The microRNA expression profiles in extracellular vesicles from HeLa cancer cells in response to cationic lipid- or polyethylenimine-mediated gene delivery.

Intercellular communication is known to be involved in various stages of tumour development and metastasis through the secretion of extracellular vesicles (EVs) containing messengers such as microRNAs (miRNAs). Therefore, this study explored miRNA profiles in cancer cell-derived EVs after non-viral gene delivery in order to better understand the molecular information of intercellular communication in cancer cells after gene delivery. Two commonly used non-viral vectors (Lipofectamine 2000 and jet polyethylenimine) were used for the delivery of gene fluorescent protein plasmid in HeLa cancer cells. EVs were extracted and the contents of their RNA were subjected to the next-generation sequencing. In order to illustrate the common characteristics of non-viral vectors in the cancer cells, two overlapped up-regulated miRNAs (hsa-miR-143-3p and hsa-miR-193b-3p) were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction in the secreted EVs in response to both lipoplexes and polyplexes. The prediction of target genes and molecular pathways involved in these two miRNAs were determined, and the protein expressions related to the pathways of cell death and stress in HeLa cells were identified. Hsa-miR-143-3p and hsa-miR-193b-3p were found to be up-regulated by the use of different non-viral vectors and can thus serve as potential targets of non-viral cancer gene therapy.

The vector-related influences of autophagic microRNA delivery by Lipofectamine 2000 and polyethylenimine 25K on mouse embryonic fibroblast cells.

Despite the greater potential for clinical applications of autophagic microRNA (miRNA) delivery, the vector-related effects of such delivery on cells have not been fully explored. In this study, autophagic mmu-miR-494-3p (miR-494) in mouse embryonic fibroblast (MEF) cells was selected as a cargo miRNA, and two commonly used non-viral carriers (Lipofectamine 2000 (Lipo) and polyethylenimine 25K (PEI)), were used as delivery vectors to mechanistically elucidate its vector-related effects. The cellular uptake, nuclear localization, and quantitative miR-494 levels of the complexes of miR-494 with Lipo (miR-494 lipoplexes) were lower than those of the complexes of miR-494 with PEI (miR-494 polyplexes) in MEF cells. The indicator of autophagic activity (LC3 (microtubule-associated protein 1 light chain 3)-II/LC3-I ratio) in cells treated with miR-494 lipoplexes was higher than that in cells treated with miR-494 polyplexes. Lipo alone and PEI alone induced slight increases in the quantitative levels of miR-494 in cells, but Lipo resulted in higher gene and protein expressions of target Igf1, higher LC3-II/LC3-I ratios, and higher autophagosome formation than PEI. We also demonstrated that the delivery of miR-494 by Lipo was more involved in apoptotic caspase-3 pathways than such delivery by PEI. By applying knock-out atg5 gene in MEF cells, we found that autophagy played a protective role in cell survival and also affected cellular uptake, the quantitative level of miR-494, and target gene Igf1 regulation of delivery systems. Taken together, these results indicate that there are different degrees of responses in MEF cells for autophagic miR-494 delivery through the use of Lipo or PEI vectors that also induce autophagy in cells. Therefore, Lipo and PEI vectors cannot be treated as inert molecules, and their effects must be known and evaluated when they are used in autophagic miRNA delivery systems. Most importantly, understanding these vector-related effects on cells will be helpful in achieving optimal delivery of autophagic miRNAs.

Exploring MicroRNA Expression Profiles Related to the mTOR Signaling Pathway in Mouse Embryonic Fibroblast Cells Treated with Polyethylenimine.

