A novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery

Aim:

To develop a novel non-viral vector with high transfection efficiency and low cytotoxicity.

Methods:

Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) was incorporated into polymer-lipid hybrid nanoparticles (PLN) to construct a PEG-DSPE modified long circulating PLN (L-PLN). The L-PLN was prepared by the emulsifying-solvent evaporation method, L-PLN and L-PLN/DNA complexes were characterized. Both HEK293 and MDA-MB-231 cells transfected by L-PLN/DNA complexes were observed under a fluorescence microscope. The transfection efficiency of the complexes to HEK293 cells was further evaluated by flow cytometry.

Results:

The GFP fluorescence intensity in HEK293 cells transfected by the L-PLN/DNA complexes (N/P=10) was about 37.2%, which was higher than those transfected by PLN alone or commercial Lipofectamine^TM 2000. The L-PLN exhibited minimal toxicity at a low N/P ratio compared with other vectors.

Conclusion:

L-PLN as a novel gene delivery system, has higher transfection efficiency and acceptable cytotoxicity compared to the corresponding PLN, which is beneficial for the development of non-viral gene transfer vectors and may offer an alternative strategy for the future gene therapy.     

Engineered Polyallylamine Nanoparticles for Efficient In Vitro Transfection

Purpose Cationic polymers (i.e. polyallylamine, poly-L-lysine) having primary amino groups are poor transfection agents and possess high cytotoxicity index when used without any chemical modification and usually entail specific receptor mediated endocytosis or lysosomotropic agents to execute efficient gene delivery. In this report, primary amino groups of polyallylamine (PAA, 17 kDa) were substituted with imidazolyl functions, which are presumed to enhance endosomal release, and thus enhance its gene delivery efficiency and eliminate the requirement of external lysosomotropic agents. Further, systems were cross-linked with polyethylene glycol (PEG) to prepare PAA-IAA-PEG (PIP) nanoparticles and evaluated them in various model cell lines. Materials and Methods The efficacy of PIP nanoparticles in delivering a plasmid encoding enhanced green fluorescent protein (EGFP) gene was assessed in COS-1, N2a and HEK293 cell lines, while their cytotoxicity was investigated in COS-1 and HEK293 cell lines. The PAA was chemically modified using imidazolyl moieties and ionically cross-linked with PEG to engineer nanoparticles. The extent of substitution was determined by ninhydrin method. The PIP nanoparticles were further characterized by measuring the particle size (dynamic light scattering and transmission electron microscopy), surface charge (zeta potential), DNA accessibility and buffering capacity. The cytotoxicity was examined using the MTT method. Results In vitro transfection efficiency of synthesized nanoparticles is increased up to several folds compared to native polymer even in the presence of serum, while maintaining the cell viability over 100% in COS-1 cells. Nanoparticles possess positive zeta potential between 5.6–13 mV and size range of 185–230 nm in water. The accessibility experiment demonstrated that nanoparticles with higher degree of imidazolyl substitution formed relatively loose complexes with DNA. An acid-base titration showed enhanced buffering capacity of modified PAA. Conclusions The PIP nanoparticles reveal tremendous potential as novel delivery system for achieving improved transfection efficiency, while keeping the cells at ease.     

Histidylated cationic polyorganophosphazene/DNA self-assembled nanoparticles for gene delivery

Cationic polyorganophosphazene has shown the ability to deliver gene. To obtain more efficient transfection, His(Boc)-OMe bearing histidine moiety was introduced to synthesize a new derivative of cationic polyphosphazenes with another side group of 2-dimethylaminoethylamine (DMAEA). The poly(DMAEA/His(Boc)-OMe)phosphazene (PDHP) and DNA could self-assemble into nanoparticles with a size around 110 nm and zeta potential of +15 mV at the PDHP/DNA ratio of 10:1 (w/w). The maximum transfection efficiency of PDHP/DNA self-assembled nanoparticles (PHSNs) against 293 T cells was much higher than that of poly(di-2-dimethylaminoethylamine) phosphazenes (PDAP)/DNA self-assembled nanoparticles (PASNs) and PEI 25/DNA self-assembled nanoparticles (PESNs) at the polymer/DNA ratio of 10:1, but the cytotoxicity of PDHP assayed by MTT was much lower than that of PDAP and PEI 25. These results suggested that PDHP could be a good candidate with high transfection efficiency and low cytotoxicity for gene delivery.     

