Encapsulation in cationic liposomes enhances antitumour efficacy and reduces the toxicity of etoposide, a topo-isomerase II inhibitor

Etoposide exerts its antineoplastic effect by forming a ternary complex with topo-isomerase II and DNA, leading to DNA breaks and cell death. However, it causes myelosuppression and its lipophilicity poses a major limitation during administration. Liposomes have been reported to increase the efficacy and reduce the toxicity of antineoplastic agents. Recent evidence suggests that cationic liposomes bind efficiently to tumours. The present study was thus designed to encapsulate etoposide in cationic liposomes and to evaluate its antitumour efficacy and systemic toxicity in comparison with a conventional parenteral formulation. Etoposide encapsulated in liposomes was synthesised by thin film hydration followed by an extrusion method. Fibrosarcoma was induced in mice by subcutaneous administration of 20-methylcholanthrene. Chemotherapy was started when the tumour reached 200 mm(3) in volume. Liposomal etoposide (10 mg/m(2)/day for 5 days) significantly delayed tumour growth as compared to non-liposomal etoposide. The median time of death was calculated to be 19.5, 26.25 and 56 days in vehicle-treated controls, non-liposomal-etoposide- and liposomal-etoposide-treated groups, respectively. A transient reduction in body weight was seen in both the liposomal- and non-liposomal-etoposide-treated groups. The maximum tolerated dose was however significantly higher in the group treated with liposomal etoposide, which also exhibited a lesser degree of myelosuppression than the animals treated with non-liposomal etoposide. The present findings suggest that cationic liposomes could be considered as potential for delivery of etoposide to tumours.     

Swainsonine,a glycosylation inhibitor,enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity

Swainsonine (SW) inhibits the formation of N-linked complex oligosaccharides and has previously been shown to inhibit experimental metastasis in nude mice models. The present studies with human effector cells have shown that SW enhanced both lymphokine activated killer cell (LAK) and natural killer (NK) cytotoxicity in standard ⁵¹Cr-release assays. SW also increased the susceptibility of human K562 and Colo 320 target cells to NK and LAK cytotoxicity. The peak response of both LAK effectors and targets to SW occurred at 1–2 μg/ml SW. A novel finding was that SW enhanced the interleukin 2 (IL-2) β chain receptor subunit expression on both LAK and NK cells to a greater extent than its enhancement of the IL-2Rα (CD25 or TAC) receptor expression on LAK effectors. In addition, increases in both these receptors occurred at the doses of SW which augmented LAK cytotoxicity. We conclude that the anti-metastatic effects of SW have an immunological component which is maximal at 1–2 μg/ml SW. This suggests that dosage may be an important consideration to obtain optimal potential of SW in any future human cancer therapy.     

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.     

Contribution of hydrophobicity,DNA and proteins to the cytotoxicity of cationic PAMAM dendrimers

In most articles, cytotoxicity of cationic polyamidoamine (PAMAM) dendrimers toward red blood cells has been exclusively explained by their surface charge. We have focused on dendrimer hydrophobicity as a second possible factor that determines this cytotoxicity. Using PAMAM-NH₂ dendrimers from the 3rd to the 6th generations and PAMAM-NH₂-C₁₂(25%) dendrimer of the 4th generation bearing 25% acyl groups, these induced hemolysis that increased with their surface charge and hydrophobicity. Interaction of PAMAM-NH₂-C₁₂(25%) G4 dendrimer with blood proteins (γ-globulin, α-thrombin, human serum albumin) and calf thymus DNA (ctDNA) significantly reduced their cytotoxicity toward red blood cells.     

Asialoganglioside enhances the efficiency of gene transfection mediated by cationic liposomes with a cationic cholesterol derivative

