Improved Transfection Efficiency Research Articles

Rational design of acid-labile branched polycation with superior gene transfection capacity

There is much need in advancing rational design of synthetic cationic polymers as gene carriers. Inspired by the molecular blueprint of the famed branched polyethylenimine (bPEI), here we describe a new series of acid-labile branched poly(ortho ester amino alcohol)s (bPOEAAs) that embodies multiple design principles about polymer degradability, chain branching, and type of cationic amino groups. The bPOEAAs were facilely synthesized via expoxide-amine reaction of monomers with various ratios, allowing a range of branching degrees and amine compositions to be explored. A polymer (bPOEAA2) with an intermediate degree of branching and approximately equal fraction of secondary and tertiary amines was identified to be optimal in forming stable polyplexes with plasmid DNA. Rapid release of DNA from the polymer was achieved by accelerated backbone degradation at endosomal acidic pH. Together, these unique features of bPOEAA2 enabled markedly improved transfection efficiency in cells in physiological medium and much lower cytotoxicity than bPEI. Insights gained from the rational design and screening of bPOEAAs may contribute to deeper understanding of structure-activity relationship of polymeric gene carriers, which may ultimately lead to safer and more effective gene therapies.

Composite liposome-PEI/nucleic acid lipopolyplexes for safe and efficient gene delivery and gene knockdown

Cytotoxicity is a major drawback impeding the therapeutic use of gene delivery and gene down-regulation vehicles. Apart from cytotoxicity, rapid degradation and low cellular uptake are other major factors affecting therapeutic use. Considering the above factors, formulation and development of PEI (Polyethylenimine) based, liposome encapsulated delivery vehicles with improved transfection efficiency and low cytotoxicity which can be used for gene delivery and gene knockdown. DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (Dipalmitoylphosphatidylcholine) and cholesterol have been considered as lipids of choice bearing in mind various factors such as rigidness and surface charge which greatly influence the formation of liposomes, polyplex encapsulation and transfection efficiency. For the condensation of plasmid DNA (pDNA) and short interfering RNA (siRNA), branched PEI 25kDa (bPEI) and deacylated linear PEI 22kDa (lPEI) were employed. lPEI and siRNA polyplexes encapsulated within DOPE/DPPC/Cholesterol (DDC) liposomes exhibited higher luc (luciferase) gene knockdown in vitro compared to the controls. They also showed superior transfection efficiencies compared to polyplexes in experiments using pCMV-luc (luciferase reporter plasmid) and pEGFP-N1 (Green Fluorescence protein reporter plasmid). This can partly be attributed to the improved integrity imparted by the liposomal layer which was confirmed by complex stability and integrity assays. Cytotoxicity and coagulation time assays of DDC-lPEI based lipopolyplexes showed decreased cytotoxic potential and negligible influence on coagulation respectively for compared to polyplexes, thus rendering them suitable for gene therapy.

Experiment study on the transfection of exogenous genes promoted by ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal

Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery(UTMD) mediated a peptide nucleic acid (PNA) binding nuclear localization signal (NLS). Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor. The SV40T antigen receptors were expressed on the membranes of 293T cells. The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA, which increased nuclear localization. Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm, respectively. The study was divided into five groups: Contrast (group A), Common microbubble+ DNA (group B), Antibody-targeted microbubbles+ DNA (group C), Common microbubbles+ NLS-PNA-DNA (group D), Antibody-targeted microbubbles+ NLS-PNA-DNA (group E). Fluorescence microscope was used to observe the fluorescent light in each group; flow cytometry to test the transfection; RT-PCR and Western blot to detect genes′ mRNA and protein expression level. Results Ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability(>80%). Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully.The transfection results of flow cytometry were 0, (9.30±0.46)%, (26.46±2.01)%, (29.54±0.62)%, (45.72±1.86)%, respectively, and the differences were statistically significant(P<0.05). The relative mRNA and protein expression in group E were greater than those in group C and D respectively (P<0.05). Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nuclear DNA import. Key words: Sonication; Microbubbles; Peptide nucleic acid; Nuclear localization signal; Gene transfection

Efficient megalin targeted delivery to renal proximal tubular cells mediated by modified-polymyxin B-polyethylenimine based nano-gene-carriers