Solid lipid nanoparticles: formulation factors affecting cell transfection capacity

Since solid lipid nanoparticles (SLNs) were introduced as non-viral transfection systems, very few reports of their use for gene delivery have been published. In this work different formulations based on SLN-DNA complexes were formulated in order to evaluate the influence of the formulation components on the "in vitro" transfection capacity. SLNs composed by the solid lipid Precirol ATO 5, the cationic lipid DOTAP and the surfactant Tween 80, and SLN-DNA complexes prepared at different DOTAP/DNA ratios were characterized by studying their size, surface charge, DNA protection capacity, transfection and cell viability in HEK293 cultured cells. The incorporation of Tween 80 allowed for the reduction of the cationic lipid concentration. The formulations prepared at DOTAP/DNA ratios 7/1, 5/1 and 4/1 provided almost the same transfection levels (around 15% transfected cells), without significant differences between them (p>0.05). Other assayed formulations presented lower transfection. Transfection activity was dependent on the DOTAP/DNA ratio since it influences the DNA condensation into the SLNs. DNA condensation is a crucial factor which conditions the transfection capacity of SLNs, because it influences DNA delivery from nanoparticles, gene protection from external agents and DNA topology.     

Preparation and evaluation of chitosan-DNA-FAP-B nanoparticles as a novel non-viral vector for gene delivery to the lung epithelial cells

Gene delivery using cationic polymers such as chitosan shows good biocompatibility, but reveals low transfection efficiency. Fibronectin Attachment Protein of Mycobacterium bovis (FAP-B) which is responsible for the attachment of many Mycobacteria on the Fibronectin molecule of epithelial cell membrane can be considered as a new targeting ligand and can improve transfection rates in epithelial cells. In this study, chitosan-DNA nanoparticles were prepared using coacervation process. The effect of stirring speed and charge ratio (N/P) on the size and zeta potential of nanoparticles were evaluated. FAP-B ligand was added to nanoparticles at the specific condition to form chitosan-DNA-FAP-B nanoparticles via electrostatic attraction. Transfection efficiency of the final nanoparticles was investigated in A549 (alveolar epithelial cells). Cell viability was investigated using MTT assay. The optimum speed of stirring which was yielded the smallest chitosan-DNA nanoparticles with a narrow distribution (227±43 nm), was 500 rpm with the corresponding N/P ratio of 20. Chitosan-DNA-FAP-B nanoparticles presented the size of 279±27 nm with transfection efficiency about 10-fold higher than chitosan-DNA nanoparticles and resulted in 97.3% cell viability compared to 71.7% using Turbofect controls. Chitosan-DNA-FAP-B nanoparticles showed good transfection efficiency without cell toxicity. They have small particle size around 279 nm which make them a promising candidate as a novel non-viral gene vector for gene delivery to lung epithelial cells.     

鱼精蛋白／DNA复合物的构建与生物性能评价

目的研究非病毒基因载体鱼精蛋白／DNA复合物的制备方法及对其细胞毒性、不同量鱼精蛋白对pDNA的结合能力、体外细胞转染率。方法不同量鱼精蛋白与DNA在室温孵育后,得到鱼精蛋白／DNA复合物;用MTT法检测鱼精蛋白对HeLa子宫颈癌细胞的毒性作用,同时与PEI进行比较;利用琼脂糖电泳实验测定不同N／P比形成复合物时对DNA的阻滞情况;用结合沉淀试验比较不同量鱼精蛋白对包裹DNA的能力的影响;在荧光显微镜下观察比较它们对BEL-7402肝癌细胞转染率的大小。结果当鱼精蛋白／DNA复合物浓度升高时,对He—La子宫颈癌细胞的毒性均为0级,而聚乙烯亚胺（PEI）浓度升高时,对细胞的毒性明显增加;鱼精蛋白与质粒DNA形成复合物时所需的N／P比为1．5：1,较PEI／DNA复合物形成时所需的N／P比2：1要小;鱼精蛋白包裹DNA的能力随N／P比增大而增强,并且包裹DNA的能力要比PEI／DNA复合物转变得快;鱼精蛋白／DNA复合物对BEL-7402肝癌细胞的转染率较PEI／DNA复合物低,但毒性较低。结论鱼精蛋白／DNA复合物是一种制备工艺简单、细胞毒性小、对pDNA包裹能力高、转染率相对较高,具有一定应用潜力的非病毒基因载体。     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.     

Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis

Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid-DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.     

