PEGylation potentiates hepatoma cell targeted liposome-mediated in vitro gene delivery via the asialoglycoprotein receptor.

Hepatocellular carcinoma is a burgeoning health issue in sub-Saharan Africa and East Asia where it is most prevalent. The search for gene medicine treatment modalities for this condition represents a novel departure from current treatment options and is gaining momentum. Here we report on nonPEGylated and on sterically stabilized PEGylated cationic liposomes decorated with D-galacto moieties linked to 24.1 Å spacers for asialoglycoprotein receptor (ASGP-R)-targeted vehiculation of pCMV-luc plasmid DNA. Cargo DNA is fully liposome associated at N/P ratio=3:1 and is partially protected from the effects of serum nucleases. Moreover, at this ratio, lipoplex dimensions (89-97 nm) are compatible with the requirements for extravasation in vivo. Ethidium displacement assays show that the reporter DNA is in a less condensed state when bound to PEGylated liposomes than with nonPEGylated liposomes. PEGylated lipoplexes were well tolerated by both HEK293 (ASGP-R-negative) and HepG2 (ASGP-R-positive) cell lines and delivered DNA to the human hepatoma cell line HepG2 by ASGP-R mediation at levels three-fold greater than nonPEGylated lipoplexes. PEGylated ASGP-R-targeted liposomes reported in this study possess the required characteristics for hepatotropic gene delivery and may be considered for further application in vivo.

Effect of Poly(ethylene glycol) Spacer on Peptide-Decorated Hepatocellular Carcinoma-Targeted Lipoplexes In Vitro.

Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths and is particularly prevalent in regions where Hepatitis B is highly endemic. Classical treatment options are largely limited to surgical measures and transcatheter chemoembolization. However biopanning of phage display libraries has identified very selective HCC-targeting peptides, which may be tethered to cationic liposomes for non-viral delivery of therapeutic DNA to affected cells thus providing a foundation for the development of new gene therapy treatment approaches. In this investigation, a cysteinylated HCC-tropic peptide has been linked directly to cationic liposomes, containing the cytofectin 3ß-[N-(N',N'-dimethlaminopropylamino)-carbamoyl]-cholestero (Chol-T) and dioleolphosphatidyl ethanolamine, via a novel membrane-embedded maleimido cholesteryl derivative 3-[N-(hydrazino-y-maleimidobutyryl)-carbamoyl] cholesterol (Chol-Mal), or with an intervening poly(ethylene glycol) spacer element. The effect that inclusion of the hydrophilic polymer has on targeted lipoplex-mediated gene delivery in the human HCC cell line HepG2 has been investigated. Results show that both systems bind DNA and transfect this cell line with equal efficiency, while transgene expression levels in human embryo kidney cells HEK293 were low and comparable to those achieved in competition assays in HepG2 cells and by lipoplexes decorated with scrambled peptides. Although inclusion of poly(ethylene glycol) in formulations should be considered for in vivo studies, we show here that it offers no advantage in this application in vitro.

Stealth lipoplex decorated with triazole-tethered galactosyl moieties: a strong hepatotropic gene vector.

Mono-antennary galacto derivatives of cholesterol are being actively developed to direct lipoplexes to the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Here we report on a novel ASGP-R ligand cholest-5-en-3-yl [1-(ß-D-galactopyranosyl)-1H-1,2,3-triazol-4-yl]methylcarbamate (4), assembled by a copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry), and compare it with cholest-5-en-3-yl-ß-D-galactopyranoside (2) and cholest-5-en-3-yl [1-(ß-D-galactopyranosyl-1'-oxy)phen-4-yl]carbamate (3), in liposome formulations with or without 5 mol% distearoylphosphatidylethanolamine poly(ethylene glycol)2000, intended for DNA delivery to ASGP-R-positive hepatocyte-derived HepG2 cells and the ASGP-R-negative embryo kidney cell line HEK293. Transfection levels attained with lipoplex 4 were 100 and 300% greater than those for lipoplexes 2 and 3 respectively in HepG2 cells, while competition assays reduced transfection levels by up to 98%. Transfection activities achieved in HEK293 cells were up to three orders of magnitude lower. Therefore, 4 is representative of a new class of promising hepatotropic ligands for gene delivery.

HER2/neu Oncogene Silencing in a Breast Cancer Cell Model Using Cationic Lipid-Based Delivery Systems.