Non-viral vectors have attracted great interest, as they are simple to prepare, easy to modify and relatively safe, compared to viral vectors. Kidney-targeted gene delivery systems depict a promising technology to improve drug efficacy in renal diseases treatments. In order to develop a novel kidney-targeted gene delivery system, we synthesized polyamine-PEI conjugates using polymyxin B as ligand and investigated their potential targeting efficiency. After grafting either PEI25 kDa or PEI10 kDa with polymyxin B through amidation reaction, the modified-polymyxin-PEI/DNA-nanoplexes were produced via electrostatic attraction between the cationic polymers and EGFP plasmid. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. Results revealed that the average size of -modified-polymyxin- PEI25kDa was about 143–180nm and modified-polymyxin-PEI10kDa 115–194nm. The zeta potentials were in the range of 16.4±1.87 to 23.43±1.25mV and 11.3±1.4 to 19.3±2.1mV for conjugates based on PEI25 and PEI10 respectively. The AFM images revealed that the complexes were spherical and nano-sized at C/P=4. The buffering capacity of both PEI 10 and 25kDa increased as the percentage of polymyxin B grafting increased. In vitro study demonstrated that modified-polymyxin-PEI conjugates could remarkably improve the gene transfection efficiency to kidney cells. The transfection efficiency of the polyplexes was dependent on the weight ratio of ligand in the formulation (~12 and 8 fold increase for PEI25 and PEI10kDa, respectively) and was significantly higher than that of unmodified PEIs/DNA nanoparticles. These results suggest that modified-polymyxin-PEI /DNA nanoparticles can effectively target megalin-expressing kidney cells and show improved transfection efficiency and low cytotoxicity in In vitro and In vivo studies. Animal studies confirmed the in vivo study. Thus, these conjugates can be considered as a safe and efficient non-viral therapeutic therapy vector for kidney diseases.

Erratum to: Inhibition of the Innate Immune Receptors for Foreign DNA Sensing Improves Transfection Efficiency of Gene Electrotransfer in Melanoma B16F10 Cells.

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells’ survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

Hydroxyl versus permethylated glycopolymers as gene carriers

The main parameters that contribute to non-viral gene delivery are chemical structure and charge distribution. Indeed, saccharide units have been reported to have specific interactions with proteins located in the outer leaflet of the plasma cell membrane that facilitate the cellular internalization of plasmid-DNA vector complexes. In this work, glycopolymers based on statistical copolymers were synthesized through radical copolymerization of a cationic unit, N-ethyl pyrrolidine methacrylamide (EPA), with two styrenic monomers derived from the hydroxylated and permethylated forms of α-glucose. These copolymers were evaluated as possible non-viral gene carriers, and their ability to complex DNA was evaluated. The transfection efficiency and cytocompatibility of the polyplexes, in both fibroblastic and tumoral murine cell lines, was evaluated. Systems derived from α-glucose (GLCSt), over a monomer concentration range of 5–70mol%, exhibited high toxicity and low transfection efficiency, and were not able to significantly improve on results obtained from positive poly-EPA (PEPA) and polyethyleneimine (PEI) controls. However, systems derived from the permethylated form of α-glucose (MGLCSt), formed stable complexes with DNA or polyplexes, which showed improved transfection efficiency and cytocompatibility in comparison to positive controls. The high transfection efficiency can be clearly attributed to their cytocompatibility, which was notably found to be different for Swiss fibroblasts and B16 melanoma cells, high for Swiss and low for B16. As such, we present permethylated MCLCSt copolymers as good candidates for the possible development of therapies against melanoma.

Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes

Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI25 (25kDa PEI) or carboxyalkylated-PEI10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143–173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity.

Inhibition of the Innate Immune Receptors for Foreign DNA Sensing Improves Transfection Efficiency of Gene Electrotransfer in Melanoma B16F10 Cells.

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells' survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells

Abstract In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (qp), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fc-fusion protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system.

Gene delivery into isolated Arabidopsis thaliana protoplasts and intact leaves using cationic, α-helical polypeptide

The application of gene delivery materials has been mainly focused on mammalian cells while rarely extended to plant engineering. Cationic polymers and lipids have been widely utilized to efficiently deliver DNA and siRNA into mammalian cells. However, their application in plant cells is limited due to the different membrane structures and the presence of plant cell walls. In this study, we developed the cationic, α-helical polypeptide that can effectively deliver DNA into both isolated Arabidopsis thaliana protoplasts and intact leaves. The PPABLG was able to condense DNA to form nanocomplexes, and they exhibited significantly improved transfection efficiencies compared with commercial transfection reagent Lipofectamine 2000 and classical cell penetrating peptides such as poly(L-lysine), HIV-TAT, arginine9, and poly(L-arginine). This study therefore widens the utilities of helical polypeptide as a unique category of gene delivery materials, and may find their promising applications toward plant gene delivery.

Evaluation of chemical modification effects on DNA plasmid transfection efficiency of single-walled carbon nanotube-succinate- polyethylenimine conjugates as non-viral gene carriers.

