so far show very low efficiency of gene delivery, there is a need to develop nanotechnology-based strategies to overcome current barriers in gene delivery. Selected nanostructures can incorporate several genetic materials, such as plasmid DNA, mRNA, and siRNA. In the field of nanotechnologies, there are still some limitations yet to be resolved for their use as gene delivery systems, such as potential toxicity and low transfection efficiency. Undeniably, novel properties at the nanoscale are essential to overcome these limitations. In this paper, we will explore the latest advances in nanotechnology in the gene delivery field.     

Low-Frequency Ultrasound Effects on Intracellular Barriers in Nonviral Gene Delivery Processes

Gene therapy, the expression in cells of genetic material with therapeutic activity, has emerged as a promising approach for the treatment or prevention of human diseases. At the present time, major somatic gene-transfer approaches employ either viral or nonviral vectors. Nonviral vectors are less efficient at introducing and maintaining foreign gene expression, but have the profound advantage of being nonpathogenic and nonimmunogenic. In this study, we aimed to develop an efficient nonviral gene delivery system in which low-frequency ultrasound (LFUS) was applied to enhance gene expression of polyplexes formed with poly(2-dimethylaminoethyl methacrylate) and plasmid encoding for green fluorescent protein. Ultrasound (US), and in particular LFUS, can cause temporary membrane permeabilization and thereby enhance drug and gene entrance into viable cells. We evaluated possible additional favorable effects of LFUS on the polyplex transfection process, such as overcoming intracellular barriers. We found that pDMAEMA protected the plasmid DNA from ultrasonic degradation. Atomic force microscopy analysis also confirmed that the LFUS did not change the polyplexes’ morphology. We also attained an insight into the structure of polyplexes during LFUS exposure and found that LFUS induced a temporary partial detachment between the polymer chains and the plasmid. In addition, LFUS application on ovarian carcinoma cells transfected with the polyplexes induced a 27% enhancement in transfection efficiency. Based on these results, we propose that LFUS enhances the decomplexation of the polyplexes, and therefore, can be used to optimize transfection efficiency.     

A sight on the current nanoparticle-based gene delivery vectors

Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/in vivo transformation efficiency.     

Guanidinylation: A simple way to fabricate cell penetrating peptide analogue‐modified chitosan vector for enhanced gene delivery

In view of the analogous transmembrane function to cell penetrating peptides, guanidine group was incorporated into chitosan by chemical modification to enhance the transfection performance of chitosan vectors. Guanidinylated chitosan (GCS) was shown to be well soluble in neutral aqueous solution. The interaction between GCS with plasmid DNA was characterized by agarose retardation experiment and ethidium bromide displacement assay. GCS formed more stable complexes with DNA under physiological pH than chitosan. The transfection efficiency of GCS was evaluated employing COS‐7 cell line—GCS polyplexes demonstrated higher transfection efficiency and lower cytotoxicity relative to chitosan. The optimum efficiency of GCS was achieved in the vicinity of the critical complexing ratio. The results of flow cytometry indicated that guanidinylation promoted an eightfold increase in the cell uptake. The study revealed that guanidinylated chitosan is a promising candidate as an effective nonviral vector for in vivo gene delivery. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors

Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current “gold standard”, namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.     

Layer by layer surface engineering of poly(lactide-co-glycolide) nanoparticles for plasmid DNA delivery

The administration of exogenous DNA has been proposed as a promising therapeutic approach for a variety of diseases. Unfortunately, exogenous DNA is unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery at high transfection efficiency, they are restricted for in vivo applications as they could potentially induce immunogenicity and mutagenesis. To overcome the clinical challenge of viral delivery, a strategy for the encapsulation of plasmid DNA on the surface of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is shown. Plasmid green fluorescence protein (pEF-GFP) or piggybac transposon (PBCAG-eGFP) are assembled on the surface of PLGA NPs through layer by layer technique. The assembly of pEF-GFP with biopolyelectrolytes is monitored on a planar support using a quartz crystal microbalance with dissipation. The assembly of the biopolymer multilayers on PLGA NPs is followed by ζ-potential measurements. Encapsulation of plasmid DNA within the multilayers coating is confirmed by gel electrophoresis. Cellular uptake studies on HEK293 cells revealed that PLGA NPs are taken up by cells within the first 5 hr of co-culturing. Intracellular release of cargo is confirmed by GFP expression in HEK293 cells. PLGA NPs encapsulating pEF-GFP on their surface are able to transfect ~20% of HEK293 cells, while those encapsulating PBCAG-eGFP can transfect up to 75% of cells after 72 hr, causing minimum to non-cytotoxic effects.     