The overexpression of the human epidermal growth factor 2 (HER2/neu) oncogene is predictive of adverse breast cancer prognosis. Silencing the HER2/neu overexpression using siRNA may be an effective treatment strategy. Major requirements for siRNA-based therapy are safe, stable, and efficient delivery systems to channel siRNA into target cells. This study assessed the efficacy of cationic lipid-based systems for the delivery of siRNA. Cationic liposomes were formulated with equimolar ratios of the respective cholesteryl cytofectins, 3ß-N-(N', N'-dimethylaminopropyl)-carbamoyl cholesterol (Chol-T) or N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), with the neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE), with and without a polyethylene glycol stabilizer. All cationic liposomes efficiently bound, compacted, and protected the therapeutic siRNA against nuclease degradation. Liposomes and siRNA lipoplexes were spherical, <200 nm in size, with moderate particle size distributions (PDI < 0.4). The siRNA lipoplexes exhibited minimal dose-dependent cytotoxicity and effective HER2/neu siRNA transfection in the HER2/neu overexpressing SKBR-3 cells. The non-PEGylated Chol-T-siRNA lipoplexes induced the highest HER2/neu silencing at the mRNA (10000-fold decrease) and protein levels (>111.6-fold decrease), surpassing that of commercially available Lipofectamine 3000 (4.1-fold reduction in mRNA expression). These cationic liposomes are suitable carriers of HER2/neu siRNA for gene silencing in breast cancer.

Biogenic Synthesis of Silver-Core Selenium-Shell Nanoparticles Using Ocimum tenuiflorum L.: Response Surface Methodology-Based Optimization and Biological Activity.

Bimetallic nanoparticles (BNPs) have shown better biological potential compared to their monometallic counterparts owing to the synergistic effect produced by these alloys. In this study, selenium-capped silver nanoparticles (Ag@Se NPs) were synthesized using an Ocimum tenuiflorum extract. These BNPs were characterized using UV-visible, Fourier transform infrared spectroscopy, nanoparticle tracking analysis, electron microscopy and energy dispersive x-ray analysis. Response surface methodology was used to understand how extract volume and temperature influenced the zeta potential, hydrodynamic size and NP concentration. The phytoconstituents were identified using gas chromatography-mass spectrometry (GC-MS) and molecular docking studies were performed on B-DNA to determine possible genotoxicity. Antioxidant activities, in vitro cytotoxicity (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay), and genotoxicity (Allium cepa root cells) of these BNPs, were also evaluated. A surface plasmon resonance band around 420 nm confirmed BNP formation with significant quantities of silver and selenium. The Ag@Se NPs displayed good stability, dispersity, antioxidant activity, and compatibility at low concentrations but showed significant cytotoxicity and genotoxicity at high concentrations. Molecular docking analysis showed weak interactions between the plant constituents and B-DNA, suggesting no genotoxicity. These results provide an insight into the conditions required for optimal production of eco-friendly Ag@Se NPs with interesting biological properties.

Anti- c-myc cholesterol based lipoplexes as onco-nanotherapeutic agents in vitro.

Background: Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNA-based, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated siRNA delivery systems. Methods: This study has focused on cationic liposomes prepared with equimolar quantities of the cytofectin, N,N-dimethylaminopropylamido-succinylcholesteryl-formylhydrazide (MS09), and cholesterol (Chol) for the development of a simple, but effective anti- c-myc onco-nanotherapeutic agent. Liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) in place of Chol as the co-lipid were included for comparative purposes. Results: Liposomes successfully bound siRNA forming lipoplexes of less than 150 nm in size, which assumed bilamellar aggregrates. The liposome formulations were well tolerated in the human breast adenocarcinoma (MCF-7) and colon carcinoma (HT-29) cells, which overexpress c-myc. Lipoplexes directed against the c-myc transcript mediated a dramatic reduction in c-myc mRNA and protein levels. Moreover, oncogene knockdown and anti-cancer effects were superior to that of Lipofectamine™ 3000. Conclusion: This anti- c-myc MS09:Chol lipoplex exemplifies a simple anticancer agent with enhanced c-myc gene silencing potential in vitro.

Anti-c-myc RNAi-Based Onconanotherapeutics.