We investigated the transfection efficiency mediated by asialoganglioside-containing cationic liposomes. Previously we reported that monosialoganglioside GM(1) (GM(1a)) enhanced transfection efficiency. In this study, we investigated the effects of sialic acid in gangliosides on transfection efficiency. Two mammalian culture cell lines HeLa and HepG2 were transfected with luciferase plasmids (pGL3) using cationic liposomes which contain monosialoganglioside GM(1) (GM(1a)) or its asialic counterpart, asialoganglioside GM(1) (GA(1)). Both GM(1a) and GA(1) enhanced the efficiency of transfection mediated by cationic liposomes, and GA(1) exhibited higher efficiency than GM(1a) in both cell lines. Transfection efficiency of ganglioside-containing liposomes was also assessed by the effects of antisense oligonucleotides (AS-ODN) for bcl-2 gene, which suppresses apoptotic cell death. Western blotting analysis revealed that the expression of Bcl-2 was decreased by AS-ODN, and the reduction of protein expression in cells treated with GA(1)-containing liposomes was more remarkable than that with GM(1a)-containing liposomes. Furthermore, the induction rate of apoptosis was higher in cells treated with AS-ODN with GA(1)-containing liposomes. Together with the results obtained by luciferase assay mentioned above, the removal of sialic acid from ganglioside causes the enhancement of efficiency of transfection mediated by cationic liposomes.     

Pre-exposure of cells to cationic lipids enhances transgene delivery and expression in a tissue culture cell line

Several factors influence non-viral transfection in tissue culture models including nature of the cationic lipid, plasmid construction, and DNA lipid complex, among others. The cell line itself is another confounding variable. Each subcellular population may respond independently to the transgene or specific delivery vector with regards to toxicity or transgene expression. In this study, the SKnSH (human neuroblastoma) and COS-1 (African green kidney) cells were exposed to three different treatments A, B, and C. Treatment A refers to cells obtained from American Type Culture Collection (ATCC) and cultivated as recommended, treatment B to cells that were grown in presence of cationic lipids for two weeks, and treatment C to cells that were grown in presence of cationic lipids for two weeks followed by normal media for two weeks to determine if lipid mediated effects were reversible. Treatment B resulted in a three-fold increase in transgene expression of a reporter gene as compared to the other treatments. This increase in transgene expression appeared not to be related to alterations in toxicity. Interestingly, the fluid phase endocytic uptake of fluorescently labeled oligonucleotides was increased in treatment B. However, there was no significant difference in the cellular-associated signal when fluorescently labeled plasmid-DNA was evaluated. In COS-1 cells, no difference in transfection was observed with treatment B illustrating that cell lines respond independently. In conclusion, pre-exposure of SKnSH cells to cationic liposomes (treatment B) resulted in higher transgene production.     

Comparison of cationic and amphipathic cell penetrating peptides for siRNA delivery and efficacy

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.     

A fluorescence study of the structure and accessibility of plasmid DNA condensed with cationic gene delivery vehicles

The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4',6-diamidino-2-phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation-induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation-induced structural changes in DNA, as well as polycation-mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed.     

Targeted delivery by pH-responsive mPEG-S-PBLG micelles significantly enhances the anti-tumor efficacy of doxorubicin with reduced cardiotoxicity

Stimuli-responsive nanotherapeutics hold great promise in precision oncology. In this study, a facile strategy was used to develop a new class of pH-responsive micelles, which contain methoxy polyethylene glycol (mPEG) and poly(carbobenzoxy-l-glutamic acid, BLG) as amphiphilic copolymer, and β-thiopropionate as acid-labile linkage. The mPEG-S-PBLG copolymer was synthesized through one-step ring-opening polymerization (ROP) and thiol-ene click reaction, and was able to efficiently encapsulate doxorubicin (DOX) to form micelles. The physicochemical characteristics, cellular uptake, tumor targeting, and anti-tumor efficacy of DOX-loaded micelles were investigated. DOX-loaded micelles were stable under physiological conditions and disintegrated under acidic conditions. DOX-loaded micelles can be internalized into cancer cells and release drugs in response to low pH in endosomes/lysosomes, resulting in cell death. Furthermore, the micellar formulation significantly prolonged the blood circulation, reduced the cardiac distribution, and selectively delivered more drugs to tumor tissue. Finally, compared with free DOX, DOX-loaded micelles significantly improved the anti-tumor efficacy and reduced systemic and cardiac toxicity in two different tumor xenograft models. These results suggest that mPEG-S-PBLG micelles have translational potential in the precise delivery of anti-cancer drugs.     

A comparison of the effectiveness of cationic polymers poly-L-lysine (PLL) and polyethylenimine (PEI) for non-viral delivery of plasmid DNA to bone marrow stromal cells (BMSC).