Polyethylenimine (PEI) is a widely used non-viral vector for DNA delivery. One major obstacle of higher molecular weight PEIs is the increased cytotoxicity despite the improved transfection efficiency and numerous chemical modifications that have been reported to overcome this problem. Carbon nanotubes (CNT) are carbon nanomaterials capable of penetrating into cell membranes with no cytotoxic effects. Covalent and noncovalent functionalization methods have been used to improve their solubility in aqueous media. The idea of conjugating PEIs and CNT through different chemical bonds and linkers seems promising as it may result in highly effective carriers due to combination of the transfection ability of PEI with cell internalization of CNT. In this study, six different water-soluble PEI conjugates of single-walled carbon nanotubes (SWNTs) were prepared by grafting PEI with one of three molecular weights (1.8, 10 and 25 kDa) through succinate as a linker which refers to "an organic moiety through which a SWNT is conjugated to PEI." The succinate linker was introduced to the surface of SWNTs through two different chemical strategies: a) ester and b) acyl linkages. The resulting SWNT-PEI vectors were characterized by IR spectroscopy, thermogravimetric analysis (TGA) and SEM imaging. All synthesized carriers were evaluated and compared for their cytotoxicity and transfection efficiency in murine neuroblastoma cells as polyplexes with plasmid DNA for luciferase and green fluorescent protein (GFP). The most efficient carriers were prepared by attaching PEI with the lowest molecular weight (1.8 kDa) through acyl linkage, which gave a transfection efficiency 190-fold greater than that of the corresponding free PEI. Transfection efficiency was the highest in polyplexes prepared with acyl-linked conjugates in all the plasmid/vector ratios studied.

Enhanced effect of nuclear localization signal peptide during ultrasound‑targeted microbubble destruction‑mediated gene transfection.

Ultrasound-targeted microbubble destruction (UTMD) can promote the entry of plasmid DNA (pDNA) into the cell cytoplasm, by increasing the permeability of the cell membrane. But the transfection efficiency remains low due to inability of the pDNA to enter the nucleus. Various methods have been explored to improve the UTMD transfection efficiency, but with little success. In cells, the classic nuclear localization signal (cNLS) peptide is an amino acid sequence that signals proteins that are due for nuclear transport. The present study aimed to investigate whether binding of a cNLS peptide to the pDNA may improve the transfection efficiency of UTMD. Four experimental groups were analyzed: Control group (UTMD + pDNA), group with cNLS (UTMD + pDNA + cNLS), group with mutated NLS (mNLS; UTMD + pDNA + mNLS), and group with cNLS and the nuclear import blocker, wheat germ agglutinin (WGA; UTMD + pDNA + cNLS + WGA). The NLS was labeled by fluorescein isothiocyanate, whereas pDNA was labeled with Cy3. Different molar ratios were tested for the NLS and pDNA combination in order to achieve optimal binding of the two molecules. Human umbilical vein endothelial cells were then transfected using the optimum ultrasonic irradiation parameters and NLS/pDNA molar ratio. At 6 h post-transfection, the rates of Cy3-labeled pDNA inside the cells and their nuclei were detected by flow cytometry and laser confocal microscopy, and the cellular vs. nuclear uptake of pDNA was calculated. In order to further evaluate the effect of NLS on UTMD-mediated gene transfection, the transfection efficiency and relative expression levels of mRNA and protein were detected at 48 h post-transfection. The results demonstrated that the optimal molar ratio of NLS with pDNA was 104:1. The rates of pDNA successful entry into the cell and nucleus were significantly higher in the cNLS group compared with the control group. The transfection efficiency, and relative expression levels of mRNA and protein from the plasmid were significantly increased in the cNLS group compared with the control group. The mNLS group displayed no significant difference compared with the control group, while the WGA group exhibited significant inhibition in most indicators of transfection efficiency compared to the cNLS group. These results suggest that combining a cNLS peptide with pDNA during UTMD-mediated transfection significantly improved transfection efficiency. Thus, a cNLS peptide may be an important mediator and a new strategy in enhancing the efficiency of UTMD-mediated gene transfection.