Overexpression of the c-myc proto-oncogene features prominently in most human cancers. Early studies established that inhibiting the expression of oncogenic c-myc, produced potent anti-cancer effects. This gave rise to the notion that an appropriate c-myc silencing agent might provide a broadly applicable and more effective form of cancer treatment than is currently available. The endogenous mechanism of RNA interference (RNAi), through which small RNA molecules induce gene silencing by binding to complementary mRNA transcripts, represents an attractive avenue for c-myc inhibition. However, the development of a clinically viable, anti-c-myc RNAi-based platform is largely dependent upon the design of an appropriate carrier of the effector nucleic acids. To date, organic and inorganic nanoparticles were assessed both in vitro and in vivo, as carriers of small interfering RNA (siRNA), DICER-substrate siRNA (DsiRNA), and short hairpin RNA (shRNA) expression plasmids, directed against the c-myc oncogene. We review here the various anti-c-myc RNAi-based nanosystems that have come to the fore, especially between 2005 and 2020.

HER-2/neu and MYC gene silencing in breast cancer: therapeutic potential and advancement in nonviral nanocarrier systems.

Globally, breast cancer is the second leading cause of cancer-related mortality among women, with approximately 1.4 million new cases diagnosed annually. Associated genetic perturbations are emerging in the face of intense scientific enquiry, facilitating its classification, prognostication and treatment. RNAi, utilizing siRNA, is a powerful treatment strategy to silence disease-causing genes. However, therapeutic siRNA instability and poor cellular uptake have limited its clinical application, necessitating the use of nanocarriers. In this review, we highlight the RNAi mechanism, HER-2/neu and MYC as breast cancer gene targets, and nonviral nanocarriers as potentially safe and efficient delivery systems.

Cholesteryl cytofectins with primary amino head groups transfect transformed human epithelial cell lines efficiently.

Two novel cholesterol-based cytofectins containing primary amino head groups, glycylcholesteryl formylhydrazide (MS10) and beta-alanylcholesterylformylhydrazide (MS11), have been prepared and incorporated into unilamellar cationic liposomes in equimolar amounts with dioleoylphosphatidylethanolamine (DOPE) as colipid. Stable lipoplexes were formed with pGL3 DNA which afforded protection to the DNA from serum nuclease digestion. Packing of the DNA was shown by ethidium displacement to be more effective in MS11 lipoplexes. High transfection levels in three human transformed epithelial cell lines HeLa (cervical), SNO (esophageal), HepG2 (hepatocyte-derived), and the murine fibroblast line NIH-3T3 were achieved by both lipoplexes at liposome: DNA ratios of 6:1 and 7:1 ((w)/(w)) corresponding to +/- charge ratios of 1.6:1 and 1.9:1. MS11 lipoplexes, in particular, afforded high transfection activities in the presence of fetal bovine serum.

A cationic cytofectin with long spacer mediates favourable transfection in transformed human epithelial cells.

The synthesis and transfection potential of a novel cationic cholesterol cytofectin with a dimethylamino head group and a long 12 atom, 15A spacer incorporating relatively polar amido and dicarbonyl hydrazine linkages are reported. Thus N,N-dimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09) in equimolar admixture with dioleoylphosphatidylethanolamine (DOPE) forms stable unilamellar liposomes (80-150 nm) which cluster into very effective transfecting, serum nuclease-resistant, lipoplexes with DNA (180-200 nm) at a liposome+/DNA- molar charge ratio of 2.8:1 (12:1, w/w). Gel retardation and ethidium displacement assays confirmed that DNA was fully liposome-associated and maximally compacted at this ratio. Transfection levels in three human transformed epithelial cell lines, as established by luciferase transgene activity, was found to be optimal at this charge ratio and in the following order: cervical carcinoma (HeLa)>oesophageal carcinoma (SNO)>hepatoblastoma (HepG2). Activity in the murine fibroblast line NIH-3T3 was comparable to that in HepG2 cells. MS09 lipoplexes achieved approximately three-times and two-times greater activity than Lipofectin complexes in HeLa and SNO cells, respectively, whilst comparable levels were recorded in HepG2 and NIH-3T3 cells. MS09 lipoplexes were well tolerated by HepG2, HeLa and SNO cells with cell numbers found to be 80, 85 and 75% of untreated cultures, respectively, at the optimal transfection concentration. These lipoplexes also exhibited high activity in the presence of 10% foetal bovine serum (FBS) in HeLa (17% inhibition) and HepG2 (33% inhibition) cells.

Lipoplexes with biotinylated transferrin accessories: novel, targeted, serum-tolerant gene carriers.