Bone marrow stromal cells (BMSC) represent an important cell phenotype for pursuit of successful gene therapy. Non-viral methods to enable expression of exogenous genes in BMSC will accelerate clinical application of gene therapy, without the concerns associated with the viral means of gene transfer. Towards this end, this study investigated the potential of cationic polymers poly-L-lysine (PLL) and branched polyethylenimine (PEI) as gene carriers for modification of BMSC. Both polymers rapidly (approximately 30 min) condensed a 4.2 kb Enhanced Green Fluorescent Protein (pEGFP-N2) plasmid into 100-200 nm particles. PLL and PEI were both readily internalized with BMSC with >80% of BMSC exhibiting polymer uptake by flow cytometric analysis. The relative uptake of PEI, however, was significantly higher as compared to the PLL. The majority of the BMSC (>60%) exhibited nuclear presence of the polymers as analyzed by fluorescent microscopy. Although both polymers were able to deliver the pEGFP-N2 into the cells under microscopic evaluation, only a small fraction of the cells (<10%) displayed nuclear localization of the plasmid. Consistent with better uptake, PEI gave a higher delivery of pEGFP-N2 into the BMSC, which resulted in a more sustained expression of the model gene EGFP in short-term (7-day) culture. We conclude that both PLL and PEI readily displayed cellular uptake, but PEI was more effective in delivering plasmid DNA intracellularly, which was likely the underlying basis for a more sustained gene expression.     

PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy

Background: Gene targeting by RNA interference (RNAi) is mediated through small interfering RNA (siRNA), which, as plasmid DNA molecules, can be delivered into cells by polyethylenimines (PEI). Grafting with poly(ethylene glycol) has been introduced previously to improve PEI biocompatibility; however, data on the effects of PEGylation have been somewhat contradictory and various PEI(-PEG) need to be evaluated independently for DNA transfection and siRNA gene targeting efficacies.

Aim: We directly compare plasmid DNA transfection and siRNA-mediated gene targeting efficacies, employing a larger set of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG; PEI(-PEG)) with different molecular weights and degrees of PEG substitution.

Method: We performed tissue culture-based bioassays on DNA transfection and siRNA-mediated targeting efficacies as well as on toxicity and cellular nucleic acid uptake, and, using sensitive assays based on radioactive labelling, physicochemically characterize the complexes regarding the degree of nucleic acid complexation and complex stabilities under various conditions.

Results: In contrast to the DNA transfection efficacy, siRNA-mediated gene targeting is much less dependent on the PEGylation of PEI or on the N/P (= PEI nitrogen/nucleic acid phosphate) ratio. A more detailed analysis reveals that, in order to define optimal N/P ratios for DNA transfection, complex toxicities and nucleic acid uptake are the most critical parameters. In contrast, at optimal N/P ratios, complex stabilities and complexation efficacies determine PEI(-PEG)/DNA transfection efficacies and the major differences between various PEI(-PEG) are observed. All these parameters are less critical for PEI(-PEG)/siRNA gene targeting efficacy. Thus, our data lead to the distinction between three PEI(-PEG) groups, which relies on the differences in transfection rather than gene targeting efficacies, and which is correlated with the molecular weights and degrees of PEG substitution.

Conclusion: In contrast to PEI(-PEG)/DNA complexes, a broader panel of PEI-PEG are capable of siRNA-mediated gene targeting. Thus, PEG grafting of PEI requires a separate evaluation of siRNA and DNA complexes, which expands the portfolio of available PEI(-PEG) for the preparation of non-toxic, biocompatible siRNA delivery reagents for the induction of RNAi.     

PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy

BACKGROUND: Gene targeting by RNA interference (RNAi) is mediated through small interfering RNA (siRNA), which, as plasmid DNA molecules, can be delivered into cells by polyethylenimines (PEI). Grafting with poly(ethylene glycol) has been introduced previously to improve PEI biocompatibility; however, data on the effects of PEGylation have been somewhat contradictory and various PEI(-PEG) need to be evaluated independently for DNA transfection and siRNA gene targeting efficacies. AIM: We directly compare plasmid DNA transfection and siRNA-mediated gene targeting efficacies, employing a larger set of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG; PEI(-PEG)) with different molecular weights and degrees of PEG substitution. METHOD: We performed tissue culture-based bioassays on DNA transfection and siRNA-mediated targeting efficacies as well as on toxicity and cellular nucleic acid uptake, and, using sensitive assays based on radioactive labelling, physicochemically characterize the complexes regarding the degree of nucleic acid complexation and complex stabilities under various conditions. RESULTS: In contrast to the DNA transfection efficacy, siRNA-mediated gene targeting is much less dependent on the PEGylation of PEI or on the N/P ( = PEI nitrogen/nucleic acid phosphate) ratio. A more detailed analysis reveals that, in order to define optimal N/P ratios for DNA transfection, complex toxicities and nucleic acid uptake are the most critical parameters. In contrast, at optimal N/P ratios, complex stabilities and complexation efficacies determine PEI(-PEG)/DNA transfection efficacies and the major differences between various PEI(-PEG) are observed. All these parameters are less critical for PEI(-PEG)/siRNA gene targeting efficacy. Thus, our data lead to the distinction between three PEI(-PEG) groups, which relies on the differences in transfection rather than gene targeting efficacies, and which is correlated with the molecular weights and degrees of PEG substitution. CONCLUSION: In contrast to PEI(-PEG)/DNA complexes, a broader panel of PEI-PEG are capable of siRNA-mediated gene targeting. Thus, PEG grafting of PEI requires a separate evaluation of siRNA and DNA complexes, which expands the portfolio of available PEI(-PEG) for the preparation of non-toxic, biocompatible siRNA delivery reagents for the induction of RNAi.     

Preparation, characterization, cytotoxicity and transfection efficiency of poly(DL-lactide-co-glycolide) and poly(DL-lactic acid) cationic nanoparticles for controlled delivery of plasmid DNA

The objective of this study was to investigate the effect of formulation parameters (i.e. polymer molecular weight and homogenization speed) on various physicochemical and biological properties of cationic nanoparticles. Cationic nanoparticles were prepared using different molecular weights of poly(DL-lactide-co-glycolide) (PLGA) and poly(DL-lactic acid) (PLA) by double emulsion solvent evaporation at two different homogenization speeds, and were characterized in terms of size, surface charge, morphology, loading efficiency, plasmid release, plasmid integrity, cytotoxicity, and transfection efficiency. Cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used to provide positive charge on the surface of nanoparticles. Reporter plasmid gWIZ Beta-gal was loaded on the surface of nanoparticles by incubation. Use of higher homogenization speed and lower molecular weight polymer led to a decrease in mean particle size, increase in zeta potential, increase in plasmid loading efficiency, and a decrease in burst release. The nanoparticles displayed good morphology as evident from scanning electron micrographs. In vitro cytotoxicity study by MTT assay showed a low toxicity. Structural integrity of the pDNA released from nanoparticles was maintained. Transfecting human embryonic kidney (HEK293) cells with nanoparticles prepared from low molecular weight PLGA and PLA resulted in an increased expression of beta-galactosidase as compared to those prepared from high molecular weight polymer. Our results demonstrate that the PLGA and PLA cationic nanoparticles can be used to achieve prolonged release of pDNA, and the plasmid release rate and transfection efficiency are dependent on the formulation variables.     

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, the authors developed nanovesicles containing bioactive cationic lysine-based amphiphiles and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. Different cytotoxic responses were found among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalised by HeLa cells and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behaviour after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute in reducing the uncertainty surrounding their potential health hazards.     

Improving the direct penetration into tissues underneath the skin with iontophoresis delivery of a ketoprofen cationic prodrug

Current topical nonsteroidal anti-inflammatory drugs (NSAIDs) showed marginal efficacy in treatment of musculoskeletal disorders due to their fast clearance by skin blood flow and thus little direct penetration into the underlying muscle and joint tissues. Using ketoprofen (Kt) as a model NSAID and converting it to a cationic ester prodrug ketoprofen choline chloride (KCC), this study was to investigate the iontophoresis delivery of the prodrug KCC for improving the drug retention in the skin and the direct penetration into underlying tissues. From in vitro flux study, anodal iontophoresis of KCC showed 5 times higher flux than cathodal iontophoresis of Kt across human epidermis skin, and also 1.5 times higher across full thickness rat skin. From in situ dual agar gel model rat study, anodal iontophoresis of KCC showed 35 times more drug penetrating across the live skin into underlying agar gel and 22 times more drug retained in the skin than those from cathodal iontophoresis of Kt. Co-iontophoresis of a vasoconstrictor phenylephrine with KCC did not show better result than the iontophoresis of KCC alone. Overall, iontophoresis delivery of the cationic prodrug KCC showed great potential for direct penetration into local tissues underneath the skin.     

