Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies

In the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.     

Evaluation of PEG-transferrin-PEI nanocomplex as a gene delivery agent

Polyethylenimine (PEI) has been shown to be an efficient nonviral delivery vector. To improve its specificity and reduce its cytotoxicity, PEI should be modified. Transferrin (Tf) is a cell-binding ligand and Tf-receptors are expressed in malignant cells. Modification of cationic polymer by polyethylene glycol (PEG) can reduce the protein interaction and cell cytotoxicity of delivery vectors. We have synthesized PEG-Tf-PEI conjugate as an efficient and safe carrier of plasmid DNA (pDNA). Nanocomplexes of conjugates with pDNA were characterized by measuring the particle size and the surface charge. Transfection efficiency of nanocomplexes in Jurkat cells was improved and cytotoxicity was decreased compared with those of PEI complex. This was due to a reduction in the membrane damaging effect via shielding of the positive charge on the nanocomplex surface by PEG.     

A Highly Emissive Conjugated Polyelectrolyte Vector for Gene Delivery and Transfection

An intrinsically fluorescent cationic polyfluorene ( CCP ) has been designed, synthesized, characterized, and examined as a plasmid DNA (pDNA) delivery vector. This material facilitates nucleic acid binding, encapsulation and efficient cellular uptake. CCP can effectively protect pDNA against nuclease degradation, which is necessary for gene carriers. Green fluorescent protein (GFP) expression experiments reveal that CCP can achieve efficient delivery and transfection of pDNA encoding GFP gene with 92% efficiency, which surpasses that of commercial transfection agents, lipofectamine 2000 (Lipo) and polyethylenimine (PEI). CCP is also highly fluorescent, with 43% quantum yield in water, and exhibits excellent photostability, which allows for real‐time tracking the location of gene delivery and transfection. These features and capabilities represent a major step toward designing and applying conjugated polymers that function in both imaging and therapeutic applications.     

Encapsulation of Plasmid DNA by Nanoscale Metal–Organic Frameworks for Efficient Gene Transportation and Expression

The intracellular delivery and functionalization of genetic molecules play critical roles in gene‐based theranostics. In particular, the delivery of plasmid DNA (pDNA) with safe nonviral vectors for efficient intracellular gene expression has received increasing attention; however, it still has some limitations. A facile one‐pot method is employed to encapsulate pDNA into zeolitic imidazole framework‐8 (ZIF‐8) and ZIF‐8‐polymer vectors via biomimetic mineralization and coprecipitation. The pDNA molecules are found to be well distributed inside both nanostructures and benefit from their protection against enzymatic degradation. Moreover, through the use of a polyethyleneimine (PEI) 25 kD capping agent, the nanostructures exhibit enhanced loading capacity, better pH responsive release, and stronger binding affinity to pDNA. From in vitro experiments, the cellular uptake and endosomal escape of the protected pDNA are greatly improved with the superior ZIF‐8‐PEI 25 kD vector, leading to successful gene expression with high transfection efficacy, comparable to expensive commercial agents. New cost‐effective avenues to develop metal–organic‐framework‐based nonviral vectors for efficient gene delivery and expression are provided.     

Charge shielding effects on gene delivery of polyethylenimine/DNA complexes: PEGylation and phospholipid coating

Polyethylenimine (PEI) is an efficient cationic polymer for gene delivery, but defective in biocompatibility. In this study, we developed two different strategies to shield the positively charged PEI/DNA complexes: PEGylation and lipid coating. The physicochemical properties, cytotoxicity and transfection efficiency of the two gene delivery systems were investigated. Both PEGylation and lipid coating succeeded in reducing the zeta-potential of the complexes. Lipid-coated PEI/DNA complexes (LPD complexes) and PEI/DNA complexes exhibited similar cytotoxicity, whereas PEG-PEI/DNA complexes showed lower cytotoxicity, especially at high N/P ratios. LPD complexes were less efficient in transfection compared to PEG-PEI/DNA complexes. The transfection efficiency was influenced remarkably by cytotoxicity and surface charge of the complexes. Intracellular processes studies revealed that endosomal release might be one of the rate-limiting steps in cell transfection with PEI as a gene delivery carrier.     

Polymeric micelles comprising stearic acid-grafted polyethyleneimine as nonviral gene carriers

Non-viral vectors composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, 10 k PEI was modified by grafting stearic acid (SA) and formulated to polymer micelles with positive surface charge and evaluated for pDNA delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil in water (o/w) solvent evaporation method. The success of PEI-SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential determined by photon correlation spectroscopy was 149.6 +/- 1.2 nm and 64.1 +/- 1.5 mV, respectively. These self-assemble positive charge micelles showed effective binding to pDNA for transfection. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results demonstrated that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Stearic acid grafted PEI micelles can be promising polymer carriers in genetic therapy.     