Ultrasound-Targeted Microbubbles Combined with a Peptide Nucleic Acid Binding Nuclear Localization Signal Mediates the Transfection of Exogenous Genes by Improving both Cytoplasmic and Nuclear Import

The development of an efficient delivery systems is critical for non-viral gene therapies used to treat cardiovascular diseases. The cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the import of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes could improve gene therapies. We have employed a novel strategy involving ultrasound-targeted microbubbles and a PNA (Peptide Nucleic Acid) binding NLS (Nuclear Localization Signal). Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm, respectively. We conducted experiments using antibody-targeted microbubbles that specifically recognize the SV40T antigen receptor expressed on the membranes of 293t cells so that ultrasound microbubbles could be gathered around the 293t cell membranes. Furthermore, we inserted the PNA containing the nuclear localization signal (NLS) in the egfp-n3 plasmid DNA, which increased nuclear localization. The nuclear import and gene expression efficiency of the antibody-microbubbles with the PNA binding NLS were then compared with antibody-targeted microbubbles alone or a PNA binding NLS. We specifically investigated the effect of the antibody-targeted microbubbles containing the PNA binding NLS on transfection. The results indicate that ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. We found the nuclear import and gene expression of combined microbubble and PNA transfected cells were significantly higher than cells transfected with antibody-targeted microbubbles or DNA with only the PNA binding NLS.The quantity of pEGFP-N3 plasmids present in the nucleus increased by more than 3.2-fold relative to the control mircrobubbles and NLS-free plasmids.The data indicate that the gene expression was 2.0-fold greater. These results demonstrate that UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid significantly improved transfection efficiency by enhancing both cytoplasmic and nuclear DNA import. It's a very promising method for noninvasive and effective delivering target genes or drugs to the targeted to treat cardiovascular diseases.

Optimization of conditions for gene delivery system based on PEI

Objective(s): PEI based nanoparticle (NP) due to dual capabilities of proton sponge and DNA binding is known as powerful tool for nucleic acid delivery to cells. However, serious cytotoxicity and complicated conditions, which govern NPs properties and its interactions with cells practically, hindered achievement to high transfection efficiency. Here, we have tried to optimize the properties of PEI/ firefly luciferase plasmid complexes and cellular condition to improve transfection efficiency. Materials and Methods: For this purpose, firefly luciferase, as a robust gene reporter, was complexed with PEI to prepare NPs with different size and charge. The physicochemical properties of nanoparticles were evaluated using agarose gel retardation and dynamic light scattering. MCF7 and BT474 cells at different confluency were also transfected with prepared nanoparticles at various concentrations for short and long times. Results: The branched PEI can instantaneously bind to DNA and form cationic NPs. The results demonstrated the production of nanoparticles with size about 100-500 nm dependent on N/P ratio. Moreover, increase of nanoparticles concentration on the cell surface drastically improved the transfection rate, so at a concentration of 30 ng/il, the highest transfection efficiency was achieved. On the other side, at confluency between 40-60%, the maximum efficiency was obtained. The result demonstrated that N/P ratio of 12 could establish an optimized ratio between transfection efficiency and cytotoxicity of PEI/plasmid nanoparticles. The increase of NPs N/P ratio led to significant cytotoxicity. Conclusion: Obtained results verified the optimum conditions for PEI based gene delivery in different cell lines.

Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene to DA neurons with improved transfection efficiency. Finally, the study will lead to a significant advancement in the field of non-viral PD gene therapy treatment.

Dissolving microneedles for DNA vaccination: Improving functionality via polymer characterization and RALA complexation

ABSTRACTDNA vaccination holds the potential to treat or prevent nearly any immunogenic disease, including cancer. To date, these vaccines have demonstrated limited immunogenicity in vivo due to the absence of a suitable delivery system which can protect DNA from degradation and improve transfection efficiencies in vivo. Recently, microneedles have been described as a novel physical delivery technology to enhance DNA vaccine immunogenicity. Of these devices, dissolvable microneedles promise a safe, pain-free delivery system which may simultaneously improve DNA stability within a solid matrix and increase DNA delivery compared to solid arrays. However, to date little work has directly compared the suitability of different dissolvable matrices for formulation of DNA-loaded microneedles. Therefore, the current study examined the ability of 4 polymers to formulate mechanically robust, functional DNA loaded dissolvable microneedles. Additionally, complexation of DNA to a cationic delivery peptide, RALA, prior to incorporation into the dissolvable matrix was explored as a means to improve transfection efficacies following release from the polymer matrix. Our data demonstrates that DNA is degraded following incorporation into PVP, but not PVA matrices. The complexation of DNA to RALA prior to incorporation into polymers resulted in higher recovery from dissolvable matrices, and increased transfection efficiencies in vitro. Additionally, RALA/DNA nanoparticles released from dissolvable PVA matrices demonstrated up to 10-fold higher transfection efficiencies than the corresponding complexes released from PVP matrices, indicating that PVA is a superior polymer for this microneedle application.

Polyethyleneimine-Coated Gold Nanoparticles: Straightforward Preparation of Efficient DNA Delivery Nanocarriers.