Although the toxicology of poly(ethylenimine) (PEI) in gene expression levels has been previously investigated, little is known about the effects of PEI on the expression of microRNAs (miRNAs) that regulate gene expression at the post-transcriptional level. In this study, we explored miRNA expression profiles related to cell death mechanisms in mouse embryonic fibroblast (MEF) cells treated with PEI by applying microarray analysis. Based on the analysis of the mTOR signaling pathway, three upregulated miRNAs (mmu-miR-3090-5p, mmu-miR-346-3p, and mmu-miR-494-3p) were verified in MEF cells treated with PEI at 24 h using real-time quantitative reverse transcriptase-polymerase chain reaction. We further demonstrated that these three upregulated miRNAs resulted in the decrease of gene and protein expressions of the target gene growth factor Igf1 in MEF cells treated with PEI or transfected with three upregulated miRNA mimics. However, these three upregulated miRNAs are not all cell-specific. Finally, we demonstrated that the mTOR signaling pathway is inhibited by autophagy induction and that the cell viability decreases in MEF cells treated with PEI or transfected with these three miRNA mimics. Collectively, our data suggested that PEI may affect the regulation of miRNAs in target cells.

Autophagy-related gene expression analysis of wild-type and atg5 gene knockout mouse embryonic fibroblast cells treated with polyethylenimine.

The molecular mechanisms of autophagy in polyethylenimine (PEI)-treated cells are not well understood because of the use of nonspecific autophagy inhibitors. Here, we applied autophagy-related gene expression analysis to pinpoint the molecular mechanisms of autophagy in PEI-treated wild-type and atg5 gene knockout (atg5(-/-)) mouse embryonic fibroblast (MEF) cells. It was demonstrated that the majority of induced genes are downregulated in wild-type and atg5(-/-) MEF cells, indicating that autophagy exhibits a trend toward downregulation after treatment with PEI. In addition to regulating genes encoding autophagy machinery components, genes related to coregulation of autophagy and apoptosis were induced in wild-type and atg5(-/-) cells treated with PEI. These data indicate that autophagy and apoptosis are closely related in the PEI-induced mechanism of cell death. In the absence of autophagy, the regulation of apoptosis was enhanced in atg5(-/-) MEF cells treated with PEI, indicating that inhibition of autophagy may lead to higher levels of apoptosis. Our study may provide deeper insight into the molecular mechanisms of cell death caused by PEI.

Cellular uptake on N- and C-termini conjugated FITC of Rath cell penetrating peptides and its consequences for gene-expression profiling in U-937 human macrophages and HeLa cervical cancer cells.

Rath peptide has been introduced as a delivery vector that transports various membrane-impermeable cargoes in a non-covalent fashion. In this paper, we present a study on Rath peptide conjugated with fluorescein-5-isothiocynate (FITC) differing in its N- and C-termini. We conducted cellular toxicity and uptake experiments in U-937 and HeLa cells to analyze biocompatibility profiles and translocation efficiencies of Rath peptide with FITC serving as both a cargo and a fluorescent marker. We found that the conjugation of FITC on Rath peptide at N-terminus (FITC-Rath) led to more rapid cellular uptake in U-937 cells and significantly higher cellular uptake in HeLa cells than that which occurred at C-terminus. From DNA microarray analysis, FITC-Rath induced gene expression changes in both U-937 and HeLa cells. Five overlapping regulated genes were identified, and this overlap indicated that FITC-Rath displayed some degree of generality regarding gene responses in the two cell lines used. A real-time quantitative reverse transcriptase-polymerase chain reaction was used to confirm which regulated genes were affected by FITC-Rath. Cell communication, signal transduction, cell surface receptor signaling pathway, signal transducer activity and cellular process, were identified as overlapping biological themes. These data provide useful information on molecular mechanisms for using Rath-based delivery systems.

Protective role of autophagy in branched polyethylenimine (25K)- and poly(L-lysine) (30-70K)-induced cell death.