A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.     

The characteristics and transfection efficiency of PEI modified by biodegradable poly(β-amino ester)

To improve the cytotoxicity of PEI25k and the transfection efficiency of poly(β-amino ester) with DNA, we synthesized a poly(β-amino ester), PEDP, bearing ester linkages in the backbone and tertiary amines in the backbone and side chain and prepared a binary mixture, PEDP–PEI25k, using physical blending meyhod. Both poly(β-amino ester) PEDP and binary mixture PEDP–PEI25k, readily self-assembled with plasmid DNA (pCMV-β gal) in a HEPES buffer, were characterized by dynamic light scattering. The results reveal that PEDP–PEI25k was able to self-assemble plasmid DNA into PEDP–PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PEDP and PEDP–PEI25k. The COS-7 cell viabilities in the presence of PEDP and PEDP–PEI25k were studied. At low mass ratio of PEDP/PEI25k (1/1), it is found that the transfection curve of PEDP–PEI25k/DNA bearing a maximum peak is similar to that of PEI25k/DNA. In addition, the PEDP–PEI25k/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier PEI25k/DNA. The results indicate that binary mixture PEDP–PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.     

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.     

Enhanced gene transfer with multilayered polyplexes assembled with layer-by-layer technique

Successful gene therapy asks for multifunctional vectors which can not only protect DNA from degradation but also transfer it into nuclear and subsequently express the loaded gene. Here we reported a novel multilayered delivery system constructed with DNA, protamine (Pro) and polyethylenimine (PEI) via lay-by-layer (LbL) technique, which posed multifunctions. DNA was previously condensed into a compact core with Pro which also contained nuclear localisation signals (NLS) domains for nuclear transfer. Then additional DNA was deposited as the first layer onto the cationic core via the electrostatic attraction which would increase the loading dose of DNA. At last, PEI was absorbed as the outmost layer to achieve the endosomal escape. Therefore a quaternary polyplexes which offered high loading of DNA, nuclear transfer ability and endosomal escape capability was constructed with the LbL technique. The obtained quaternary polyplexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA protection capability. Compared with commercially available PEI/DNA complexes, the novel multifuctional vector exhibited not only lower cytotoxicity (P<0.05) but also higher transfection efficiency in HepG2 and HeLa cells (P<0.05) in vitro test.     

Smart Carbon Nanotubes with Laser‐Controlled Behavior in Gene Delivery and Therapy through a Non‐Digestive Trafficking Pathway

Near‐infrared (NIR) laser‐controlled gene delivery presents some benefits in gene therapy, inducing enhanced gene transfection efficiency. In this study, a “photothermal transfection” agent is obtained by wrapping poly(ethylenimine)‐cholesterol derivatives (PEI‐Chol) around single‐walled carbon nanotubes (SWNTs). The PEI‐Chol modified SWNTs (PCS) are effective in compressing DNA molecules and protecting them from DNaseI degradation. Compared to the complexes formed by PEI with DNA (PEI/DNA), complexes of PCS and DNA that are formed (PCS/DNA) exhibit a little lower toxicity to HEK293 and HeLa cells under the same PEI molecule weight and weight ratios. Notably, caveolae‐mediated cellular uptake of PCS/DNA occurs, which results in a safer intracellular transport of the gene due to the decreased lysosomal degradation in comparison with that of PEI/DNA whose internalization mainly depends on clathrin rather than caveolae. Furthermore, unlike PEI/DNA, PCS/DNA exhibits a photothermal conversion ability, which promotes DNA release from PCS under NIR laser irradiation. The NIR laser‐mediated photothermal transfection of PCS_10K/plasmid TP53 (pTP53) results in more apoptosis and necrosis of HeLa cells in vitro than other groups, and achieves a higher tumor‐growth inhibition in vivo than naked pTP53, PEI_25K/pTP53, and PCS_10K/pTP53 alone. The enhanced transfection efficiency of PCS/DNA can be attributed to more efficient DNA internalization into the tumor cells, promotes detachment of DNA from PCS under the mediation of NIR laser and higher DNA stability in the cells due to caveolae‐mediated cellular uptake of the complexes.     

Cationic liposomes-polyallylamine-plasmid nanocomplexes for gene delivery

The aim of the present study was to prepare, characterise and evaluate the transfection efficiency of ternary complexes (lipopolyplexes) composed of cationic liposome, polyallylamine (PAA), plasmid DNA (pDNA). PAA was reacted with a varying amount of a linker, 6-bromohexanoic acid (6-bromo-HA), to prepare a series of modified polymers. Lipopolyplexes consisting of cationic liposome, PAA (or modified PAA), pDNA were prepared. The nanoparticles, so formed, were characterised by their size and zeta potential and were subsequently evaluated for their cytotoxicity and transfection ability on Neuro2A cells. Mean size of prepared complexes ranged from 170 to 280 nm. All lipopolylexes showed a positive zeta potential. Highest transfection efficiency was for lipopolyplex containing PAA 15 kDa-modified polymer and liposome at C/P ratio of 0.5. High molecular weight PAA was more toxic than PAA 15 kDa for Neuro2A cells especially in higher C/P ratio. The results indicate that using the hydrophobic modified PAA in the structure of lipopolyplexes is an effective strategy for improving transfection efficiency.     

