Enhanced Gene Delivery Research Articles

Improving drug loading efficiency and delivery performance of micro- and nanoparticle preparations through optimising formulation variables

In this report, we briefly introduce our recent studies related to drug delivery systems using biodegradable materials, PLGA, PLA or chitosan to prepare submicroparticles or microparticles for carrying vaccine, protein or small molecule drugs. These preparations were prepared by spray dried or solvent extraction methods. In addition, liposomal formulations containing various lipids and pegylated lipids were also investigated. Gene–loaded liposomal preparations were prepared and evaluated in ARPE19 (retinal pigment epithelium cell). These preparations were significantly internalised by ARPE19 and confirmed by confocal microscopy. Pegylated lipid linked with RGD peptide was synthesised for preparing siRNA delivery systems; these delivery systems could significantly enhance gene delivery and reduce the VEGF expression of ARPE19 for achieving anti–angiogenic effects. Another, poorly soluble compound was loaded in liposomal preparation which was further investigated in A549 (non–small cell lung cancer cell line). The preparation had higher cellular uptake to improve anti–cancer activity. Pegylated lipid with conjugated–ligand plays an important role to achieve efficient drug delivery. The strategy in preparing conjugated–ligand liposomal formulations can be a potential application for targeting delivery in cancer therapy or specific delivery to achieve therapeutic effects.

Developing a chitosan supported imidazole Schiff-base for high-efficiency gene delivery

A chitosan supported imidazole Schiff-base (CISB) has been developed to be used as the vector for high performance gene delivery. Introducing the imidazole Schiff-base to the branch of chitosan could improve its water solubility and gene binding ability under physiological conditions, and thus significantly enhance gene delivery capability due to the formation of Schiff-bases (azomethines) and the substitution of imidazole functional groups along chitosan backbones. Gel electrophoresis and light scattering results show that the CISB could effectively bind plasmid DNA (pDNA) and protect pDNA from DNase I digestion in solution. The CISB does not induce remarkable cytotoxicity against HEK 293 cells and can enhance delivery of pDNA into cytoplasm and nucleus efficiently. A transfection efficiency of 70% can be reached after systematically optimizing cell transfection conditions. Therefore, the CISB is a promising gene delivery vector due to its high solubility in physiological pH, strong gene binding and protection capability, low cytotoxicity, good biodegradability, and high efficiency in gene delivery and cell transfection.

Grafting zwitterionic polymer chains onto PEI as a convenient strategy to enhance gene delivery performance

Herein, we demonstrated a one-step method for preparing a new gene delivery vector by grafting poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) onto the 25 kDa polyethylenimine (PEI 25k) via a TBHP-initiated polymerization. The graft ratio of PMPC on the PEI polymer can be readily controlled, and in this study three PEI-PMPC polymers (PEI-PMPC6%, PEI-PMPC11% and PEI-DMAAPS24%) have been prepared with their graft ratios of 6 wt%, 11 wt% and 24 wt%, respectively. The PEI-PMPC polymers exhibit lower protein adsorption and cytotoxicity compared to PEI 25k. Moreover, the PEI-PMPC polymers display satisfactory DNA condensation capability. In the absence and presence of serum, PEI-PMPC6%/pEGFP and PEI-PMPC11%/pEGFP complexes work well and exhibit satisfactory gene transfection efficiencies, which are in general higher than that of PEI 25k/pEGFP, especially in the presence of 10% serum. With these improved gene delivery properties, the PEI-PMPC polymers hold great potential for being used as efficient gene delivery vectors. This strategy may provide a facile and effective way for constructing some other biocompatible materials.

Polycations-functionalized water-soluble gold nanoclusters: a potential platform for simultaneous enhanced gene delivery and cell imaging

Noble metal nanoclusters have emerged as a fascinating area of widespread interest in nanomaterials. Herein, we report the synthesis of the PEI-templated gold nanoclusters (PEI-AuNCs) as an efficient carrier for gene delivery. The PEI-AuNCs integrate the advantages of PEI and AuNCs: the presence of AuNCs can effectively decrease the cytotoxicity of PEI, making it possible to apply them in biological systems, while the cationic polymer layer PEI with positive charges is essential for enhanced gene transfection efficiency. In addition, with excellent photoluminescent properties, the AuNCs also endow our system with the versatility of fluorescent imaging, indicating a great potential as an ideal fluorescent probe to track the transfection behavior. Our studies provide strong evidence that the PEI-AuNCs can be utilized as efficient gene delivery agents.