Polyethylenimine (PEI) and poly(L-lysine) (PLL), which are cationic polymers used for gene therapy, are known to be cytotoxic, but their molecular mechanisms of cell death are not fully understood. In this study, we provide evidence that PEI and PLL induced autophagy in HeLa cervical cancer cells. In cells overexpressed with green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) fusion protein, PEI and PLL induced fluorescent puncta formations that represent LC3 recruitment to autophagosomes. In Western blot analysis, conversions of the LC3-I to LC3-II were significant, and p62 degradation was observed in cells treated with PEI and PLL. At higher doses, the ability of endosomal escape by PEI facilitates the conversion of LC3-I to LC3-II without the use of lysosomal protease inhibitors. From the analysis of annexin V-flourescein isothiocyanate (FITC) and propidium iodide (PI) staining by flow cytometry, both apoptosis and necrosis occurred in PEI- and PLL-treated cells. Significant activated caspase-3 expression was detected in PLL- and PEI-treated cells. By applying Z-VAD apoptotic inhibition, apoptosis and autophagy may occur independently or autophagy may be in the upstream of apoptosis on PEI- and PLL-treated cells. The degree of cell death was higher in incubated HeLa cells treated with PEI or PLL plus autophagy inhibitors (3-methyladenine (3-MA) and wortmannin). Treatment with these autophagy inhibitors, however, did not inhibit LC3-II formation specifically. In addition, PEI and PLL induced higher degree of cell death in atg5(-/-) mouse embryonic fibroblast (MEF) cells than in wild-type cells. Autophagy was also induced in PEI- and PLL-treated MEFs, as evidenced by the formation of LC3-II in wild-type-but not in atg5(-/-) MEFs. These results indicate that PEI and PLL can trigger both death and survival pathways simultaneously, and autophagy played a role in cell survival in PEI- and PLL-treated cells. Our study therefore provides deeper insight into the molecular mechanisms of cell death caused by cationic polymers.

The global gene-expression profiles of U-937 human macrophages treated with Tat peptide and Tat-FITC conjugate.

There is increased interest in using cell penetrating peptides such as HIV Tat-derived peptide (Tat) to deliver biologically active cargo to cells. However, little is known about the precise molecular mechanism in cells after the uptake of Tat and after it conjugates with the cargo. Using high-density microarray analysis, we evaluated the alteration of whole genomic responses in U-937 macrophages that had been treated with Tat and Tat-fluorescein-5-isothiocynate (FITC), which served as model cargo, and then incubated for 24 h. Sixteen genes were upregulated and 28 downregulated in Tat-treated cells. After FITC had conjugated to Tat, 13 genes were upregulated and 23 downregulated. Ten upregulated and 13 downregulated genes were overlapped by both Tat and Tat-FITC. The molecular functions of regulated genes affected by Tat and Tat-FITC are described. A real-time quantitative reverse transcriptase-polymerase chain reaction was used to confirm three regulated genes (IFNAR2, CASP8, and CRLF1) affected by both Tat and Tat-FITC. We demonstrated that regulating the three genes was time-dependent and that CASP8 is also kinetically regulated in HeLa cells. Understanding the influences and consequences of Tat (Tat-cargo)-induced gene changes should help us design and develop efficient and safe delivery systems into target cells.

Evaluating the gene-expression profiles of HeLa cancer cells treated with activated and nonactivated poly(amidoamine) dendrimers, and their DNA complexes.

Using dendrimers in cancer therapy as nonviral vectors for gene delivery seems promising. The biological performance of a dendrimer-based gene delivery system depends heavily on its molecular architecture. The transfection activity of dendrimers is significantly improved by processes activated in the heat degradation treatment of solvolysis. However, very little is known about the molecular mechanisms that dendrimers produce in cancer cells. We studied the changes in global gene-expression profiles in human cervical cancer HeLa cells exposed to nonactivated and activated poly(amidoamine) (PAMAM) dendrimers, alone or in complexes with plasmid DNA (dendriplexes). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to confirm four regulated genes (PHF5A, ARNTL2, CHD4, and P2RX7) affected by activated dendrimers and dendriplexes. Activated and nonactivated dendrimers and dendriplexes alike induced multiple gene expression changes, some of which overlapped with their dendriplexes. Dendrimer activation improved transfection efficiency and induced additional gene expression changes in HeLa cells. Dendrimers and dendriplexes principally affect genes with the molecular functions of nucleic acid binding and transcription activity, metal-ion binding, enzyme activity, receptor activity, and protein binding. Our findings provide a deeper insight into the changes in gene expression patterns caused by the molecular structure of PAMAM dendrimers for gene-based cancer therapy.

Interactions between octaarginine and U-937 human macrophages: global gene expression profiling, superoxide anion content, and cytokine production.