Distance of hydroxyl functionality from the quaternized center influence DNA binding and in vitro gene delivery efficacies of cationic lipids with hydroxyalkyl headgroups

In vitro gene delivery efficacies of cationic amphiphiles 1-7 (Scheme 1) were measured by both the reporter gene expression assays in CHO, COS-1, HepG2, and MCF7 cells and by the whole cell histochemical X-gal staining of representative Chinese hamster ovary cells. Our results demonstrated that in vitro gene delivery efficiencies of cationic lipids with hydroxyalkyl headgroups are adversely affected by increased covalent distances between the hydroxyl functionality and the cationic centers. Findings in the DNase I protection experiments and transmission electron microscopic study support the notion that such compromised gene delivery efficacies may originate from poor lipid-DNA binding interactions and significantly increased lipoplex nanosizes.     

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity

We used well-characterized and positively charged nanoparticles (NP⁺) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP⁺ physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP⁺ surface charge was significantly reduced, proportionally to NP⁺/serum and NP⁺/BSA ratios, while NP⁺ size was not modified. Microscopy studies showed high endocytosis of NP⁺ in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP⁺. No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP⁺ genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP⁺ might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.     

Spacer-arm modulated gene delivery efficacy of novel cationic glycolipids: design, synthesis, and in vitro transfection biology

Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids 1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described. The results of the present structure-activity investigation convincingly demonstrate that the in vitro gene delivery efficacies of galactosylated cationic glycolipids are strikingly dependent on the absence of a spacer-arm between the open-form galactose and the positively charged nitrogen atom in their headgroup region. While the cationic glycolipids 1-3 with no headgroup spacer unit between the positively charged nitrogen and galactose showed high in vitro gene transfer efficacies in all four cells (lipids 1 and 2 with myristyl and palmityl tails, respectively, being the most efficacious), lipids 4-6 with five-carbon spacer units between the quaternized nitrogen and galactose heads were essentially transfection incompetent. The transfection inhibiting role of the five-carbon spacer unit in the headgroup region of the present novel class of cationic lipids was demonstrated by both beta-galactosidase reporter gene expression and histochemical X-gal staining assays. Results of MTT assay-based cell viability measurements in representative MCF7 cells show that cell viabilities of lipoplexes (lipid:DNA complexes) prepared from all the lipids 1-6 are remarkably high. Thus, possibilities of differential cellular cytotoxicities playing any key role behind the strikingly contrasting transfection properties of lipids 1-3 with no spacer and lipids 4-6 with a spacer unit in the headgroup regions was ruled out. Electrophoresis gel patterns in DNase I sensitivity assays are consistent with more free DNA (accessible to DNase I) being present in lipoplexes of lipids 4-6 than in lipoplexes of lipids 1-3. Thus, the results of our DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 may play an important role in abolishing their in vitro gene transfer efficacies.     

Inhibition of protein synthesis reduces the cytotoxicity of 4'-(9-acridinylamino)methanesulfon-m-anisidide without affecting DNA breakage and DNA topoisomerase II in a murine mastocytoma cell line

Stimulation of cleavable complex formation by 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) and related anticancer drugs is an important initial event in drug action which correlates with cytotoxicity. However, it was recently suggested that factors in addition to cleavable complex formation are needed to express lethality. Therefore we investigated the effects of inhibitors of DNA replication and RNA and protein synthesis on mAMSA-induced cell killing in the K21 subline of the P815 murine mastocytoma cell line. This showed that RNA and protein synthesis, but not DNA replication, was necessary for maximal mAMSA cytotoxicity. Moreover, inhibition of RNA synthesis with cordycepin or protein synthesis with cycloheximide protected cells from the cytotoxic action of mAMSA without reducing DNA breakage or cleavable complex formation and there was no decrease in DNA topoisomerase II activity in nuclear extracts from cells treated with cordycepin or cycloheximide. We conclude that cleavable complex formation is independent of RNA and/or protein synthesis and we propose that the subsequent conversion into a lethal event requires an additional labile protein factor.