Novel transfecting assemblies comprising biotinylated cationic liposomes, DNA and tribiotinylated transferrin-streptavidin (streptavidin(bio3-transferrin)) accessories have been prepared, characterized and evaluated for toxicity and DNA delivery capability in human cervical carcinoma cells (HeLa). Two new lipophilic cholesteryl-based biotin derivatives, biotinylcholesterylformylhydrazide (MSB1) and aminohexanoylbiotinylcholesterylformylhydrazide (MSB2) provided docking points for streptavidin(bio3-transferrin) on cationic liposomes which were formulated with N,N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09) and dioleoylphosphatidylethanolamine (DOPE) in a 2:48:50 molar ratio. Ethidium dye displacement assays and gel retardation studies suggest that in ternary complexes, the DNA is electrostatically bound to the cationic liposomes while transferrins remain liposome-bound through streptavidin-biotin interactions. Assemblies fully protected plasmid DNA from serum nuclease digestion over a range of liposome:pGL3 DNA ratios (3-8:1, w/w) and exhibited low growth inhibition of HeLa cells (circa 5%) at the optimal transfection composition for streptavidin(bio3-transferrin):liposome:pGL3 DNA of 10:6:1 (w/w/w). Transfection levels, which were twice those of untargeted lipoplexes containing MSB1 or MSB2, were not significantly diminished in the presence of 10% foetal bovine serum. Excess transferrin (200 microg per well) reduced transfection levels to those of untargeted complexes, supporting the notion that at least 50% of ternary complexes gained entry into the cervical carcinoma cells by receptor mediation. Conversely, transfection levels with untargeted lipoplexes were only slightly reduced in the presence of transferrin at the same concentration.

Targeted Delivery of Peptide-Tagged DNA Lipoplexes to Hepatocellular Carcinoma Cells.

The application of homing peptides to direct DNA and RNA lipoplexes to target cells is a rapidly evolving area of study, which may find application in corrective gene therapy for the treatment of neoplasms and other disorders of a genetic origin. Here, a step-wise account of the assembly and characterization of hepatocellular carcinoma cell-specific DNA lipoplexes and their cytotoxicity assessment in and delivery to the human hepatocellular carcinoma cell line HepG2 is given.

Spacer Length: A Determining Factor in the Design of Galactosyl Ligands for Hepatoma Cell-Specific Liposomal Gene Delivery.

BACKGROUND: Use of nucleic acids to treat acquired or inherited hepatic diseases has considerable potential. Although recombinant viruses are popular vectors, interest in cheaper, often less immunogenic, non-viral modalities, is increasing. Thus hepatotropic, galactosylated lipoplexes directed to the hepatic asialoglycoprotein receptor (ASGP-R) are promising candidates. OBJECTIVE: Here we examine the effect that galactosyl ligand spacer length has on the transfection activity of ASGP-Rtargeted lipoplexes in the human hepatoma cell line HepG2. METHODS: Galactosyl ligands with spacer lengths in the range 2.4-24.1 Å were prepared and formulated into lipoplexes that were characterized by cryo-TEM, band shift, dye displacement and nuclease digestion assays. Cytotoxicity and transfection profiles were determined in liver-derived HepG2 cells and the renal ASGP-R-negative HEK293 line. RESULTS: Lipoplexes, which formed at endpoint +/- charge ratios in the range 1:1-3:1, accorded cargo DNA good protection from serum nuclease digestion and were well-tolerated by both cell lines. Transfection activities in the hepatoma cell line decreased markedly in the presence of a competing ASGP-R cognate ligand and also as the ligand spacer length increased, while activities in HEK293 cells were significantly lower (P <0.05-0.001). CONCLUSION: Targeted lipoplexes enter HepG2 cells by receptor mediation and the uptake of transfecting complexes and those displaying more rigid short and medium length spacers is more efficient. This observation will inform the design of hepatotropic lipoplexes that are suitable for applications in vivo.

Liposomal Formulation of Monovalent Cholesteryl Cytofectins with Acyclic Head Groups and Gene Delivery: A Systematic Review.