Physicochemical characterization of poly(L-lactic acid) and poly(D,L-lactide-co-glycolide) nanoparticles with polyethylenimine as gene delivery carrier

Polymer nanoparticles have been used as non-viral gene delivery systems and drug delivery systems. In this study, biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) and poly(D,L-lactide-co-glycolide) (PLGA)/PEI nanoparticles were prepared and characterized as gene delivery systems. The PLA/PEI and PLGA/PEI nanoparticles, which were prepared by a diafiltration method, had spherical shapes and smooth surface characteristics. The size of nanoparticles was controlled by the amount of PEI, which acted as a hydrophilic moiety, which effectively reduced the interfacial energy between the particle surface and the aqueous media. The nanoparticles showed an excellent dispersive stability under storage in a phosphate-buffered saline solution for 12 days. The positive zeta-potentials for the nanoparticles decreased and changed to negative values with increasing plasmid DNA (pDNA) content. Agarose gel electrophoresis showed that the complex formation between the nanoparticles and the pDNA coincided with the zeta-potential results. The results of in vitro transfection and cell viability on HEK 293 cells indicated that the nanoparticles could be used as gene delivery carriers.     

Novel biomimetic vectors with endosomal-escape agent enhancing gene transfection efficiency

Low cytotoxicity and high transfection efficiency are critical issues in designing current non-viral gene delivery vectors. In the present study, a novel biomimetic lipid-polycation copolymer, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-graft-poly(l-lysine)-block-poly(ethylene glycol) (DOPE-g-PLL-b-PEG) was first synthesized and the potential of this novel hybrid lipid-polycation as efficient gene vector was further evaluated. DOPE-g-PLL-b-PEG and DNA could self-assemble into lipid modified polyion complex micelles (LPCM) through electrostatic interactions. Compared with PEG-b-PLL/DNA polyion complex micelles (PIC), LPCM could protect DNA from plasma, nuclease degradation in vitro and showed lower cytotoxicity to HepG2 and HeLa cells (P<0.05). The results of transfection study in vitro indicated that LPCM exhibited higher gene expression than PIC. Especially, the corresponding LPCM displayed the highest transfection efficiency in HeLa cells (P<0.05) when DOPE grafting ratio reached up to 30%. These results suggested that LPCM could facilitate gene transfer in cultured cells and might alleviate the drawbacks of the conventional cationic vector/DNA complexes. As a novel hybrid lipid-polycation, DOPE-g-PLL-b-PEG was valuable to be evaluated for its further application as gene carrier in vivo.     

Guanidinium-grafted polyethylenimine: an efficient transfecting agent for mammalian cells.

Polyethylenimine (PEI) is one of the most efficient polycationic non-viral gene delivery vectors. Its efficiency and cytotoxicity depends on molecular weight, with the 25-kDa PEI being most efficient but accompanied with cytotoxicity. In the present study, enhancement in gene delivery efficiency along with reduction in cytotoxicity by attachment of guanidinium side group was explored. The hypothesis was that the guanidination would lead to the delocalization of charge present on primary amines of the polymer thereby leading to enhancement in gene delivery efficiency along with reduction in cytotoxicity. The polymer was guanidinated using O-methylisourea hemisulfate and the chemical linkage characterized by FTIR spectroscopy. The hydrodynamic diameter of guanidinated PEI-DNA complexes was determined using DLS. Subsequently, these complexes were used for DNA binding assay and zeta-potential measurements, taking native PEI as reference. Further, guanidinated PEI-DNA complexes were investigated for their gene delivery efficacy on HEK 293 cells. The hydrodynamic diameter of guanidinated PEI-DNA complexes was found to be in the range of 176-548 nm. As expected, the zeta potential values increased, on increasing the N/P ratios. It was found that guanidinated PEI had higher transfection efficiency at the majority of the N/P ratios tested as compared to commercially available transfecting agent lipofectin and native PEI itself. The toxicity of guanidinated PEI-DNA complexes was also reduced considerably in comparison to PEI polymer, as determined by MTT colorimetric assay. Out of the various derivatives prepared, gPEI 56% was found to be the most efficient in in vitro transfection.     

