Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 ～ 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Poly(L-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 approximately 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods

Controlled release of plasmid DNA (pDNA) from biodegradable poly lactic-co-glycolic acid (PLGA) microparticles has the potential to enhance transgene expression. However, barriers to this approach include limited encapsulation efficiency, pDNA damage during fabrication and confinement of the microparticles inside phagolysosomal compartments. Combining PLGA with poly ethyleneimine (PEI) can improve protection of pDNA during fabrication, increase encapsulation efficiencies and impart the PLGA microparticles with the capacity to escape the phagolysosomal compartments. This study compares three promising formulation methods for preparing PLGA PEI pDNA microparticles and evaluates for buffering capacity, cellular uptake, transfection efficiency and toxicity. In the first method, PLGA PEI pDNA microparticles are prepared by entrapping pDNA in blended PLGA/PEI using the double emulsion water-in-oil-in-water solvent evaporation technique (PA). In a second approach, PEI-pDNA polyplexes are prepared and then entrapped in PLGA microparticles using a double emulsion solvent evaporation method (PB). Microparticles prepared using formulation methods PA and PB are then compared against PLGA microparticles with PEI conjugated to the surface using carbodiimide chemistry (PC); 0.5% PVA is identified as the optimum concentration of surfactant for generating the strongest transfection efficiencies. N:P ratios of 5 and 10 are selected for preparation of each group. Gel electrophoresis demonstrates that all PLGA microparticle formulations have strong pDNA binding capacity. An MTT assay shows that in vitro cytotoxicity of PLGA PEI microparticles is significantly lower than PEI alone. PLGA PEI pDNA microparticles mediate higher cellular uptake efficiency and consequently higher transgene expression than unmodified PLGA microparticles in COS7 and HEK293 cells. Preparing PEI-pDNA polyplexes prior to entrapment in PLGA microparticles (PB) results in the highest pDNA loading. This is 2.5-fold higher than pDNA loading in unmodified PLGA microparticles. PLGA PEI pDNA microparticles prepared using method PB generates the strongest transfection efficiencies, which are 500-fold higher than unmodified PLGA pDNA microparticles in HEK293 cells and 1800-fold higher in COS-7 cells. The highest transfection efficiencies generated from microparticles prepared using method PB is achieved using an N:P ratio of 5.     

Spray-drying preparation of microparticles containing cationic PLGA nanospheres as gene carriers for avoiding aggregation of nanospheres

Preparation of nano-sized particles using lyophilization, which is a standard drying technique for high-molecular-weight compounds such as bioactive peptides, proteins, plasmid DNA and siRNA, often results in particle aggregation. In this study, spray-drying was applied for preparation of cationic PLGA nanospheres as gene delivery vectors in order to minimize aggregation and loss of gene transfection efficiency. PLGA nanoparticle emulsions were prepared by dropping an acetone/methanol mixture (2/1) containing PLGA and a cationic material, such as PEI, DOTMA, DC-Chol or CTAB, into distilled water with constant stirring. The PLGA nanosphere emulsion was dried with mannitol by spray-drying, and mannitol microparticles containing PLGA nanospheres were obtained. Mean particle diameter of spray dried PLGA particles was 100-250 nm, which was similar to that of the nano-emulsion before drying, whereas the lyophilized PLGA particles showed increased particle diameter due to particle aggregation. PEI, DOTMA and DC-Chol were useful for maintaining nanoparticle size and conferring positive charge to nanospheres. Transfection of pDNA (pCMV-Luc) using these spray-dried cationic PLGA nanospheres yielded high luciferase activity in COS-7 cells, particularly with PLGA/PEI nanospheres. The present spray-drying technique is able to provide cationic PLGA nanospheres, and may improve redispersal and handling properties.     