Application of Ferriferous Oxide Modified by Chitosan in Gene Delivery

New approaches to improve the traditional gene carriers are still required. Here we explore Fe3O4 modified with degradable polymers that enhances gene delivery and target delivery using permanent magnetic field. Two magnetic Fe3O4 nanoparticles coated with chitosan (CTS) and polyethylene glycol (PEG) were synthesized by means of controlled chemical coprecipitation. Plasmid pEGFP was encapsulated as a reported gene. The ferriferous oxide complexes were approximately spherical; surface charge of CTS-Fe3O4 and PEG-Fe3O4 was about 20 mv and 0 mv, respectively. The controlled release of DNA from the CTS-Fe3O4 nanoparticles was observed. Concurrently, a desired Fe3O4 concentration of less than 2 mM was verified as safe by means of a cytotoxicity test in vitro. Presence of the permanent magnetic field significantly increased the transfection efficiency. Furthermore, the passive target property and safety of magnetic nanoparticles were also demonstrated in an in vivo test. The novel gene delivery system was proved to be an effective tool required for future target expression and gene therapy in vivo.

Oligobenzylethylenimine enriches linear polyethylenimine with a pH-sensitive membrane-disruptive property and leads to enhanced gene delivery activity

We report here the synthesis of a diblock linear polymer of oligo(benzylethylenimine)-b-polyethylenimine (OBzEI-PEI) and investigate its gene delivery properties. The linear copolymer OBzEI-PEI was prepared in a straightforward manner by acidic hydrolysis of a diblock polyoxazoline, which had been made by sequential polymerization of 4-benzyl-2-ethyl-2-oxazoline followed by 2-ethyl-2-oxazoline. pH titration and DNA complexation profiles of the new polymer are similar to regular linear PEIs, but with higher gene transfection efficiencies in various cell lines despite a decreased cellular uptake of plasmid DNA. Further experiments suggest that the OBzEI tail complements the intrinsic proton-sponge endosomolytic activities of PEI with an acid pH-sensitive membrane-perturbing activity.

Modeling the proton sponge hypothesis: examining proton sponge effectiveness for enhancing intracellular gene delivery through multiscale modeling

Dendrimers have been proposed as therapeutic gene delivery platforms. Their superior transfection efficiency is attributed to their ability to buffer the acidification of the endosome and attach to the nucleic acids. For effective transfection, the strategy is to synthesize novel dendrimers that optimize both of these traits, but the prediction of the buffering behavior in the endosome remains elusive. It is suggested that buffering dendrimers induce an osmotic pressure sufficient to rupture the endosome and release nucleic acids, which forms to sequestrate most internalized exogenous materials. Presented here are the results of a computational study modeling osmotically driven endosome burst or the ‘proton sponge effect.’ The approach builds on previous cellular simulation efforts by linking the previous model with a sponge protonation model, then observing the impact on endosomal swelling and acidification. Calibrated and validated using reported experimental data, the simulations offer insights into defining the properties of suitable dendrimers for enhancing gene delivery as a function of polymer structure.

Hydrogel macroporosity and the prolongation of transgene expression and the enhancement of angiogenesis

The utility of hydrogels for regenerative medicine can be improved through localized gene delivery to enhance their bioactivity. However, current systems typically lead to low-level transgene expression located in host tissue surrounding the implant. Herein, we investigated the inclusion of macropores into hydrogels to facilitate cell ingrowth and enhance gene delivery within the macropores in vivo. Macropores were created within PEG hydrogels by gelation around gelatin microspheres, with gelatin subsequently dissolved by incubation at 37 °C. The macropores were interconnected, as evidenced by homogeneous cell seeding in vitro and complete cell infiltration in vivo. Lentivirus loaded within hydrogels following gelation retained its activity relative to the unencapsulated control virus. In vivo, macroporous PEG demonstrated sustained, elevated levels of transgene expression for 6 weeks, while hydrogels without macropores had transient expression. Transduced cells were located throughout the macroporous structure, while non-macroporous PEG hydrogels had transduction only in the adjacent host tissue. Delivery of lentivirus encoding for VEGF increased vascularization relative to the control, with vessels throughout the macropores of the hydrogel. The inclusion of macropores within the hydrogel to enhance cell infiltration enhances transduction and influences tissue development, which has implications for multiple regenerative medicine applications.

