High Gene Transfection Efficiency Research Articles

Targeted delivery of biodegradable nanoparticles with ultrasound-targeted microbubble destruction-mediated hVEGF-siRNA transfection in human PC-3 cells in vitro

A potentially viable approach for treating late-stage prostate cancer is gene therapy. Successful gene therapy requires safe and efficient delivery systems. In this study, we report the efficient delivery of small interfering RNA (siRNA) via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. On the basis of previous findings, cyclic Arg-Gly-Asp (cRGD) peptides were conjugated to NPs to recognize the target site, integrinαvβ3, expressed in high levels in PC-3 prostate cancer cells. The suppression of angiogenesis by the downregulation of vascular endothelial growth factor (VEGF) expression has been widely used to inhibit the growth of malignant tumors. In our study, human VEGF (hVEGF)-siRNA was encapsulated in NPs to inhibit VEGF expression in PC-3 cells. Concurrently, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for the delivery of siRNA-loaded NPs. Our results showed low cytotoxicity and high gene transfection efficiency, demonstrating that the targeted delivery of biodegradable NPs with UTMD may be potentially applied as new vector system for gene delivery.

Gene delivery of PEI incorporating with functional block copolymer via non-covalent assembly strategy

A novel functional diblock polymer P(PEGMA-b-MAH) is prepared and incorporated to improve the gene delivery efficiency of poly(ethyleneimine) PEI via non-covalent assembly strategy. First, P(PEGMA-b-MAH) is prepared from l-methacrylamidohistidine methyl ester (MAH) by reversible addition fragmentation chain transfer polymerization, with poly[poly(ethylene glycol) methyl ether methacrylate] (P(PEGMA)) as the macroinitiator. Then P(PEGMA-b-MAH) is assembled with plasmid DNA (pDNA) and PEI (Mw=10kDa) to form PEI/P(PEGMA-b-MAH)/pDNA ternary complexes. The agarose gel retardation assay shows that the presence of P(PEGMA-b-MAH) does not interfere with DNA condensation by the PEI. Dynamic light scattering tests show that PEI/P(PEGMA-b-MAH)/pDNA ternary complexes have excellent serum stability. In vitro transfection indicates that, compared to the P(PEGMA-b-MAH) free PEI-25k/pDNA binary complexes, PEI-10k/P(PEGMA-b-MAH)/pDNA ternary complexes have lower cytotoxicity and higher gene transfection efficiency, especially under serum conditions. The ternary complexes proposed here can inspire a new strategy for the development of gene and drug delivery vectors.

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.

Amino acid-modified bioreducible poly(amidoamine) dendrimers: Synthesis, characterization and In vitro evaluation

Poly(amidoamine) (PAMAM) dendrimers are synthetic polymers commonly used as carriers in gene and drug delivery. PAMAMs have the ability to transfect DNA, but their transfection efficiency is currently insufficient for clinical use. Here, we demonstrate the synthesis and evaluation of cationic dendrimers consisting of a cystamine core PAMAM generation 3 (cPAM G3) and amino acids. Introduction of histidine (His) and arginine (Arg) residues to cPAM G3 resulted in high transfection efficiency and low cytotoxicity. cPAM G3-His-Arg formed stable polyplexes at a weight ratio of 6:1, and the mean polyplex diameter was 99.03±1.68 nm. Nano-sized polyplexes increased in diameter up to 132.93±1.79 nm when treated with a reducing agent, dithiothreitol (DTT). cPAM G3-His-Arg showed much higher transfection efficiency than native cPAM G3 and polyethylenimine (PEI, 25KD). In addition, cPAM G3-His-Arg displayed negligible toxicity, even at high polymer concentrations. Finally, confocal laser microscopy results showed that cPAM G3-His-Arg effectively internalized plasmid DNA into the cells. Therefore, we believe that cPAM G3-His-Arg could be a promising bioreducible vector for non-viral gene delivery with high gene transfection efficiency and low cytotoxicity.