Fabrication,characterization and in vitro evaluation of poly(D,L-lactide-co-glycolide) microparticles loaded with polyamidoamine–plasmid DNA dendriplexes for applications in nonviral gene delivery

We report, for the first time, on the preparation, characterization and in vitro testing of poly(D,L-lactide-co-glycolide) (PLGA) microparticles loaded with polyamidoamine (PAMAM)–plasmid DNA (pDNA) dendriplexes. Loading of pDNA into the PLGA microparticles increased by 150% when pDNA was first complexed with PAMAM dendrimers relative to loading of pDNA alone. Scanning electron microscopy (SEM) showed that the presence of PAMAM dendrimers in the PLGA microparticles created porous features and indentations on the surface of the microparticles. Loading PLGA microparticles with PAMAM–pDNA dendriplexes lowered the average PLGA microparticle size and changed the surface charge of the microparticles from negative to positive when compared to PLGA microparticles loaded with pDNA alone. The zetapotential and buffering capacity of the microparticles increased as the generation of the PAMAM dendrimer loaded in the PLGA microparticles increased. Gel electrophoresis assays showed that all the PLGA microparticle formulations were able to entrap the pDNA within the PLGA matrix. There was no significant difference in the cytotoxicity of PLGA microparticles loaded with PAMAM–pDNA dendriplexes when compared to PLGA microparticles loaded with pDNA alone. Furthermore, and in contrast to PAMAM dendrimers alone, the generation of the PAMAM dendrimer loaded in the PLGA microparticles had no significant impact on cytotoxicity or transfection efficiencies in human embryonic kidney (HEK293) or Monkey African green kidney fibroblast-like (COS7) cells. The transfection efficiency of PLGA microparticles loaded with generation 3 (G3) PAMAM–pDNA dendriplexes was significantly higher than PLGA microparticles loaded with pDNA alone in HEK293 and COS7 cells. PLGA microparticles loaded with G3 PAMAM–pDNA dendriplexes generated equivalent transfection efficiencies as (G3 to G6) PAMAM–pDNA dendriplexes alone in COS7 cells when the transfection was carried out in serum containing media. The delivery system developed in this report has low toxicity, high pDNA loading efficiencies and high transfection efficiencies that are not reduced in the presence of serum. A delivery system with these characteristics is expected to have significant potential for translational applications. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:368–384, 2010     

Skin photoprotection improvement: synergistic interaction between lipid nanoparticles and organic UV filters

The main objective of this study was to prepare two types of nanoparticles with poly(d,l-lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) polymers. Plasmid DNA (pDNA) was adsorbed either on PLGA/PEI nanoparticles, or as PEI/DNA complex onto the surface of PLGA nanoparticles. Both types of nanoparticles were prepared by the double emulsion method. The nanoparticles were characterized by their size, zeta potential and pDNA or PEI/DNA complex adsorption. The PEI/DNA complex adsorption was confirmed with ethidium bromide assay. pDNA adsorption onto PLGA/PEI nanoparticles (PLGA/PEI-DNA) was studied by electrophoresis on agarose gel. Cytotoxicity and transfection efficiency of both types of nanoparticle and PEI/DNA complexes formulations were studied in head and neck squamous carcinoma cell line (FaDu). To improve endosomal release, photochemical internalization (PCI) was used. The zeta potential increased when the PEI/DNA complex adsorbed onto PLGA nanoparticles (PLGA-PEI/DNA). Optimal pDNA adsorption efficiency was achieved for nitrogen/phosphorous ratio≥20/1. In vitro transfection and cells viability on FaDu cells with or without PCI were found to be variable depending on the type and concentration of nanoparticles. The results showed that transfection efficiency for PLGA/PEI-DNA or PLGA-PEI/DNA nanoparticles ranged between 2 and 80%, respectively. PCI was found to slightly improve the transfection efficiency for all formulations.     