A novel one-pot method for the synthesis of polyethyleneimine (PEI)-coated gold nanoparticles (AuPEI-NPs) that combines the reductant-stabilizer properties of PEI with microwave irradiation starting from hydrogen tetrachloroaurate acid (HAuCl4 ) and branched PEI 25 kDa (b25kPEI) was explored. The method was straightforward, green, and low costing, for which the Au/PEI ratio (1:1 to 1:128 w/w) was a key parameter to modulate their capabilities as DNA delivery nanocarriers. Transfection assays in CHO-k1 cells demonstrated that AuPEI-NPs with 1:16 and 1:32 w/w ratios behaved as effective DNA gene vectors with improved transfection efficiencies (twofold) and significantly lower toxicity than unmodified b25kPEI and Lipofectamine 2000. The transfection mediated by these AuPEI-NP-DNA polyplexes preferentially used the caveolae-mediated route for intracellular internalization, as shown by studies performed by using specific internalization inhibitors as well as colocalization with markers of clathrin- and caveolae-dependent pathways. The AuPEI-NP polyplexes preferentially used the more efficient caveolae internalization pathway to promote transfection, a fact that supports their higher transfection efficiency relative to that of Lipofectamine 2000. In addition, intracellular trafficking of the AuPEI-NPs was studied by transmission electron microscopy.

Improved formulation of cationic solid lipid nanoparticles displays cellular uptake and biological activity of nucleic acids

Non-viral delivery using cationic solid lipid nanoparticles (SLNs) represents a useful strategy to introduce large DNA and RNA molecules to target cells. A careful selection of components and their amounts is critical to improve transfection efficiency. In this work, a selected and optimized formulation of SLNs was used to efficiently transfect circular DNA and linear RNA molecules into cells. We characterized the main physicochemical characteristics and binding capabilities of these SLNs and show that they deliver DNA and RNA molecules into cells where they display full bioactivity at nontoxic concentrations using fluorescence- and luminescence-based methodologies. Hence, we established a novel and simple SLN formulation as a powerful tool for future therapeutic use.

Treatment of Parkinson's disease in rats by Nrf2 transfection using MRI-guided focused ultrasound delivery of nanomicrobubbles

Parkinson's disease (PD) is a very common neurological disorder. However, effective therapy is lacking. Although the blood-brain-barrier (BBB) protects the brain, it prevents the delivery of about 90% of drugs and nucleotides into the brain, thereby hindering the development of gene therapy for PD. Magnetic resonance imaging (MRI)-guided focused ultrasound delivery of microbubbles enhances the delivery of gene therapy vectors across the BBB and improves transfection efficiency. In the present study, we delivered nuclear factor E2-related factor 2 (Nrf2, NFE2L2) contained in nanomicrobubbles into the substantia nigra of PD rats by MRI-guided focused ultrasound, and we examined the effect of Nrf2 over-expression in this animal model of PD. The rat model of PD was established by injecting 6-OHDA in the right substantia nigra stereotactically. Plasmids (pDC315 or pDC315/Nrf2) were loaded onto nanomicrobubbles, and then injected through the tail vein with the assistance of MRI-guided focused ultrasound. MRI-guided focused ultrasound delivery of nanomicrobubbles increased gene transfection efficiency. Furthermore, Nrf2 gene transfection reduced reactive oxygen species levels, thereby protecting neurons in the target region.

Guanidinylated bioresponsive poly(amido amine)s designed for intranuclear gene delivery.

Guanidinylated poly(amido amine)s with multiple disulfide linkages (Gua-SS-PAAs) were designed and constructed as nonviral gene carriers. The main chains of these novel carriers were synthesized based on monomers containing guanidino groups (guanidine hydrochloride and chlorhexidine), which could avoid complicated side-chain-modification reactions while introducing the guanidino groups. The synthesized Gua-SS-PAAs polymers were characterized by 1H nuclear magnetic resonance, molecular weight, and polydispersity. Furthermore, Gua-SS-PAAs polymers were complexed with pDNA, and the properties of the complexes were determined, including entrapment efficiency, particle size, ζ-potential, atomic force microscopy images, stability, DNA complexation ability, reduction sensitivity, cytotoxicity, and transfection efficiency. The new Gua-SS-PAAs carriers exhibited higher transfection efficiency and lower cytotoxicity compared with two widely used gene delivery carriers, polyethylenimine and lipofectamine 2000. Furthermore, the relationship between the side-chain structure and morphological/biological properties was extrapolated, and the results showed that guanidine in the side chain aids in the improvement of transfection efficiency. In addition, the introduction of guanidino group might confer the new carriers with nuclear localization function compared to carriers without it.