Gene‐carried chitosan‐linked polyethylenimine induced high gene transfection efficiency on dendritic cells

A dendritic cell (DC) networking system has become an attractive approach in cancer immunotherapy. Successful DC gene engineering depends on the development of transgene vectors. A cationic polymer, chitosan-linked polyethylenimine (PEI) (CP), possessing the advantages of both PEI and chitosan, has been applied in nonviral transfection of DCs. Physicochemical evaluation showed that CP/DNA complexes could form cationic nanoparticles. Compared with DCs transfected with commercial reagent, Lipofectamine2000, it showed higher transfection efficiency and lower cytotoxicity when DCs were transfected with CP/DNA complexes. A nuclear trafficking observation of CP/DNA complexes by a confocal laser scanning microscope further revealed that the CP could help DNA enter into the cytoplasm and finally into the nucleus of a DC. Finally, vaccination of DCs transfected with CP/DNA encoding gp100 slightly improved resistance to the B16BL6 melanoma challenge. This is the first report that CP polymer is used as a nonviral vector for DC gene delivery and DC vaccine. Essentially, these results might be helpful to design a promising nonviral vector for DC gene delivery.

Cationic niosomes composed of spermine-based cationic lipids mediate high gene transfection efficiency

Non-ionic surfactant-based vesicles (niosomes) composed of non-ionic surfactants (i.e., Tween and Span) and cholesterol were formulated, and their turbidity and particle size assayed. The most appropriate niosomes formulation was mixed with novel synthesized spermine-based cationic lipids to prepare cationic niosomes that could act as gene carriers. Factors affecting gene transfection and cell viability including differences in the acyl chain length (C14, C16 and C18) of cationic lipids and the weight ratio of niosomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge of the niosomes were also characterized, and a gel retardation assay to determine complex formation was performed. The results revealed that the transfection efficiency of the Span 20-niosomes was the highest for the spermine-C14 formulation and decreased as follows: spermine-C14 > spermine-C16 > spermine-C18. In addition to the highest transfection efficiency, there was also no serum effect on transfection efficiency of the spermine-C14 niosomes at a weight ratio of 10. This formulation was safe in vitro and had good physical stability for at least 1 month at 4°C. In conclusion, the cationic niosomes may constitute a good alternative carrier for gene transfection.

Fabrication of functionalized double-lamellar multifunctional envelope-type nanodevices using a microfluidic chip with a chaotic mixer array.

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>105) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency.

Ternary Complexes of Poly(Vinyl Pyrrolidone)-Graft-Poly(2-Dimethylaminoethyl Methacrylate), DNA and Bovine Serum Albumin for Gene Delivery

Novel well-defined hydrophilic cationic polymers with different length of polycation chain for gene delivery, poly(vinyl pyrrolidone)-graft-poly(2-dimethylaminoethyl methacrylate)s (PPDs), were synthesized by atom transfer radical polymerization (ATRP). The chemical structures and compositions of these polymers were characterized by FT-IR, 1H-NMR and GPC. The experimental results of dynamic light scattering (DLS), ζ-potential and transmission electron microscopy (TEM) indicated that PPD could condense plasmid DNA (pDNA) to form nanocomplexes. Agarose gel retardation assays demonstrated that PPD could encapsulate plasmid DNA completely when the N/P ratio is equal to or above 3. MTT assay and in vitro gene transfection results indicated that PPD/pDNA complexes exhibited high transfection efficiency concomitant with obvious cytotoxicity. Furthermore, bovine serum albumin (BSA) was utilized to assembly with the binary complexes of PPD/pDNA to screen the residual surface positive charges of complexes in order to decrease cytotoxicity of the binary complexes. Physicochemical properties were characterized and the results indicated that the coating of BSA was able to decrease the zeta potential of the nano-sized PPD/pDNA complexes nearly to electroneutrality without interfering with DNA condensation ability. The ternary complexes of BSA/PPD/pDNA demonstrated no cytotoxicity and also maintained high gene transfection efficiency in HepG2 cells in 10% serum compared with that in serum-free condition.