Cell penetrating peptides such as octaarginine (R8) have been widely used as intracellular delivery vectors to import biologically active membrane-impermeable molecules. However, before using these peptides clinically, human immune responses to them must be fully understood. Because macrophages are important for immune responses, we evaluated the interactions between R8 and a human U-937 cell line. Cytotoxicity, binding, internalization, genome-wide profiling of gene expression, intracellular superoxide anion content, and cytokine release were assessed after U-937 cells had been incubated with different amounts of R8. Cytotoxicity was limited for up to 40 microM of R8 and 24 h of incubation. Kinetic analysis of the binding and uptake of cells treated with fluorescein-5-isothiocynate-R8 showed time- and concentration-dependent increases. Microarray analysis identified 4386 genes time-dependently regulated when U-937 macrophages were exposed to 10 microM of R8 for 0.5 h and 4 h; the majority of these genes were upregulated for each time point. Thirty-five upregulated genes responded to the stimuli with immune functions, and, using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, five genes - FOS, OSM, C1R, TNF, IL1R1 - were confirmed. R8 induced superoxide anion production after 0.5 h, but not after longer incubations. Incubating U-937 cells with R8 for up to 24 h did not release the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6. In summary, exposing U-937 macrophages to R8 did not induce proinflammatory cytokine release; however, it generated superoxide anion and affected gene expression.

Interactions between U-937 human macrophages and tyloxapol.

Tyloxapol is reported to prevent macrophages from reacting to endotoxin. However, the intracellular responses that tyloxapol induces in macrophages are still not fully explored. Hence, the objective of this study was to evaluate the intracellular events in macrophages treated with tyloxapol and assess the antioxidant properties of tyloxapol in endotoxin-activated macrophages. Using flow cytometry, we examined intracellular responses in macrophages: reactive oxygen species (ROS) content, mitochondria membrane potential, and cell cycle profiles. We also assessed the antioxidant properties of tyloxapol in endotoxin-activated macrophages. Kinetic hydrogen peroxide production tended to decline with increasing doses. Tyloxapol produced a progressive increase followed by a decline in superoxide anion production in macrophages with increasing doses. Tyloxapol also caused unstable fluctuations in mitochondrial membrane potential. Apoptosis had developed at higher doses after 4h of incubation time. After 2h of tyloxapol-pretreatment, tyloxapol acted as an antioxidant only at lower doses. Most tyloxapol-pretreated cells at lower doses fully recovered from the changes in superoxide anion and hydrogen peroxide production. Our findings contribute to a better understanding of the molecular action of tyloxapol in macrophages and how it protects macrophages against endotoxin.

Flow cytometric characterization of interactions between U-937 human macrophages and positively charged catanionic vesicles.

Catanionic vesicles are considered a potential alternative to liposomes for drug delivery systems because of their greater stability and lower cost. Before using catanionic vesicles in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. Using flow cytometry, we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondrial membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with positively charged catanionic vesicles. Kinetic hydrogen peroxide production initially increased at lower concentrations (4-10nM) but declined at higher concentrations (40 nM and 80 nM) over the entire incubation period. Superoxide content generation, however, increased over the entire concentration range and incubation period. Catanionic vesicles decreased mitochondrial membrane potential for every concentration after 4h of incubation but caused a significant fluctuation in mitochondrial membrane potential at 6h. After 6h of incubation, catanionic vesicles produced more changes in cell size and complexity than after 4h. The increase in the subG1 population of cells treated with catanionic vesicles at higher doses indicated that apoptosis progressed. Positively charged catanionic vesicles induced different activated patterns of ROS generation and changes in mitochondrial membrane potential than did cationic liposomes. The nature of the interactions between macrophages and catanionic vesicles is of great importance for the design of safer and more effective delivery systems for macrophages. Our findings contribute to a better understanding of the molecular action of catanionic vesicles in the cellular system.

Interactions between U-937 human macrophages and poly(propyleneimine) dendrimers.