Insights into the mechanism of magnetofection using MNPs-PEI/pDNA/free PEI magnetofectins

Magnetofection is an efficient new physical gene transfection technology. Despite its effective gene delivery capability, till now relatively little work has been conducted on the mechanism of magnetofection, especially the intracellular fates of the components of magnetofectins and their effects on magnetofection. In this study, we investigated the mechanism of magnetofection using magnetofectins that were prepared via electrostatic self-assembly of the three components: polyethyleneimine (PEI)-coated magnetic nanoparticles (MNPs-PEI), plasmid DNA (pDNA) and PEI in the free form (free PEI). TEM observation and agarose gel electrophoresis assays have indicated MNPs play the role of driving magnetofectins to the cell surface without entering into the nucleus. Confocal microscopic tracking of fluorescence-labeled PEI has shown that the free PEI (green) can be found in the nucleus but almost all of the MNPs-PEI (red) are confined in the cytoplasm in COS-7 cells 30 min post-transfection or in SPC-A1 cells 90 min post-transfection, implying that the pDNA/PEI complex must separate from MNPs-PEI before entering into the nucleus. In addition, reporter gene assays showed the magnetofectins, in which the free PEI was absent, failed to transfect SPC-A1 or COS-7 cell lines; and there was an optimal ratio of the constituents of magnectofectins to achieve optimal transfection efficiency by balancing stable complex formation and facile release of PEI/pDNA from the complex. In summary, our findings further the knowledge of magnetofection and can be helpful for the design and preparation of gene delivery vehicles for effective magnetofection.     

Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine

Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.     

Self-assembly of cyclodextrin complexes: aggregation of hydrocortisone/cyclodextrin complexes

Oral delivery of gene therapeutics would facilitate treatment of local intestinal disease, including colon cancer and inflammatory bowel disease, thus avoiding invasive surgery. The aims of this study were to investigate; if the orientation of the lipid tail on the cyclodextrin (CD) influenced the efficacy of a novel poly-6-cationic amphiphilic CD to transfect intestinal enterocytes; the endocytotic uptake pathway(s), and, the intracellular trafficking of the CD·DNA complexes. Inhibitors of clathrin- and caveolae-mediated endocytosis and macropinocytosis were used to determine the mechanism(s) of CD·DNA uptake by both undifferentiated and differentiated Caco-2 cells. Cell surface heparan sulphate proteoglycans were involved in the association of CD·DNA complexes with undifferentiated Caco-2 cells. Complexation of pDNA with CD facilitated significant levels of pDNA uptake and gene expression (comparable to PEI) in both undifferentiated and differentiated Caco-2 cells. Disruption of intracellular vesicular trafficking reduced transfection activity. CD was also capable of transfecting the more physiologically relevant differentiated Caco-2 model. Macropinocytosis was responsible for the uptake of CD·DNA transfection complexes by both undifferentiated and differentiated Caco-2 cells. The ability of this novel CD to transfect differentiated intestinal cells indicates the potential of this vector for oral gene delivery.     

Microparticle-mediated transfection of non-phagocytic cells in vitro

DNA-loaded microparticles represent an attractive delivery system to target professional antigen presenting cells (APC) for the delivery of DNA vaccines. Microparticles exhibiting a positively charged surface were prepared by the incorporation of two selected cationic polymers into a poly(D, L-lactide-co-glycolide) polymer (PLGA) core. The toxicity of the different formulations was checked in two cell lines and was found to be comparable to plain PLGA particles. Increased toxicity of some formulations was observed in primary macrophages (Mphi) with high phagocytosis activity. Plasmid DNA was efficiently adsorbed to the microparticle surfaces, and the different formulations were checked for their transfection efficiency in phagocytic and non-phagocytic cells. Interestingly, the most pronounced gene transfer efficiency was observed in a non-phagocytic 293 cell line when compared to a macrophage cell line and primary Mphi. Possible mechanisms include the dissociation of DNA-polymer complex and subsequent transfection of the cells. Microscopic observation of fluorescent-labeled DNA in primary Mphi revealed large amounts of DNA entering the cells, but no detectable DNA inside the nuclei. We conclude that phagocytic professional APC represent a group of cells, which is especially difficult to transfect when compared to other cell types. The administration of DNA in vivo is likely to predominantly result in the transfection of non-lymphoid cells unless there is a possibility to provide efficient targeting and trafficking of the DNA to the nucleus of professional APC. Although DNA-loaded PEI and DAEM microparticles resulted in significant transfection of cells, toxicity and transfection efficiency was not superior to that of DNA complexed with soluble PEI and DAEM.     