Calcium phosphate embedded PLGA nanoparticles: A promising gene delivery vector with high gene loading and transfection efficiency

In the purpose of increasing incorporation efficiency and improving the release kinetics of plasmid DNA (pDNA) from poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles, a facile method for the fabrication of calcium phosphate (CaPi) embedded PLGA nanoparticles (CaPi-pDNA-PLGA-NPs) was developed. The effect of several preparation factors on the particle size, incorporation efficiency, pDNA release and transfection efficiency in vitro was studied by Single Factor Screening Method. These preparation factors included the molecular weight (MW), hydrolysis degree (HD) of polyvinyl alcohol (PVA), sonication power and time, composition of organic phase, initial concentration of calcium phosphate and calcium (Ca) to phosphate ion (P) ratio (Ca/P ratio), etc. The CaPi-pDNA-PLGA-NPs made according to the optimal formulation were spherical in shape observed by transmission electron microscopy (TEM) with a mean particle size of 207±5nm and an entrapment efficiency of 95.7±0.8%. Differential scanning calorimetry (DSC) suggested that there existed interaction between the DNA-calcium-phosphate (CaPi-pDNA) complexes and the polymeric matrices of PLGA. X-ray diffractometry (XRD) further proved the conclusion and indicated that the CaPi-pDNA was in weak crystallization form inside the nanoparticles. The Brunauer–Emmett–Teller (BET) surface area measurement demonstrated that the CaPi-pDNA-PLGA-NPs are mesoporous with specific surface area of 57.5m2/g and an average pore size of 96.5Å. The transfection efficiency of the CaPi-pDNA-PLGA-NPs on human embryonic kidney 293 (HEK 293) cells in vitro was 22.4±1.2%, which was much higher than those of both the pDNA loaded PLGA nanoparticles (pDNA-PLGA-NPs) and the CaPi-pDNA embedded PLGA microparticles (CaPi-pDNA-PLGA-MPs). The CaPi-pDNA-PLGA-NPs are promising vectors for gene delivery.

Abstract 5653: A novel cationic peptide nanoparticle-based gene therapy with angiotensin II type 2 receptor gene significantly attenuates growth of lung adenocarcinoma xenografts in mouse model

Abstract A successful gene therapy requires targeted gene delivery, high gene transfection efficiency at the target tissue, and low toxicity of the vectors. We have demonstrated that the HIV-1 TAT peptide is suggested to be an effective gene delivery vector in various cultured cells (Baoum, et al., Pharm Res, 2009; Baoum, et al., Int J Pharm, 2011). The objectives of the present study were to examine the gene transfection efficacy of modified TAT peptide nanoparticle (two TAT peptides connected in tandem i.e. dTAT NP) as a vector, and to examine the efficiency of the tumor-targeted gene delivery after intratracheal administration. Expression efficiency of the dTAT NP encapsulating luciferase or angiotensin II type 2 receptor (AT2R) plasmid DNA (pDNA) was evaluated using Lewis Lung carcinoma (LLC) cells in culture and orthotopic autografts in C57BL/6 mice lungs. Cell culture studies clarified that the dTAT NP caused effective gene transfection with negligible cytotoxicity until approximately 4 mg dTAT/mL (IC50= 4.075 mg/mL), whereas polyethyleneimine (PEI) showed much stronger cytotoxicity (IC50= 0.028 mg/mL). The in vitro study also revealed that the addition of calcium and glucose to the dTAT/pDNA NP caused effective DNA transfection. The in vitro transfection of pAT2R by dTAT NP caused marked attenuation of the growth of LLC cells. Immunohistochemical analysis of the in vitro transfection and in vivo mouse gene expression studies with dTAT/luciferase pDNA NP revealed that dTAT NP vector successfully delivered the dTAT/pDNA complex to the tumor tissues and caused gene expression primarily in the tumor cells and bronchial epithelium. Gene expression in the tumor tissues lasted for at least 14 days post-administration. Bolus intratracheal administration of dTAT/pAT2R as well as dTAT/pTRAIL markedly attenuated tumor growth of LLC autografts in the mouse lungs. Immunohistochemical analysis of dissected tumors revealed that the AT2R expression was predominantly located in the tumor cells of the LLC tumor bearing mouse lung. Apoptotic index was significantly higher in the treated tumors than PBS or luciferase treated control tumors, suggesting that AT2R pDNA was successfully delivered to the tumor tissues by dTAT NP vector and caused apoptosis of tumor cells. Taken together, the current study suggests that a gene delivery system using intratracheal administration of dTAT/pAT2R NP offers an effective strategy for lung cancer gene therapy. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH P20 RR017686, p20 RR1556, R21CA135599 and Kansas Bioscience Authority Collaborative Cancer Research grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5653. doi:1538-7445.AM2012-5653