Nanoparticles for Targeted Delivery of Active Agents against Tumor Cells

Cancer therapy may still be considered as one of the most challenging issues in pharmaceutical sciences. Various new technologies and molecules have been developed to meet the needs of this devastating disease, but there is a long way to go. One of the most important challenges is finding a drug molecule or drug delivery system which only influences the cancer cells. This has not been achieved so far even though a great deal of research has focused on this goal. Most of the cytotoxic agents used for cancer treatment are toxic in nature and not only have an effect on tumor cells, but also normal cells are damaged when exposed with them. As a result, patients may experience intensive side effects which may lead to an early termination in chemotherapy due to their severity. Targeting the cancer cells specifically and selectively seems to be the best solution for this obstacle. Targeting the cancer cells occurs via two different strategies: passive targeting and active targeting. The conventional approach used for cancer therapy may be considered a sort of passive targeting. Cytotoxic agents used for cancer treatment select the cancer cells due to their faster growth rate. Inevitably, normal fast growing cells and normal fast dividing cells may damage following conventional chemotherapy. In 1986, Matsumura and Maeda brought up a new concept in passive targeting called enhanced permeation and retention (EPR) effect which was widely used in many researches as an approach for targeting tumor cells. In this strategy, the drug delivery system benefits from defective vasculature, fenestrations, and poor lymphatic drainage in tumor tissues. Utilizing molecular targeting moieties is another strategy which is widely used in new researches for targeting tumor cells actively and diminishing chemotherapy side effects. This special issue will primarily address new approaches for passive and active targeted cancer therapy with a focus on nanoparticle formulations specially those ones that specifically and selectively target tumors. There is a peper which describes using a natural protein, atelocollagen for oligonucleotide delivery, and evaluating its biological function. The researchers found that when oligonucleotides were used concomitantly with atelocollagen, paracellular flux was enhanced, but when each of these two constituents of the formulation was used separately, no effect was observed. Another paper reviews poly(lactic-co-glycolic acid)- (PLGA-) based nanoparticles especially echogenic ones to enhance gene delivery. Ultrasound may be used to enhance localized gene delivery in tumors. On the other hand, nanoparticles themselves may utilize targeting mechanisms to enhance the targeting potential of the current system. Another paper describes microemulsions in the form of nanocarriers for passive tumor targeting. In this study, an experimental design layout was used to optimize the excipients selection for microemulsion nanocarriers. These nanocarriers possess the ability of solubilizing hydrophobic drugs and physical stability alongside with their capability of passive targeting. In another paper human serum albumin was used as a naturally occurring protein to create nanoparticles for enhanced drug delivery in breast cancer. Albumin-based nanoparticles were further cationized by polyethylenimine. It was shown that delivery efficacy was improved by passive EPR strategy. One of the papers also reviews carbon nanotubes for cancer therapy and designing drug delivery systems. Carbon nanotubes present a great canvas for surface engineering and creating novel targeted nanoparticles. Another paper presents glucan particles which actively target cancer cells. Glucan particles which benefit from β-glucan derived from the cell wall of Saccharomyces cerevisiae provide receptor-mediated uptake by phagocytic macrophage cells who express β-glucan receptors. Different types of nanoparticles were incorporated or surface bounded to these glucan particles to deliver doxorubicin to macrophages. Macrophages further migrate into solid tumors carrying the cytotoxic agent to the target tissues like a Trojan horse, while they are resistant to the effects of doxorubicin due to their nondividing differentiated nature. Another paper reviews noble metal nanoparticles with a wide variety of biomedical applications which are used for both therapeutic and diagnostic purposes. They also present facile surface chemistry and the possibility of functionalizing on their surfaces. Finally, in one of the papers, a lipoplatin formulation consisting of liposomal encapsulated cisplatin is reviewed. The purpose of this formulation is to become a substitute for the original cisplatin and has gained positive results in clinical trials. Lipoplatin gives 200-fold higher concentrations in tumor tissues in comparison with cisplatin once more because of the compromised endothelium of tumor tissues. To sum up, this special issue tries to provide the readers some insight about the targeted nanoparticles which are designed for cancer therapy both for small molecular drugs and gene delivery systems. Rassoul Dinarvand Paulo Cesar de Morais Antony D'Emanuele

Factors influencing regeneration and Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris L.)