Hyperbranched polymeric "star vectors" for effective DNA or siRNA delivery

Although gene therapy offers an attractive strategy for treating inherited disorders, current techniques using viral and nonviral delivery systems have not yielded many successful results in clinical trials. Viral vectors such as retroviruses, lentiviruses, and adenoviruses deliver genes efficiently; however, the possibility of negative outcomes from viral transformation cannot be completely ruled out. In contrast, various types of nonviral vectors are attracting considerable attention because they are easier to handle and induce weak immune responses. Cationic polymers, such as polyethylenimine (PEI) and poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm), can generate nanoparticles through the formation of polyion complexes, "polyplexes" with DNA. These nonviral systems offer many advantages over viral systems. The primary obstacle to implementing these cationic polymers in an effective gene therapy remains their comparatively inefficient gene transfection in vivo. We describe four strategies for the development of hyperbranched star vectors (SVs) for enhancing DNA or siRNA delivery. The molecular design was performed by living radical polymerization in which the chain length can be controlled by photoirradiation and solution conditions, including concentrations of the monomer or iniferter (a molecule that serves as a combination of initiator, transfer agent, and terminator). The branch composition is controlled by the types of monomers that are added stepwise. In our first strategy, we prepared a series of only cationic PDMAPAAm-based SVs with no branches or 3, 4, or 6 branching numbers. These SVs could form polyion complexes (polyplexes) by mixing with DNA only in aqueous solution. The relative gene expression activity of the delivered DNA increased according to the degree of branching. In addition, increasing the molecular weight of SVs and narrowing their polydispersity index (PDI) improved their activity. For targeting DNA delivery to the specific cells, we modified the SV with ligands. Interestingly, the SV could adsorb the RGD peptide, making gene transfer possible in endothelial cells which are usually refractory to such treatments. The peptide was added to the polyplex solution without covalent derivatization to the SV. The introduction of additional branching by cross-linking using iniferter-induced coupling reactions further improved gene transfection activity. After block copolymerization of PDMAPAAm-based SVs with a nonionic monomer (DMAAm), the blocked SVs (BSVs) produced polyplexes with DNA that had excellent colloidal stability for 1 month, leading to efficient in vitro and in vivo gene delivery. Moreover, BSVs served as carriers for siRNA delivery. BSVs enhanced siRNA-mediated gene silencing in mouse liver and lung. As an alternative approach, we developed a novel gene transfection method in which the polyplexes were kept in contact with their deposition surface by thermoresponsive blocking of the SV. This strategy was more effective than reverse transfection and the conventional transfection methods in solution.     

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis,Characterization, and Evaluation

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; M_W 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.     

Folic acid‐conjugated depolymerized quaternized chitosan as potential targeted gene delivery vector

Ligand‐conjugated delivery vectors that target over‐expressed cell surface receptors have a potential impact on gene therapy. In the study reported, high‐molecular‐weight chitosan was depolymerized to medium and low molecular weight and trimethylated to render the polymer soluble over a wider pH range. Folate conjugation was introduced to the quarternized derivative to improve gene transfection efficiency. Complexes of the folic acid‐conjugated trimethylated depolymerized chitosan (FTMC) with plasmid DNA (pDNA) formed core–shell nanostructured particles. Gel electrophoretic band retardation showed efficient condensation of DNA. These derivatives and their complexes with pDNA were tested for toxicity and haemocompatibility and were found to be significantly less toxic and haemocompatible than polyethyleneimine. Transfection efficiency and nuclear uptake properties were tested in the human KB oral epidermoid cell line, which over‐expresses the folate receptor in the presence of 10% serum. Among the four FTMC derivatives investigated, folic acid‐conjugated chitosan having low molecular weight and medium folate conjugation was found to be a potential vector for gene delivery applications with good transfection and nuclear uptake properties, as proved by YOYO labelling of pDNA. Copyright © 2011 Society of Chemical Industry     

Calcium Phosphate—DNA Nanocomposites: Morphological Studies and Their Bile Duct Infusion for Liver-Directed Gene Therapy

In this study, liver-directed gene transfer in rats with calcium phosphate (CaP) nanoparticles and the effect of the route of administration and surgical manipulations on transfection efficiency is reported. Formulations of CaP nanoparticles entrapping plasmid DNA (pDNA) were prepared by the reverse micellar method using two different surfactants. Transmission electron microscopy, scanning electron microscopy and dynamic light scattering were used to characterize the CaP–DNA nanocomposites. The morphological characteristics of the formulations showed a strong dependency on temperature. Gel electrophoresis experiments indicated that there was no degradation of the encapsulated pDNA, and in vitro cell transfection in HEK-293 and primary hepatocytes from rats as well as in vivo intraductal delivery experiments suggested that CaP nanoparticles led to significant and prolonged transgene expression. Therefore, our methodology gives a stable and viable formulation for hepatic gene therapy. Low-DNA dosage entrapped in CaP nanoparticles makes it an effective gene delivery system for clinical applications.     