Tris[2-(acryloyloxy)ethyl]isocyanurate Cross-Linked Low-Molecular-Weight Polyethylenimine as Gene Delivery Carriers in Cell Culture and Dystrophic mdx Mice

Hyperbranched poly(ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) and low-molecular-weight polyethylenimine (LPEI, M(w) 0.8k, 1.2k, and 2.0k) and evaluated in vitro and in vivo as gene carriers. PEAs effectively condensed plasmid DNA with particle sizes below 200 nm and surface charges between 11.5 and 33.5 mV under tested doses [at the ratios 2-10:1 of polymer/pDNA(w/w)]. The PEAs showed significantly lower cytotoxicities when compared with PEI 25k in two different cell lines. The PEAs (C series) composed of PEI 2k showed higher transgene expression compared to PEAs of PEI 0.8k (A series) or 1.2k (B series). Highest gene transfection efficiency in CHO, C2C12 myoblast, and human skeletal muscle (HSK) cell lines was obtained with TAEI/PEI-2K (C12) at a ratio of 1:2. Both C12, C14(TAEI/PEI-2K at a ratio of 1:4) demonstrated 5-8-fold higher gene expression as compared with PEI 25k in mdx mice in vivo through intramuscular administration. No obvious muscle damage was observed with these new polymers. Higher transfection efficiency and lower toxicity indicate the potential of the biodegradable PEAs as safe and efficient transgene delivery vectors.

Improved biocompatibility of poly(vinylpyrrolidone)-graft-poly(2-dimethylaminoethyl methacrylate)/DNA complexes by coating with bovine serum albumin

Abstract Bovine serum albumin (BSA) was utilized to assemble with the binary complexes of poly(vinylpyrrolidone)-graft-poly(2-dimethylaminoethyl methacrylate) (PVP-g-PDMAEMA)/DNA formed by layer-by-layer electrostatic interactions to screen the residual surface positive charges of complexes. The coating of BSA was able to decrease the zeta potential of binary complexes nearly to electroneutrality without interfering with DNA condensation ability. The ternary complexes of BSA/PVP-g-PDMAEMA/DNA demonstrated lower cytotoxicity compared with the binary complexes and also maintained high gene transfection efficiency in HepG2 cells.

Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors

Development of highly efficient nonviral gene delivery vectors still remains a great challenge. In this study, we report a new gene delivery vector based on dendrimer-entrapped gold nanoparticles (Au DENPs) with significantly higher gene transfection efficiency than that of dendrimers without AuNPs entrapped. Amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers (G5.NH2) were utilized as templates to synthesize AuNPs with different Au atom/dendrimer molar ratios (25:1, 50:1, 75:1, and 100:1, respectively). The formed Au DENPs were used to complex two different pDNAs encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The Au DENPs/pDNA polyplexes with different N/P ratios and compositions of Au DENPs were characterized by gel retardation assay, light scattering, zeta potential measurements, and atomic force microscopic imaging. We show that the Au DENPs can effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines as demonstrated by both Luc assay and fluorescence microscopic imaging of the EGFP expression. The transfection efficiency of Au DENPs with Au atom/dendrimer molar ratio of 25:1 was at least 100 times higher than that of G5.NH2 dendrimers without AuNPs entrapped at the N/P ratio of 2.5:1. The higher gene transfection efficiency of Au DENPs is primarily due to the fact that the entrapment of AuNPs helps preserve the 3-dimensional spherical morphology of dendrimers, allowing for more efficient interaction between dendrimers and DNA. With the less cytotoxicity than that of G5.NH2 dendrimers demonstrated by thiazoyl blue tetrazolium bromide assay and higher gene transfection efficiency, it is expected that Au DENPs may be used as a new gene delivery vector for highly efficient transfection of different genes for various biomedical applications.