A Potential Targeting Gene Vector Based on Biotinylated Polyethyleneimine/Avidin Bioconjugates

Purpose  To improve the gene delivery efficiency and safety of non-viral vector in liver cells, avidin, which exhibited good biocompatibility and remarkable accumulation in liver, was bioconjugated with biotinylated polyethylenimine to obtain a novel gene vector. Materials and methods  Biotinylated polyethyleneimine/avidin bioconjugate (ABP) was synthesized through grafting biotin to high molecular weight branched polyethylenimine (PEI, 25 kDa) and then bioconjugating with avidin by the biotin-avidin interaction. Physiochemical characteristics of ABP/pDNA complexes were analyzed, and in vitro cytotoxicity and transfection of ABP were also evaluated in HepG2, Hela and 293 T cells by using 25 kDa PEI as the control. Results  It was found that ABP was able to condense pDNA efficiently at N/P ratio of 4. The particle sizes of ABP/pDNA complexes were less than 220 nm, and the average surface charges were around 27 mV at the N/P ratio ranging from 2 to 60. Among three different cell lines, ABP and its DNA complexes demonstrated much lower cytotoxicity and higher transfection efficacy in HepG2 cells as compared with 25 kDa PEI. Conclusion  ABP presented higher transfection efficacy and safety in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells.     

Novel cationic lipids possessing protonated cyclen and imidazolium salt for gene delivery

In this study, two novel protonated cyclen and imidazolium salt-containing cationic lipids differing only in their hydrophobic region (cholesterol or diosgenin) have been designed and synthesized for gene delivery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including DLS, gel retardation assay, ethidium bromide intercalation assay, and TEM demonstrated that these amphiphilic molecules are able to bind and compact DNA into nanometer particles that could be used as non-viral gene delivery agents. Our results from in vitro transfection showed that in association with DOPE, two cationic lipids can induce effective gene transfection in HEK293 cells. Furthermore, the gene transfection efficiencies of two cationic lipids were dramatically increased in the presence of calcium ion (Ca²⁺). It is notable that the gene transfection abilities of two cationic lipids were maintained in the presence of 10% serum. Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vector.     

Low-molecular-weight polyethylenimine enhanced gene transfer by cationic cholesterol-based nanoparticle vector

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.     

Enhanced transfection of polyplexes based on pluronic-polypropylenimine dendrimer for gene transfer

Third generation cationic dendritic polymeric polypropyleneimine (PPI) was modified by Pluronic P123 and investigated for gene delivery. The cytotoxicity of P123-PPI was evaluated by the MTT assay and shown to be much lower than that of PPI alone. P123-PPI and PPI can both condense plasmid DNA into nanoparticles with a size of approximately 100 nm and a zeta potential of about 15 mV at the N/P ratio 20:1. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 0.4 U/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 75 μg/mL sodium heparin. The transfection efficiency of SPC-A1 cells using P123-PPI/DNA nanoparticles was much higher than the transfection utilizing PPI/DNA nanoparticles. The addition of free P123 during the preparation of P123-PPI/DNA nanoparticles could significantly enhance the transfection efficiency in the presence of 10% fetal bovine serum. Therefore, P123-PPI/DNA complex nanoparticles may be a safe, efficient and promising cationic conjugate for gene delivery.     

Calcium phosphate-mediated gene delivery using simulated body fluid (SBF)

The present study aimed at developing a new approach in gene delivery of calcium phosphate nanoparticles through simulated body fluid (CaP-SBF). The physicochemical and biological characteristics of the CaP-SBF nanoparticles were compared with those made in pure water (CaP-water) via a similar procedure. The CaP-SBF and CaP-water solutions were then adjusted to two different pH values of 7.4 and 8.0, mixed with plasmid DNA (pDNA), and added in varying amounts to human embryonic kidney (HEK 293T) cells. The transfection efficiency and cell viability were studied in vitro by reporter gene (luciferase and Enhanced Green Fluorescent Protein) expression and the resazurin reduction assay, respectively, 24 and 48 h after the incubation with the nanoparticles. Our results indicated considerably high in vitro transfection efficiency for CaP-SBF/DNA complexes at physiological pH (7.4) with high amounts of CaP. Additionally, the SBF solution exhibited the ability to reduce the rapid growth of CaP particles over time, leading to higher transfection efficiency of CaP-SBF/DNA complexes than those made in water (CaP-water/DNA).     

A novel gene delivery system for stable transfection of thiopurine-S-methyltransferase gene in versatile cell types