A systematic study was carried out to optimize regeneration and Agrobacterium tumefaciens-mediated transformation of four common bean (Phaseolus vulgaris L.) cultivars; Red Hawk, Matterhorn, Merlot, and Zorro, representing red kidney, great northern, small red, and black bean commercial classes, respectively. Regeneration capacity of leaf explants, stem sections, and embryo axes were evaluated on 30 media each containing Murashige and Skoog (MS) medium and different combinations of plant growth regulators. For stem sections and leaf explants, none of the media enabled plant regeneration from any of the four cultivars tested, indicating the recalcitrance of bean regeneration from these tissues. In contrast, several media enabled multiple shoot production from embryo axis explants, although optimal regeneration media was genotype-dependent. Under optimal regeneration conditions, multiple shoots, 2.3–10.8 on average for each embryogenic explant, were induced from embryo axis explants at frequencies of 93 % for ‘Merlot’, 80 % for ‘Matterhorn’, 73 % for ‘Red Hawk’, and 67 % for ‘Zorro’. Transient expression studies monitored by an intron-interrupted gusA on explants transformed with A. tumefaciens strains GV3101, LBA4404, and EHA105 indicated that all three A. tumefaciens strains tested were efficient in gene delivery. Gene delivery depended on parameters including strain of A. tumefaciens, co-cultivation time, explant type, and bean genotype. Agroinfiltration also enhanced gene delivery. Kanamycin-resistant and GUS-positive calluses were induced from leaf, stem, and embryo axis explants. Chimeric transformants were obtained from embryo axis explants and showed partial GUS-staining. Lack of efficient regeneration from non-meristem containing tissues is the main limitation for stable transformation of common bean.

Involvement of Ca2+ and ATP in Enhanced Gene Delivery by Bubble Liposomes and Ultrasound Exposure

Recently, we reported the accelerated gene transfection efficiency of laminin-derived AG73-peptide-labeled polyethylene glycol-modified liposomes (AG73-PEG liposomes) and cell penetrating TAT-peptide labeled PEG liposomes using PEG-modified liposomes, which trap echo-contrast gas, "Bubble liposomes" (BLs), and ultrasound (US) exposure. BLs and US exposure were reported to enhance the endosomal escape of AG73-PEG liposomes, thereby leading to increased gene expression. However, the mechanism behind the effect of BLs and US exposure on endosomes is not well understood. US exposure was reported to induce an influx of calcium ions (Ca²⁺) by enhancing permeability of the cell membrane. Therefore, we examined the effect of Ca²⁺ on the endosomal escape and transfection efficiency of AG73-PEG liposomes, which were previously enhanced by BLs and US exposure. For cells treated with EGTA, the endosomal escape and gene expression of AG73-PEG liposomes were not enhanced by BLs and US exposure. Similarly, transfection efficiency of the AG73-PEG liposomes in ATP-depleted cells was not enhanced. Our results suggest that Ca²⁺ and ATP are necessary for the enhanced endosomal escape and gene expression of AG73-PEG liposomes by BLs and US exposure. These findings may contribute to the development of useful techniques to improve endosomal escape and achieve efficient gene transfection.

Ligands located within a cholesterol domain enhance gene delivery to the target tissue

Targeted gene delivery provides enormous potential for clinical treatment of many incurable diseases. Liposomes formulated with targeting ligands have been tested extensively both in vitro and in vivo, and many studies have strived to identify more efficacious ligands. However, the environment of the ligand within the delivery vehicle is generally not considered, and this study assesses the effect of ligand microenvironment by utilizing a lipoplex possessing a cholesterol domain. Our recent work has shown that the presence of the targeting ligand within the cholesterol domain promotes more productive transfection in cultured cells. In the present study, lipoplexes having the identical lipid composition were formulated with different conjugates of the folate ligand such that the ligand was included in, or excluded from, the cholesterol domain. The effect of locating the ligand within the cholesterol domain was then tested in a xenograft tumor model in mice. Lipoplexes that included the ligand within the cholesterol domain showed significantly higher luciferase expression and plasmid accumulation in tumors as compared to lipoplexes in which the ligand was excluded from the domain. These results demonstrate that the microenvironment of the ligand can affect gene delivery to tumors, and show that ligand-mediated delivery can be enhanced by locating targeting ligands within a cholesterol domain.

Protamine and Chloroquine Enhance Gene Delivery and Expression Mediated by RNA-Wrapped Single Walled Carbon Nanotubes

The use of non-viral vectors as delivery systems in gene therapy has been extensively studied recently owing to their advantages over viral vectors. Here, we propose a new gene delivery system based on the use of RNA-wrapped single-walled carbon nanotubes (SWCNTs) complexed with the cationic protein, protamine and the drug chloroquine. Protamine was selected as a cationic protein acting as bridge between negatively charged RNA-wrapped SWCNTs and plasmid DNA. Protamine also contains a nuclear localization signal which enhances the expression of the transfected gene. The drug chloroquine, a lysosomotropic compound which has been reported to increase the transfection efficiency, was attached to RNA-wrapped SWNTs by ionic interactions. The simultaneous delivery of the drug chloroquine with plasmid DNA clearly showed an enhanced gene delivery and expression. The levels of gene expression were quantified using the luciferase reporter gene as model. Optimal conditions for transfection and gene expression were obtained and cytoxicity of the carbon nanotube complexes measured. The optimal complexes were shown to efficiently deliver plasmid DNA for efficient gene expression and may thereby be useful as gene delivery systems for gene therapy.

PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid) (PLGA) or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound) composed either of polymers (PLGA, polystyrene) or other contrast agent materials (Optison, SonoVue microbubbles). The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a) echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b) PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery.

Characterization of Ku702–NLS as Bipartite Nuclear Localization Sequence for Non-Viral Gene Delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku702-NLS), a nuclear transport active mutant of Ku702-NLS (s1Ku702-NLS) and a nuclear transport deficient mutant of Ku702-NLS (s2Ku702). We examined the transfection efficiency of binary Ku702-NLS/DNA and ternary Ku702-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku702-NLS and s1Ku702-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

Hyaluronic Acid Enhances Gene Delivery into the Cochlea

Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

Single tyrosine mutation in AAV8 and AAV9 capsids is insufficient to enhance gene delivery to skeletal muscle and heart.

Site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of adeno-associated viral (AAV) capsids have been reported as a simple method to greatly enhance gene transfer in vitro and in vivo. To determine whether the Y-to-F mutation could also enhance AAV8 and AAV9 gene transfer in skeletal muscle and heart to facilitate muscular dystrophy gene therapy, we investigated four capsid mutants of AAV8 (Y447F or Y733F) and AAV9 (Y446F or Y731F). The mutants and their wild-type control AAV8 and AAV9 capsids were used to package reporter genes (luciferase or β-galactosidase) resulting in similar vector yields. To evaluate gene delivery efficiencies, especially in muscle and heart, the vectors were compared side by side in a series of experiments in vivo in two different strains of mice, the outbred ICR and the inbred C57BL/6. Because AAV8 and AAV9 are among the most effective in systemic gene delivery, we first examined the mutant and wild-type vectors in neonatal mice by intraperitoneal injection, or in adult mice by intravenous injection. To our surprise, no statistically significant differences in transgene expression were observed between the mutant and wild-type vectors, regardless of the reporter genes, vector doses, and the ages and strains of mice used. In addition, quantitative analyses of vector DNA copy number in various tissues from mice treated with mutant and wild-type vectors also showed similar results. Finally, direct intramuscular injection of the above-described vectors with the luciferase gene into the hind limb muscles revealed the same levels of gene expression between mutant and wild-type vectors. Our results thus demonstrate that a single mutation of Y447F or Y733F on capsids of AAV8, and of Y446F or Y731F on AAV9, is insufficient to enhance gene delivery to the skeletal muscle and heart.

Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells.

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.

Enhanced gene delivery by palmitic acid-conjugated low molecular weight polyethylenimine

Palmitic acid-conjugated low molecular weight polyethylenimine (PEI-g-PEG-PA) was successfully synthesized to develop an efficient non-viral gene carrier. The judicious integration of hydrophobic palmitic acid and polyethylenimine via hydrophilic polyethylene glycol (PEG) facilitated the formation of nano-sized complex (nanoplex) with plasmid DNA (pDNA) which provided the protection of pDNA against the serumic enzymatic degradation. Furthermore, the delivery system demonstrated enhanced gene transfection efficiency in comparison to unmodified low molecular weight PEI without inducing any significant cytotoxicity.

High efficiency and low toxicity of polyethyleneimine modified Pluronics (PEI–Pluronic) as gene delivery carriers in cell culture and dystrophic mdx mice

A series of low molecular weight polyethyleneimine (LPEI) conjugated Pluronic copolymers (PCMs) were synthesized and evaluated in C2C12 myoblasts and CHO cells in vitro and in dystrophic mdx mice in vivo as gene delivery carriers. Pluronics with different molecular weights (Mw) and hydrophilic–lipophilic-balance (HLB), and two LPEIs (Mw: 0.8k, 1.2k Da) as composition of PCMs have been synthesized, and the dynamics between the structures and properties were investigated. The conjugation efficiency of PEI ranged from 77.5–95.4% with relatively higher levels of PEI conjugation to the Pluronic size in PCMs, with the Pluronics of smaller size achieving relatively higher levels of PEI conjugation. Almost all of the PCM polymers were able to bind and condense plasmid DNA effectively into particles of approximately 200 nm in solution at the polymer/DNA ratio of 2 and above. The PCMs composed of relatively moderate size (Mw: 2000–5000 Da), intermediate HLB (12–23) of Pluronics, and LPEI produce much higher gene delivery efficacy and less cytotoxicity as compared with PEI 25k in C2C12 myoblasts and CHO cells in vitro. The PCMs were also able to enhance gene delivery in mdx mice in vivo, indicating their potential as biocompatible gene delivery carriers.