Interest in using poly(propyleneimine) (PPI) dendrimers for biomedical applications is increasing. Before using PPI dendrimers in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. However, few investigators have studied in detail the intracellular responses that cationic dendrimers induce in macrophages. Here we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondria membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with poly(propyleneimine) dendrimers generation 2 (DAB 2.0) and 3 (DAB 3.0). Our study focused on the concentration ranges within which cell viability was greater than 90% after PPI dendrimers had been incubated for 16 h. For spontaneous ROS generation, DAB 2.0 did not consistently generate hydrogen peroxide production with increasing dosages over the entire culture period while it was capable of generating superoxide content except during the 12 h of incubation. In contrast, DAB 3.0 did not induce any hydrogen peroxide and superoxide production except for an abrupt increase of superoxide content at 60 microg/mL after 6 h of incubation. Our results showed that ROS responses in macrophages were strongly influenced by the nature of the dendrimer surface. Except at 3 h, DAB 2.0 increased mitochondrial membrane potential for every dose and culture period. In contrast, DAB 3.0 caused a significant fluctuation in mitochondrial membrane potential only at 6 h, compared with other incubation times. Exposing macrophages to PPI dendrimers caused dramatic and significant changes in macrophage cell size and complexity, and DAB 3.0 caused greater changes than DAB 2.0 did. For incubation times longer than 1 h, propidium iodide staining showed that cells treated with DAB 2.0 and 3.0 had a higher subG1 phase (indicative of apoptosis) than did untreated cells. PPI dendrimers induced different activated patterns in ROS generation and changes of mitochondrial membrane potential than did other carriers such as cationic liposomes and polyalkylcyanoacrylate. The nature of interactions between macrophages and PPI dendrimers is crucial for the design of safer and more effective delivery systems for macrophages. Our findings provide a novel insight into the cytotoxic effects at the molecular level that dendrimers cause in macrophages.

Remnant cationic dendrimers block RNA migration in electrophoresis after monophasic lysis.

Cationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) have attractive characteristics for the delivery of nucleic acid and various biomedical applications. Most studies have focused on cationic dendrimer-based intracellular delivery, and very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that PAMAM and PPI dendrimers strongly altered the mobility of RNA in the gels. In addition, the extent of dendrimer-induced alteration in RNA mobility was directly dendrimer-generation-dependent: the alteration was greater with higher-generation dendrimers. We also found that DNA/dendrimer complexes at higher dendrimer to DNA ratios interacted with RNA after isolation while gene expression was maintained. The interactions between RNA and remnant dendrimers after isolation were caused by electrostatic bindings, and we recovered total RNA using high ionic strength solvents (2M NaCl solution) to disrupt the electrostatic forces binding dendrimers to RNA. Because RNA isolation is routinely used for biological applications, such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.

Cytotoxic properties of tyloxapol.

PURPOSE: Tyloxapol, a viscous polymer of the alkyl aryl polyether alcohol type, is classified as a nonionic surfactant and is widely used in biomedical applications. Although tyloxapol has been reported to be cytotoxic in various cell lines, there is no published information about its possible mechanisms of cell death. Hence, the objective of this study was to determine whether tyloxapol causes apoptosis or necrosis. These data could be helpful for a better understanding of the action of tyloxapol in cellular systems. METHODS: RAW 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the activity of dehydrogenases in those cells, an indicator of cell viability, was assessed. The cell morphology changes induced by tyloxapol treatment were detected using propidium iodide nuclear staining. The hallmarks of apoptotic cells were characterized using DNA fragmentation assays, DNA fluorescence staining, and then flow analysis. RESULTS: Tyloxapol treatment produced dose- and time-dependent cytotoxicity. Tyloxapol treatment damaged RAW 264.7 cells more than it damaged NIH/3T3 cells. All the cells exposed to tyloxapol showed some morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from tyloxapol-treated cells. Flow cytometric analysis revealed an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after tyloxapol treatment. In addition, we showed that pretreating cells with zVAD-fmk, a general caspase inhibitor, did not prevent tyloxapol-induced apoptosis. The cytotoxicity of tyloxapol can be reduced by adding a nontoxic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine to attenuate the interaction of tyloxapol with the cell membrane. CONCLUSIONS: Our results indicate that tyloxapol induces apoptosis in RAW 264.7 and NIH/3T3 cells. These data provide a novel insight into the cytotoxic action of tyloxapol at the molecular level.