A large number of liposome-based non-viral gene and siRNA delivery systems include monovalent cholesteryl cytofectins with acyclic head groups in their formulations. Progress in their clinical development has, however, been hampered by relatively low transfection efficiencies. Structural differences between members of this class of cationic amphiphiles are located primarily in their linker, spacer and head group regions. This review examines the structural diversity encountered in each of these domains and seeks to identify those features linked to favourable transfection activity. Thus the ether linker, with its greater chemical and metabolic stability, is associated with higher transfection activity than ester, amide or carbamoyl tethers. While a medium length 6 atom spacer in the ether series is preferred over shorter spacers for enhanced activity, short (2 atom) to long (11 atom) spacers are effective in the more common carbamoyl series. For largely historic reasons, the dimethylamino head group has remained a popular cationic centre, but several studies have shown that the N-hydroxyethyl secondary amine functionality may be more effective in cytofectins. This observation has been attributed, in part, to the increased hydrophilicity of the head group and facilitated release of the nucleic acid cargo from liposomes in endosomal compartments. However, the hypothesis that the incorporation of each of these favourable chemical features into a single novel cytofectin may lead to superior transfection activity remains to be fully tested.

Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes.

Unilamellar activated cationic liposomes containing 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol, dioleoyl phosphatidylethanolamine (DOPE) and the N-hydroxysuccinimide ester of cholesteryl hemisuccinate (4:5:1, molar ratio) have been prepared and their DNA-binding capacity has been assessed in a gel retardation assay. Ternary complexes composed of activated cationic liposomes, carbodiimide-cationized asialoorosomucoid (Me+AOM) and pRSVL plasmid DNA were assembled for receptor-mediated DNA delivery into cells expressing the asialoglycoprotein receptor (ASGP-R). Binding of complexes in which Me+AOM was replaced by fluoresceinated Me+AOM (FMe+AOM) to the human hepatocellular cell line HepG2 at 4 degrees C was severely reduced by co-incubation with asialoorosomucoid (AOM). Moreover, assemblies containing liposomes, pRSVL DNA and Me+AOM (8:1:4, w/w/w) promoted high levels of luciferase activity in this cell line (1.3 x 10(7) relative light units/mg soluble cell protein). Assays conducted in the presence of a hundred-fold excess of the ligand AOM afforded considerably lower levels of transfection (2.5 x 10(5) relative light units/mg soluble cell protein). In contrast, the highest level of luciferase activity achieved with liposome, pRSVL DNA, AOM complexes was only a quarter of the best levels obtained with liposome, pRSVL DNA, Me+AOM assemblies. These findings strongly support the notion that complexes gain entry into hepatocyte-derived cells by ASGP-R mediation and that they are potentially useful gene carriers to liver hepatocytes.

A novel cationic cholesterol derivative, its formulation into liposomes, and the efficient transfection of the transformed human cell lines HepG2 and HeLa.

A novel cationic cholesterol derivative, 3beta[N-(N',N',N'-trimethylaminopropane)-carbamoyl] cholesterol iodide (Chol-Q), has been formulated with equimolar amounts of dioleoyl phosphatidylethanolamine (DOPE) into stable unilamellar liposomes up to 100 nm in size for DNA delivery into mammalian cells. When compared with similarly constituted liposomes containing the tertiary analogue 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) in a band shift assay, liposomes displayed similar DNA binding affinities and appeared to afford complete protection to plasmid DNA against serum nuclease catalysed degradation at liposome:DNA ratios (w/w) of 2.5:1, 5:1, and 10:1 in incubation mixtures containing 5% fetal bovine serum at 37 C for 90 min. Chol-Q liposomes were, however, markedly less toxic to cells in culture over a wide range of concentrations with cells numbering 76% of untreated controls at 37.5 microg/mL complete medium in the human hepatocellular carcinoma line HepG2 and 75% at 30 microg/mL in cervical carcinoma HeLa cells. At these levels of Chol-T liposomes, cell numbers were 37% and 15%, respectively. Gene transfer experiments with pSV2CAT and pRSVCAT plasmids in HepG2 cells showed maximum efficiency at a Chol-Q liposome:DNA ratio of 5:1 (w/w) and at a Chol-T liposome:DNA ratio of 10:1. In HeLa cells, both liposome preparations performed best at a ratio of 2.5:1. Differences in transfection efficiencies over the liposome range of 5-20 microg/ mL were rather less pronounced with Chol-Q lipoplexes suggesting a greater versatility of this system.

New cationized LDL-DNA complexes: their targeted delivery to fibroblasts in culture.