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.     

Peptide vectors for the nonviral delivery of nucleic acids

Over the past two decades, gene therapy has garnered tremendous attention and is heralded by many as the ultimate cure to treat diseases such as cancer, viral infections, and inherited genetic disorders. However, the therapeutic applications of nucleic acids extend beyond the delivery of double-stranded DNA and subsequent expression of deficient gene products in diseased tissue. Other strategies include antisense oligonucleotides and most notably RNA interference (RNAi). Antisense strategies bear great potential for the treatment of diseases that are caused by misspliced mRNA, and RNAi is a universal and extraordinarily efficient tool to knock down the expression of virtually any gene by specific degradation of the desired target mRNA. However, because of the hurdles associated with effective delivery of nucleic acids across a cell membrane, the initial euphoria surrounding siRNA therapy soon subsided. The ability of oligonucleotides to cross the plasma membrane is hampered by their size and highly negative charge. Viral vectors have long been the gold standard to overcome this barrier, but they are associated with severe immunogenic effects and possible tumorigenesis. Cell-penetrating peptides (CPPs), cationic peptides that can translocate through the cell membrane independent of receptors and can transport cargo including proteins, small organic molecules, nanoparticles, and oligonucleotides, represent a promising class of nonviral delivery vectors. This Account focuses on peptide carrier systems for the cellular delivery of various types of therapeutic nucleic acids with a special emphasis on cell-penetrating peptides. We also emphasize the clinical relevance of this research through examples of promising in vivo studies. Although CPPs are often derived from naturally occurring protein transduction domains, they can also be artificially designed. Because CPPs typically include many positively charged amino acids, those electrostatic interactions facilitate the formation of complexes between the carriers and the oligonucleotides. One drawback of CPP-mediated delivery includes entrapment of the cargo in endosomes because uptake tends to be endocytic: coupling of fatty acids or endosome-disruptive peptides to the CPPs can overcome this problem. CPPs can also lack specificity for a single cell type, which can be addressed through the use of targeting moieties, such as peptide ligands that bind to specific receptors. Researchers have also applied these strategies to cationic carrier systems for nonviral oligonucleotide delivery, such as liposomes or polymers, but CPPs tend to be less cytotoxic than other delivery vehicles.     

Graphene based gene transfection

Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.     

omega-Hydrazino linear polyethylenimine: a monoconjugation building block for nucleic acid delivery

Nonviral gene therapy requires efficient vectors that are able to deliver nucleic acids inside the targeted cell nucleus. Developing new tools for the synthesis of supramolecular vectors with improved transfection efficiency and better biodistribution is therefore a crucial issue. Here we describe the synthesis of a 140-mer linear polyethylenimine (L-PEI) terminated at one end by a highly nucleophilic hydrazine residue. This cationic polymer, whose backbone is well known for its remarkable gene-delivery efficiency, constitutes a building block for omega-regioselective conjugation to molecules through the formation of stable linkages such as the hydrazone bonds. To demonstrate the potential of the omega-hydrazino linear polyethylenimine, human serum transferrin, a ligand that is well know to improve gene-delivery systems, was used as a model of sensitive material. The blood protein was oxidized to generate an aldehyde function and was subsequently conjugated to hydrazino PEI. The new polyethylenimine-transferrin (PEI-Tf) vector was purified and was shown to condense plasmid DNA into compact superstructures compatible with cellular uptake. Finally, the cellular-binding and gene-delivery properties of PEI/DNA polyplexes incorporating different quantities of transferrin were evaluated by FACS analysis and luciferase assay.     