Induction of apoptosis in macrophages by air oxidation of dioleoylphosphatidylglycerol.

Dioleoylphosphatidylglycerol (DOPG) containing unsaturated sites is the target of oxidation during preparation, storage, or in vivo use of anionic liposomes. We investigated the biological effect of air oxidation of DOPG on RAW 264.7 murine macrophage-like cells. Oxidation was induced by exposing DOPG to air for 24-72 h. The extent of air oxidation was confirmed using Matrix-Assisted Laser Desorption and Ionization with Time-of-Flight (MALDI-TOF) mass spectrometry. The product of the air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG at m/z 814.77. The treatment of DOPG with air oxidation produced dose-dependent cytotoxicity in macrophages. RAW 264.7 cells exposed to oxidized DOPG exhibited morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with oxidized DOPG. Flow cytometric analysis demonstrated an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after treatment with oxidized DOPG. In addition, we showed that pretreating RAW 264.7 cells with zVAD-fmk, a general caspase inhibitor, did not prevent apoptosis induced by oxidized DOPG, suggesting that apoptosis in macrophage cells follows a caspase-independent pathway. These results point to a need for precaution in formulating DOPG liposomes for drug delivery and therapeutic purposes.

Mechanism of cell death induced by cationic dendrimers in RAW 264.7 murine macrophage-like cells.

Cationic dendrimers possess attractive nano-sized architectures that make them suitable as targeted drug/gene delivery systems. However, very little is known about their mechanisms of cell death in cellular systems. In the current study, the apoptotic and necrotic effects of starburst polyamidoamine(PAMAM) and polypropylenimine (DAB) dendrimers in cultured RAW 264.7 murine macrophage-like cells were investigated. Cationic dendrimer treatment produced a typically dose-dependent cytotoxic effect on macrophage cells. RAW 264.7 cells exposed to cationic dendrimers exhibited morphological features of apoptosis. Apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with cationic dendrimers. Analysis from flow cytometry demonstrated an increase in hypodiploid DNA population (sub-G1) and a simultaneous decrease in diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells to cationic dendrimers. Also, cells treated with DAB dendrimer induced a higher percentage of sub-G1 population than those treated with PAMAM dendrimer at the same dose. In addition, it was shown that pre-treatment of RAW 264.7 cells with the general caspase inhibitor zVAD-fmk prevented some degree of apoptosis induced by cationic dendrimers, suggesting that apoptosis in macrophage cells involves a caspase dependent pathway. Macrophage cells were also found to be sensitive to induction of apoptosis by dendrimers, whereas NIH/3T3 cells (mouse fibroblast) and BNL CL.2 (mouse liver) cells did not undergo apoptosis. These results could be helpful for optimizing the biocompatibility of dendrimers used for targeted drug/gene delivery.

Cytotoxicity characterization of catanionic vesicles in RAW 264.7 murine macrophage-like cells.

In comparison with cationic liposomes, catanionic vesicles possess more attractive properties such as stability and lower cost, and these characteristics may make them suitable as a non-viral vehicle and for other biomedical applications such as vaccine adjuvants. However, very little is known about their possible cytotoxic mechanisms in cellular system. Also, this information is vital for the future development of safe biomedical systems. In the current study, the cytotoxic effect of catanionic vesicles, consisting of anionic surfactant (SDS), cationic surfactant (HTMAB), and cholesterol, in cultured RAW 264.7 murine macrophage-like cells was determined. The treatment of catanionic vesicles produced a dose-dependent effect on macrophage cells. RAW 264.7 cells exposed to catanionic vesicles exhibited morphological features of apoptosis such as chromatin condensation. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated by catanionic vesicles. Analysis from flow cytometry demonstrated an increase of hypodiploid DNA population (sub-G1) and a simultaneous decrease of diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells with catanionic vesicles. In addition, it was shown that pretreatment of RAW 264.7 cells with the general caspase inhibitor (zVAD-fmk) did not prevent apoptosis induced by catanionic vesicles, suggesting that apoptosis in macrophage cells followed a caspase-independent pathway induced by catanionic vesicles. These data provide novel insight into the effect of catanionic vesicles on the mechanisms of cell death induced by catanionic vesicles.

Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA.

This study investigates the feasibility of using the process of spray-freeze drying (SFD) to produce DNA dry powders for non-viral gene delivery. The effect of protective agents was assessed on the stability of DNA dry powders after SFD. The process of SFD had adverse effects on the tertiary structure of DNA with the protective agents of sucrose, trehalose and mannitol. With the protection of these sugars, a band corresponding to the linear form of DNA was observed during gel electrophoresis between the supercoiled form (SC) and the open circular (OC) form. On the contrary, excess cationic condensing polyethyleneimine (PEI), in conjunction with the above sugars, had the ability to provide protection for DNA from degradation after SFD. This is indicated by the reservation in SC and OC forms of DNA during agarose gel electrophoresis. The electrostatic forces between PEI polymer and DNA are critical for providing protection against various stresses generated by the process of SFD. Furthermore, on rehydration, the particle size and zeta potential of PEI/DNA complexes at weight ratios 3:1 of SFD dry powders were well maintained. Also, no transfection activity loss of PEI/DNA complexes at weight ratios 3:1 on NIH/3T3 cells was observed for reconstituted powders as compared with untreated control solutions. These results give a better understanding of preparing stable DNA dry powders by the process of SFD.

The effect of protective agents on the stability of plasmid DNA by the process of spray-drying.

The effect of several protective agents was assessed on the stability of spray-dried plasmid DNA. The spray-drying process had adverse effects on the tertiary structure of plasmid DNA with the protective agents of sucrose, glycine and agarose. With the protection of these noncondensing agents, a band corresponding to the linear form of plasmid DNA was observed in the gel electrophoresis between the supercoiled circular (SC) form and the open circular (OC) form. On the contrary, spray-dried plasmid DNA maintained some degree of structural integrity under the protection of condensing agents. For the protection by neutral condensing polymers, such as polyethylene glycol 1000 and 4000, no linear form between the SC form and the OC form of plasmid DNA was revealed in the gel electrophoresis. Also, excess cationic condensing polymer, polyethyleneimine, had the ability to provide the plasmid DNA with protection from degradation as indicated by the preservation in SC and OC forms of plasmid DNA on the agarose gel electrophoresis. Moreover, DNA topology was unchanged after six-month storage at 4 degrees C by the protection of these neutral and cationic condensing agents. Accordingly, DNA condensation induced by condensing agents may provide a way to minimize damage to plasmid DNA by the process of spray drying.

Evaluation of the stability of polymer-based plasmid DNA delivery systems after ultrasound exposure.

Under ultrasound exposure, the stability of plasmid DNA protected by polymer-based gene delivery system is an important factor for achieving optimal transfection into cells. We have evaluated the effectiveness of various polymer-based plasmid DNA delivery systems, which are interactive polymers and cationic polymers, to avoid shear degradation induced by ultrasound exposure. Alternatively, it is shown that sonication of plasmid DNA for exposure time as low as 10s resulted in total DNA fragmentation and the loss of transfection potency in NIH/3T3 cells. Among these polymer-based plasmid DNA delivery systems, only cationic polymers had the ability to provide the protection of plasmid DNA from ultrasonic degradation as indicated by the reservation in supercoiled circular (SC) and open circular (OC) forms of plasmid DNA on the agarose gel electrophoresis. The DNA stability protected by cationic polymers decreased after ultrasound exposure in 1M sodium chloride solution. Also, higher molecular weight of cationic polymers and sufficient cationic polymer/DNA weight ratios are essential to prevent DNA from degradation under ultrasound exposure in aqueous or salt solution. These results suggest that the protective mechanism by cationic polymers is due to the attractive bonding between cationic polymer and negative plasmid DNA. Whereas, DNA condensation alone provoked by the addition of polyethylene glycols was not sufficient to resist the DNA fragmentation induced by ultrasound exposure.