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.     

Analysis of poly(D,L-lactic-co-glycolic acid) nanosphere uptake by human dendritic cells and macrophages in vitro

Purpose. The purpose of this study was to demonstrate and characterize phagocytosis of poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres by human dendritic cells (DCs). Methods. Parallel cultures of DCs and macrophages (M) were established from peripheral blood leukocytes using media supplemented with granulocyte-macrophage colony stimulator factor and interleukin-4 (for DC) or granulocyte-macrophage colony stimulator factor alone (for M). PLGA nanospheres containing tetramethylrhodamine-labeled dextran with or without an adjuvant, monophosphoryl lipid A, were prepared using a water/oil/water solvent evaporation technique. Cells were incubated with the nanospheres for 24 h. Confocal laser scanning microscopy was used to determine the intracellular location of the nanospheres and flow cytometry to measure the fraction of phagocytic cells in the culture and the amount of uptake per cell. After phagocytosis, cells were stained for MHC class II molecules, CD14, CD80, and CD86 to identify the phagocytic population. Results. DCs phagocytosed PLGA nanospheres as efficiently as M. Cell-surface marker expression conclusively established that the phagocytic cells were DC. Conclusions. DCs can take up PLGA nanospheres. Because DCs are the key professional antigen-presenting cells capable of stimulating naive T cells, our data suggest that PLGA nanospheres can be used as an efficient delivery system for vaccines designed to activate T cell-mediated immune responses.     

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.     

Plasmid DNA uptake and subsequent cellular activation characteristics in human monocyte-derived cells in primary culture

Plasmid DNA (pDNA) uptake and subsequent cellular activation characteristics were studied in three types of human monocyte-derived cells, that is, human monocytes, macrophages, and dendritic cells (DCs) in primary culture. Naked pDNA was bound to and taken up by the macrophages and DCs while only significant binding occurred in the monocytes. pDNA binding to these monocyte-derived cells was significantly inhibited by polyinosinic acid (poly[I]), dextran sulfate, maleylated bovine serum albumin (Mal-BSA) and to a lesser extent by polycytidylic acid (poly[C]), but not by dextran or galactosylated BSA (Gal-BSA), mannosylated BSA (Man-BSA), suggesting that a specific mechanism for polyanions is involved in the pDNA binding. In cellular activation studies, naked pDNA could not induce TNF-alpha production from any monocyte-derived cells, regardless of the abundant presence of CpG motifs in the pDNA. However, when complexed with cationic liposomes, pDNA produced a significant amount of TNF-alpha from the human macrophages. TNF-alpha induction was not observed in the monocytes or DCs. Moreover, calf thymus DNA (CT DNA) complexed with cationic liposomes also induced TNF-alpha production to a similar extent in the human macrophages. These results indicate that, among human monocyte-derived cells, macrophages are activated by DNA when complexed with cationic liposomes in a CpG motif-independent manner.     

Emerging significance of plasmid DNA nuclear import in gene therapy

The signal-mediated import of plasmid DNA (pDNA) into nondividing mammalian cell nuclei is one of the key biological obstacles to nonviral therapeutic pDNA delivery. Overcoming this barrier to pDNA transfer is thus an important fundamental objective in gene therapy. Here, we outline the rationale behind current and future strategies for signal-mediated pDNA nuclear import. Results obtained from studies of the nuclear delivery of pDNA coupled to experimentally defined nuclear localisation signal (NLS) peptides, in conjunction with detergent-permeabilised reconstitution cell assays, direct intracellular microinjection, cell-based transfection, and a limited number of in vivo experiments are discussed.     