A novel gene delivery system termed artificial viral particles (AVPs) containing a plasmid coding for a recombinant fusion protein of enhanced green fluorescent protein (EGFP) with thiopurine-S-methyltransferase (TPMT) was designed for transfection of selected cell lines to establish stable clones which express recombinant EGFP–TPMT protein for further in vitro investigation of toxic effect of thiopurine drugs. Various AVPs based on a complex of the cationic polymer polyethylenimine (PEI) and anionic liposomes were formulated and transfection conditions were adapted in order to transfect the human Jurkat, HepG2 and HEK 293 cell lines. An adequate transfection rate was achieved with AVP containing branched low molecular weight PEI at a PEI:DNA charge ratio of 4.5:1 and liposomes composed of DOPS, DLPE, cholesterol and an activated N-glutaryl-DOPE membrane anchor. Stably transfected clones were successfully established and expression of recombinant EGFP–TPMT in homogeneous cell populations was demonstrated by flow cytometry, fluorescence microscopy and immunoblotting. The level of the expressed protein in stable clones was highest in HEK 293, followed by HepG2 and Jurkat. The enzymatic activity of the TPMT moiety was demonstrated by decreased sensitivity to 6-thioguanine and increased sensitivity to 6-mercaptopurine in HEK 293 cells expressing EGFP–TPMT. Formulation of AVP as transfection vector succeeded in establishing human cell lines stably expressing EGFP–TPMT, thereby proving a successful delivery system and providing an initial step to enable investigation of the role of the clinically important drug metabolizing enzyme TPMT.     

Design, engineering and preparation of a multi-domain fusion vector for gene delivery

Peptide based gene carriers are among the most promising non-viral vectors for gene delivery to eukaryotic cells. We have engineered a new fusion peptide using recombinant technology with the purpose of overcoming the cell barriers to gene delivery. A His- tagged multi-domain peptide was expressed in Escherichia coli BL21 (DE3) pLysS and purified using Ni-NTA resin. The fusion peptide is composed of two repeats of truncated histone H1 peptide to condense pDNA, a fusogenic peptide to disrupt endosome membranes and a nuclear localization signal to enhance translocation of pDNA towards nucleus. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with average sizes of 200 nm. The fusogenic peptide in the vector structure also showed membrane disruptive effect in the endosomal pH. Overall, the transfection efficiency of the vector demonstrated that it holds great promise as a nontoxic and effective non-viral gene carrier.     

Cross-linked polyethylenimine-hexametaphosphate nanoparticles to deliver nucleic acids therapeutics

Branched polyethylenimine (PEI; 25 kDa) as a nonviral vector exhibits high transfection efficiency and is a potential candidate for efficient gene delivery. However, the cytotoxicity of PEI limits its application in vivo. PEI was ionically interacted with hexametaphosphate, a compact molecule with high anionic charge density, to obtain nanoparticles (PEI-HMP). Nanoparticles were assessed for their efficacy in protecting complexed DNA against nucleases. The intracellular trafficking of nanoparticles was monitored by confocal microscopy. The cytotoxicity and transfection efficiency of PEI-HMP nanoparticles were evaluated in vitro. In vitro transfection efficiency of PEI-HMP (7.7%) was ～1.3- to 6.4-folds higher than that of the commercial reagents GenePORTER 2^TM, Fugene^TM, and Superfect^TM. Also, PEI-HMP (7.7%) delivered green fluorescent protein (GFP)-specific small interfering ribonucleic acid (siRNA) in culture cells leading to >80% suppression in GFP gene expression. PEI-HMP nanoparticles protected complexed DNA against DNase for at least 2 hours. A time-course uptake of PEI-HMP (7.7%) nanoparticles showed the internalization of nanoparticles inside the cell nucleus in 2 hours. Thus, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as nonviral vectors for gene delivery.From the Clinical EditorBranched polyethylenimine (PEI) as a non-viral vector exhibits high transfection efficiency for gene delivery, but its cytotoxicity limits its applications. PEI hexametaphosphate nanoparticles (PEI-HMP) demonstrated a 1.3-6.4 folds higher transfection rate compared to commercial reagents. Overall, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as non-viral vectors for gene delivery.     

Folate-linked lipid-based nanoparticle for targeted gene delivery

Cancer gene therapy has been intensively developed using non-viral vectors, among which cationic liposomes and nanoparticles are the most thoroughly investigated. For targeted delivery to tumors, vitamin folic acid has been utilized for folate receptor (FR)-mediated drug delivery, since FR is frequently overexpressed on many types of human tumors. Liposomes conjugated to folate ligand have been used as carriers of chemotherapeutic agents and DNA to receptor-bearing tumor cells in vitro. As an alternative treatment for prostate cancer, suicide gene therapy by local injection using an adenoviral vector has been reported, but not that using non-viral vectors. The folate-linked, lipid-based nanoparticles which we developed could deliver genes extensively to FR-negative LNCaP and PC-3 cells, as well as FR-positive KB and Hela cells. In this review, we outline folate-linked liposomes and nanoparticles, and show the effectiveness of folate-linked, lipid-based nanoparticles as a vector for DNA transfection and for suicide gene therapy, to treat human nasopharyngeal and prostate tumors.