Nano‐Sized Sunflower Polycations As Effective Gene Transfer Vehicles

The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2‐dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC₅₀, greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful 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.     

Photothermally Controlled Gene Delivery by Reduced Graphene Oxide–Polyethylenimine Nanocomposite

Externally stimuli‐triggered spatially and temporally controlled gene delivery can play a pivotal role in achieving targeted gene delivery with maximized therapeutic efficacy. In this study, a photothermally controlled gene delivery carrier is developed by conjugating low molecular‐weight branched polyethylenimine (BPEI) and reduced graphene oxide (rGO) via a hydrophilic polyethylene glycol (PEG) spacer. This PEG–BPEI–rGO nanocomposite forms a stable nano‐sized complex with plasmid DNA (pDNA), as confirmed by physicochemical studies. For the in vitro gene transfection study, PEG–BPEI–rGO shows a higher gene transfection efficiency without observable cytotoxicity compared to unmodified controls in PC‐3 and NIH/3T3 cells. Moreover, the PEG–BPEI–rGO nanocomposite demonstrates an enhanced gene transfection efficiency upon NIR irradiation, which is attributed to accelerated endosomal escape of polyplexes augmented by locally induced heat. The endosomal escaping effect of the nanocomposite is investigated using Bafilomycin A1, a proton sponge effect inhibitor. The developed photothermally controlled gene carrier has the potential for spatial and temporal site‐specific gene delivery.     

Carbon fiber/epoxy composite property enhancement through incorporation of carbon nanotubes at the fiber–matrix interphase – Part I: The development of carbon nanotube coated carbon fibers and the evaluation of their adhesion

A new method to realize the uniform coating of carbon nanotubes (CNTs) to carbon fibers (CFs) has been developed, which enables the scalable fabrication of CNT containing CF/epoxy composites. In this method, CNTs are treated by cationic polymers, then, the CNTs are coated to CFs by immersion into a CNT/water suspension. Good dispersion is achieved by repulsive force between positively charged CNTs and uniform coating of the CNTs is achieved by attractive forces between positively charged CNTs and negatively charged CFs. It is found that the use of specific cationic polymers including polyethyleneimine (PEI) results in stable CNT/water suspensions, and uniform coating of the CNTs. Single fiber fragmentation tests of the CF/epoxy composites were conducted to evaluate the strength of interface and interphase under shear loading. The results show that the combination of epoxy resin sizing and PEI treated CNT coating to CFs results in high interfacial shear strength.     

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (?M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.     

Design of novel polysaccharidic nanostructures for gene delivery

The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10?kDa (HAO) and chitosan 125 (CS) or 10-12?(CSO)?kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230?nm, a positive zeta potential of +10 to +32?mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK?293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In?conclusion, HA:CS nanoparticles are promising carriers for gene delivery.     

聚乙烯亚胺改性的双介孔氧化硅基因载体构建

为了开发一种新型的非病毒无机基因载体, 采用3-氨丙基三乙氧基硅烷(APTMS)和聚乙烯亚胺(PEI)对嵌入型双介孔氧化硅球(EDMSNs)进行改性, 分别得到EDMSNs-NH₂和EDMSNs-PEI, 并比较了两种载体结合和保护pCMV-EGFP-N1质粒(pDNA)的能力及细胞转染性能。利用透射电镜、动态光散射及氮气吸附-脱附实验对材料的颗粒形态, 动力学粒径, Zeta电位及孔结构参数进行表征。结果显示, EDMSNs-NH₂和EDMSNs-PEI均表现出明显的双介孔结构, 形貌为规整的球形且平均动力学粒径分别为343.2和338.9 nm, 表面电位分别为+18和+43 mV。琼脂糖凝胶电泳、CCK-8法及荧光显微镜结果表明, EDMSNs-PEI对pDNA的担载量为8%, 远高于EDMSNs- NH₂(1%)。与PEI和lipofectamine2000相比, EDMSNs-PEI载体展示出更低的细胞毒性。EDMSNs-PEI/pDNA质量比为33 : 1时, EDMSNs-PEI/pDNA对293T细胞的转染效率在72 h达到最大值。因此, 与EDMSNs-NH₂相比, EDMSNs-PEI具有更高的正电位及pDNA担载量, 可作为一类有前景的非病毒无机类基因载体用于重大疾病如恶性胶质瘤的治疗。