CHO-DG44细胞瞬时转染条件的优化

目的优化CHO-DG44细胞瞬时转染的条件。方法采用TubeSpin一次性生物反应器,以绿色荧光蛋白(Green fluorescence protein,GFP)为报告基因,氯醚酰亚胺(Polyether imide,PEI)为转染试剂,将质粒pIRESneo3-eGFP瞬时转染CHO-DG44细胞,优化瞬时转染的基本条件[细胞密度、DNA浓度和DNA:PEI比例(w/w)]以及其他条件(换液、渗透压和温度),采用优化的条件转染质粒pIRESneo3-eGFP,流式细胞术检测细胞转染效率,生物发光仪检测相对荧光强度。结果 CHO-DG44细胞瞬时转染的最佳条件为:采用XLG-P8培养基进行转染,细胞密度为2×106个/ml,DNA浓度为6.25μg/5 ml,DNA∶PEI(w/w)比例为1∶5;转染后4 h更换新鲜培养基,添加30 mmol/L NaCl,并于31℃继续培养。在此条件下,CHO-DG44细胞的瞬时转染效率可达81.45%,相对荧光强度可达9×105RFU/106cells。结论优化了CHO-DG44细胞瞬时转染的条件,为下一步药物蛋白的研发奠定了基础。     

Recent advances in nonviral vectors for gene delivery

Gene therapy has long been regarded a promising treatment for many diseases, whether acquired (such as AIDS or cancer) or inherited through a genetic disorder. A drug based on a nucleic acid, however, must be delivered to the interior of the target cell while surviving an array of biological defenses honed by evolution. Successful gene therapy is thus dependent on the development of an efficient delivery vector. Researchers have pursued two major vehicles for gene delivery: viral and nonviral (synthetic) vectors. Although viral vectors currently offer greater efficiency, nonviral vectors, which are typically based on cationic lipids or polymers, are preferred because of safety concerns with viral vectors. So far, nonviral vectors can readily transfect cells in culture, but efficient nanomedicines remain far removed from the clinic. Overcoming the obstacles associated with nonviral vectors to improve the delivery efficiency and therapeutic effect of nucleic acids is thus an active area of current research. The difficulties are manifold, including the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, and managing the targeting ability of the carriers with respect to the cells of interest. Modifying the surface with poly(ethylene glycol), that is, PEGylation, is the predominant method used to reduce the binding of plasma proteins to nonviral vectors and minimize clearance by the RES after intravenous administration. Nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors because of the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral and anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal models. Other approaches include (i) designing and synthesizing novel cationic lipids and polymers, (ii) chemically coupling the nucleic acid to peptides, targeting ligands, polymers, or environmentally sensitive moieties, and (iii) utilizing inorganic nanoparticles in nucleic acid delivery. Recently, the different classes of nonviral vectors appear to be converging, and the ability to combine features of different classes of nonviral vectors in a single strategy has emerged. With the strengths of several approaches working in concert, more hurdles associated with efficient nucleic acid delivery might therefore be overcome. In this Account, we focus on these novel nonviral vectors, which are classified as multifunctional hybrid nucleic acid vectors, novel membrane/core nanoparticles for nucleic acid delivery, and ultrasound-responsive nucleic acid vectors. We highlight systemic delivery studies and consider the future prospects for nucleic acid delivery. A better understanding of the fate of the nanoparticles inside the cell and of the interactions between the parts of hybrid particles should lead to a delivery system suitable for clinical use. We also underscore the value of sustained release of a nucleic acid in this endeavor; making vectors targeted to cells with sustained release in vivo should provide an interesting research challenge.     

DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells.     

A supramolecular gene carrier composed of multiple cationic α-cyclodextrins threaded on a PPO-PEO-PPO triblock polymer

Cationic polymers have been studied as promising nonviral gene delivery vectors. In contrast to the conventional polycations with long sequences of covalently bonded repeating units, this work reports a supramolecular gene carrier where many cationic cyclic units are threaded over a polymer chain to form a chain-interlock structured gene carrier. A series of novel supramolecular cationic polyrotaxanes consisting of multiple α-cyclodextrin (α-CD) rings grafted with various linear or nonlinear oligoethylenimine (OEI) chains, which are threaded and capped over a reverse Pluronic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEO-PPO) amphiphilic triblock copolymer chain, were synthesized and characterized in term of their molecular and supramolecular structures, DNA binding and condensation ability, cytotoxicity, and in vitro gene transfection efficiency in cultured cells. The supramolecular cationic polyrotaxanes were found to contain 8 cationic α-CD rings that are threaded on a PPO-PEO-PPO triblock copolymer chain. They demonstrated strong ability to bind and condense plasmid DNA into nano-sized particles which are suitable for gene delivery. In both HEK293 and COS7 cells, these polyrotaxanes show low cytotoxicity and high transfection efficiency. In particular, the cationic polyrotaxanes displayed sustained gene delivery capability in HEK293 cells in both serum and serum free condition with the increasing expression duration.     

Bioreducible Guanidinylated Polyethylenimine for Efficient Gene Delivery

Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought‐after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI‐SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI‐SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI‐SS demonstrate that it has the appropriate size (～200 nm) and surface potential (～30 mV) at a nitrogen‐to‐phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI‐SS, GBPEI‐SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.