Well-defined hydroxyapatite–polycation nanohybrids via surface-initiated atom transfer radical polymerization for biomedical applications

The ability to manipulate and control the surface properties of hydroxyapatite (HA) nanoparticles is of crucial importance for the design of HA-based carriers of therapeutic agents. In this work, surface-initiated atom transfer radical polymerization (ATRP) of (2-dimethyl amino)ethyl methacrylate (DMAEMA) is first employed to tailor the functionality of HA surfaces in a well-controlled manner and to produce a series of new cationic hybrids (termed as HA-PDM). The HA parts of HA-PDM were coated by different lengths of PDMAEMA chains. The HA-PDM exhibited a good ability to condense plasmid DNA (pDNA) with suitable particle size and a zeta potential for gene transfection. Most importantly, in comparison with PDMAEMA homopolymers, the HA-PDM displayed considerably enhanced buffering capacity, and exhibited much higher gene transfection efficiencies in different cell lines, including osteoblast MC3T3 and osteosarcoma MG63 cells. In addition, the HA-PDM/pDNA complexes also could largely enhance the differentiation of preosteoblast cells. Such well-defined HA-PDM nanohybrids possess great potential applications as new drug-delivery vectors in bone tissue engineering.

Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity

BackgroundThe success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.MethodsA liver cancer-targeted specific peptide (FQHPSF sequence) was successfully synthesized and linked with chitosan-linked polyethylenimine (CP) to form a new targeted gene delivery vector called CPT (CP/peptide). The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/ DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.ResultsThe results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT was confirmed and the vector showed low cytotoxicity and strong targeting specificity to liver tumors in vitro. The in vivo study results showed that interleukin-12 delivered by the new gene vector CPT/DNA significantly enhanced the antitumor effect on ascites tumor-bearing imprinting control region mice as compared with polyethylenimine (25 kDa), CP, and other controls, which further demonstrate the targeting specificity of the new synthesized polymer.ConclusionThe synthesized CPT copolymer was proven to be an effective liver cancer-targeted vector for therapeutic gene delivery, which could be a potential candidate for targeted cancer gene therapy.

Synthesis and evaluation of N-(2,3-dihydroxypropyl)-PEIs as efficient vectors for nucleic acids

Branched polyethylenimine (bPEI, 25 kDa) has been widely used as an efficient delivery vector for nucleic acids in vitro. However, its charge-associated toxicity has limited its in vivo applications. In an attempt to control its toxicity, it was reacted with varying amounts of glycidol (2,3-epoxy-1-propanol) to obtain a small series of hydrophilic polymers, 2,3-dihydroxypropyl-grafted-polyethylenimines (DHP-g-P). The resulting polymers were characterized by (1)H-NMR and subjected to interaction with negatively charged pDNA, which yielded complexes in the size range of ~171-190 nm with a zeta potential of ∼+33-39 mV. Acid-base titration revealed no effect of substitution on the buffering capacity of the modified polymers. Grafting of 2,3-dihydroxypropyl groups on bPEI significantly improved the cell viability (i.e. almost non-toxic) as well as the DNA release properties of these modified polymers compared to native bPEI. Formation of a relatively loose DHP-g-P25/pDNA complex (the best working system in terms of transfection efficiency) resulted in the efficient nuclear release of pDNA for transcription, a prerequisite for efficient transfection. Subsequently, upon evaluation of their ability to transfer nucleic acids in vitro, the DHP-g-P/pDNA complexes exhibited higher gene transfection efficiency with one of the formulations, DHP-g-P25/DNA complex, displaying ~2.7 folds higher GFP expression than bPEI and ~2.3-3.5 folds higher than the selected commercial transfection reagents used in this study. Further to quantify the extent of GFP positive cells, FACS analysis was performed, which revealed DHP-g-P25/DNA mediated gene expression in ~51% cells outcompeting bPEI, Superfect™, Fugene™ and Lipofectamine™. Sequential delivery of GFP-specific siRNA resulted in ~78% suppression of the target gene compared to ~49% achieved by Fugene™. All these results demonstrate the potential of these polymers for in vivo gene delivery.