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.     

A one-step process in preparation of cationic nanoparticles with poly(lactide-co-glycolide)-containing polyethylenimine gives efficient gene delivery

A one-step preparation of nanoparticles with poly(lactide-co-glycolide) (PLGA) pre-modified with polyethylenimine (PEI) is better in requirements for DNA delivery compared to those prepared in a two-step process (preformed PLGA nanoparticles and subsequently coated with PEI). The particles were prepared by emulsification of PLGA/ethyl acetate in an aqueous solution of PVA and PEI. DLS, AFM and SEM were used for the size characteristics. The cytotoxicity of PLGA/PEI nanoparticles was detected by MTT assay. The transfection activity of the particles was measured using pEGFP and pβ-gal plasmid DNA. Results showed that the PLGA/PEI nanoparticles were spherical and non-porous with a size of about 0.2 μm and a small size distribution. These particles had a positive zeta potential demonstrating that PEI was attached. Interestingly, the zeta potential of the particles (from one-step procedure) was substantially higher than that of two-step process and is ascribed to the conjugation of PEI to PLGA via aminolysis. The PLGA/PEI nanoparticles were able to bind DNA and the formed complexes had a substantially lower cytotoxicity and a higher transfection activity than PEI polyplexes. In conclusion, given their small size, stability, low cytotoxicity and good transfection activity, PLGA/PEI-DNA complexes are attractive gene delivery systems.     

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.     

Uptake characteristics of NGR-coupled stealth PEI/pDNA nanoparticles loaded with PLGA-PEG-PLGA tri-block copolymer for targeted delivery to human monocyte-derived dendritic cells

We have investigated the in vitro uptake, toxicity, phenotypic consequences and transfection efficiency of a stealth NGR/PEG/PDBA-coupled-SHA-PEI/pDNA targeting polyplex loaded with PLGA-PEG-PLGA tri-block copolymer in human monocyte-derived dendritic cells (DCs). Modification with PEG effectively shielded and reduced non-specific phagocytosis by immature DCs to approximately 20%. Coupling the NGR cell-specific peptide to the PEGylated polyplex (NGR/PEG/PDBA-SHA-PEI/pDNA) however resulted in specific and enhanced phagocytosis in DCs without any observable toxicity at the optimum concentration of 0.25% of the copolymer. DNase treatment had no effect on DNA integrity in the encapsulated polyplex. Confocal microscopy confirmed intracellular localization of the targeting NGR/PEG/PDBA-SHA-PEI/pDNA microparticles, resulting in more enhanced uptake of the radiolabeled plasmid DNA and approximately 5- and 10-fold increase over the control tri-block Pluronic F68 copolymer and the non-targeting polyplex, respectively. More importantly, phagocytosis of the targeting microparticles neither altered the functionality of immature DCs nor the phenotypic expression of DC-specific cell surface molecules, CD80, CD86, CD40 and CD54 (ICAM-1), suggesting that uptake of the targeting microparticles by themselves did not induce DC maturation. Taken together, these results suggest that PLGA-PEG-PLGA encapsulation of this stealth targeting polyplex has no negative effects on key properties of immature DCs and should pave the way for targeting DCs for vaccination purposes.     

Improvement of in vivo transfer of plasmid DNA in muscle: comparison of electroporation versus ultrasound

Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods—electroporation and ultrasound—that facilitate DNA uptake into cells. Mice (C57Bl/6) were injected intramuscular using plasmid DNA encoding an intracellular protein (p53) followed by electroporation or ultrasound. Then 48 hr after the injections the mice were sacrificed. The parameter for transfection efficiency was the area of muscle expressing the transgene. The p53 expression plasmid showed a 36-fold increase (p = 0.015) in transfection efficiency with electroporation compared to ultrasound. Compared with ultrasound, electroporation significantly improves transfection efficiency of naked plasmid DNA transfer into skeletal muscle.