Novel hyperbranched polyamidoamine nanoparticle based gene delivery: Transfection, cytotoxicity and in vitro evaluation

In this study, hyperbranched polyamidoamine (hPAMAM) was developed as a novel non-viral gene vector for the first time. The hPAMAM was synthesized using a modified “one-pot” method. DNA was then bound to hPAMAM at different weight ratios (whPAMAM/wDNA). The higher weight ratio could bring larger particle size and higher zeta potential of hPAMAM–DNA complexes. The encapsulated DNA was protected by hPAMAM from degradation for over 3h. Under the optimal condition, high gene transfection efficiency could be achieved in COS7 (47.47±1.42%) and HEK293 (40.8±0.98%) cell lines. And hPAMAM showed rather minor cytotoxicity in vitro (cell viability=91.38±0.46% in COS7 and 92.38±0.61% in HEK293). The hPAMAM mediated human vascular endothelial growth factor 165 (hVEGF165) gene transfected cells could express hVEGF165 stably for 14 days, with the peak expression at day 2. In conclusion, hPAMAM based gene delivery was economical, effective and biocompatible, and may serve as a promising non-viral vehicle for gene therapy.

Exploring N-Imidazolyl-O-Carboxymethyl Chitosan for High Performance Gene Delivery

Chitosan shows good biocompatibility and biodegradability, but the poor water solubility and low transfection efficiency hinder its applications as a gene delivery vector. We here report the detailed synthesis and characterization of a novel ampholytical chitosan derivative, N-imidazolyl-O-carboxymethyl chitosan (IOCMCS), used for high performance gene delivery. After chemical modification, the solubility of the resulting polymer is enhanced, and the polymer is soluble in a wide pH range (4-10). Gel electrophoresis study reveals the strong binding ability between plasmid DNA and the IOCMCS. Moreover, the IOCMCS does not induce remarkable cytotoxicity against human embryonic kidney (HEK293T) cells. The cell transfection results with HEK293T cells using the IOCMCS as gene delivery vector demonstrate the high transfection efficiency, which is dependent on the degree of imidazolyl substitution. Therefore, the IOCMCS is a promising candidate as the DNA delivery vector in gene therapy due to its high solubility, high gene binding capability, low cytotoxicity, and high gene transfection efficiency.

In vitro and in vivo gene delivery using polyethylenimine-poly(hydroxyethyl glutamine) as a non-viral carrier

Summary Polyethylenimine-poly(hydroxyethyl glutamine) copolymer (PEI-PHEG) could effectively form positively charged nanoparticles with pDNA. The PEI-PHEG copolymer exhibited much lower cytotoxicity and higher gene transfection efficiency than PEI-25 k with different cell lines in vitro. PEI-PHEG/pDNA complex demonstrated excellent transfection efficiency in animal tests. PEI-PHEG could efficiently deliver siRNA to cells to silence the target gene. It could also complex vascular endothelial growth factor siRNA (VEGF siRNA) to achieve the aim of tumor therapy.

CNGR conjugated poly(lactic acid)-poly(ethylene glycol) nanoparticles for targeted gene delivery

Cyclic asparagines-glycine-arginine (cNGR) peptide was used to target gene loaded poly(lactic acid)-poly(ethylene glycol) nanoparticles (PLA-PEG NPs) to human umbilical vein endothelial cells (HUVEC). cNGR modified gene loaded PLA-PEG nanoparticles (cNGR-PLA-PEG NPs) could effectively condense DNA into small-sized complexes (<200nm) with positive charge (~10mV) using a novel cationic surfactant, 6-lauroxyhexyl lysinate (LHLN). cNGR-PLA-PEG NPs could be internalized into HUVEC by caveolae-mediated endocytosis with higher gene transfection efficiency and lower cytotoxicity than PLA-PEG NPs.