Low density lipoproteins (LDL) have been cationized using the water-soluble carbodiimide, N-ethyl-N'-(3-trimethylpropylammonium) carbodiimide iodide at a reagent: lipoprotein mole ratio of 10 000:1. This was shown to increase the innate DNA-binding capacity of LDL 10-fold. [125I]-labeled carbodiimide-modified LDL ([125I])-labeled ECDI-LDL) appeared to recognize the LDL receptor on normal human skin fibroblasts, although some nonspecific binding also was detected. To demonstrate the large ionic component in the lipoprotein-DNA interactions, epsilon -NH2 amino groups on the apolipoprotein B-100 (apoB-100) component of LDL were acetylated with acetic anhydride. A nitrocellulose filter-binding assay revealed that acetylated LDL bound approximately 25% of the [3H]-labeled pBR322 plasmid DNA bound by native LDL under the same conditions. ECDI-LDL-[3H]-labeled plasmid DNA complexes were considerably more stable to NaCl challenge than complexes formed between [3H]-labeled plasmid DNA and native LDL. Thus, the half dissociation of ECDI-LDL containing complexes was achieved at 0.28 M NaCl, whereas for LDL-plasmid DNA complexes this was reached at 0.18 M NaCl. Displacement studies with native LDL studies showed that ECDI-LDL-[3H]-labeled plasmid DNA complexes retained the ability to recognize the LDL receptor on normal skin fibroblasts. Finally, ECDI-LDL complexes with pSV2CAT expression plasmid were shown to transfect CV-1 fibroblasts, a cell line known to specifically recognize apoB-liposome conjugates.

A note on poly-L-lysine-mediated gene transfer in HeLa cells.

Poly-L-lysines of chain lengths varying from 70 to 300 residues are shown to bring about luciferase pRSVL DNA uptake and expression in HeLa cells. Transfection was approximately 50% that of the cationic liposome DOTAB. Expression was higher in the presence of chloroquine. Of interest was the fact that luciferase activity depended on the polysine/DNA charge ratio (+/-). Maximum activity occurred at a charge ratio (+/-) of 3, while at a charge ratio of 1 (conjugate electrically neutral) activity was much lower. At the higher charge ratios (+/-) of 4 and 5, luciferase activity decreased. The results obtained are discussed.

Low concentrations of chlorpromazine and related phenothiazines stimulate gene transfer in HeLa cells via receptor-mediated endocytosis.

Chlorpromazine and related phenothiazine antipsychotic compounds at the low concentration of 10(-5) M stimulated luciferase pRSVL DNA uptake and expression in HeLa cells. On the other hand, chloroquine at a 10(-5) M was without effect at this low concentration. However, at the higher normally used concentration of 10(-4) M (100 microM), chloroquine strongly stimulated luciferase expression and activity. Unfortunately, at 10(-4) M, the phenothiazines were toxic to the cells and could not be tested at this concentration. Further experimental work was carried out to elucidate the mechanism of action of phenothiazines and chloroquine on DNA uptake and expression. Interaction of [3H] pBR 322 DNA with chlorpromazine, perphenazine, and chloroquine was studied using these compounds as their free bases dissolved in chloroform, followed by their impregnation onto Whatman No. 1 filter paper discs. Both phenothiazines on filter paper discs bound [3H] pBR 322 DNA to a far greater extent than chloroquine. The method of assay (free base) suggests that the major contribution to binding is through intercalation. A further possible assay for studying the interaction of phenothiazines and chloroquine made use of the ethidium bromide/calf thymus DNA intercalation method. Intercalated calf thymus (CT) DNA complexes with ethidium bromide (EB) were examined for possible dissociation into free DNA and EB on the addition of either chloroquine. SO4 or chlorpromazine.HCl (soluble salts). Partial dissociation was observed with both compounds. Further experiments on the stability of pBR 322 DNA-polylysine complexes were also carried out using an alternative method of assay. Chloroquine (10(-2)-10(-4) M) and chlorpromazine (10(-4) M) did not bring about a dissociation of [3H] pBR 322 DNA-polylysine(200) complexes when reactions were studied by nitrocellulose filter assays to measure released double-stranded DNA. The results indicate that chlorpromazine and related phenothiazines stimulate luciferase DNA uptake expression at 10(-5) M. Chloroquine at this concentration had practically no effect on expression of luciferase activity. Further studies of chloroquine and chlorpromazine on their interaction with plasmid DNA as well as DNA-polylysine complexes are reported.

PEGylated galactosylated cationic liposomes for hepatocytic gene delivery.

The efficiency of liposome-mediated gene delivery is greatly enhanced by appropriate decoration of vehicles with cell-specific targeting ligands. However, liposome-DNA complexes may still be opsonized in serum thus ablating any advantage gained. A stealth aspect may therefore be conferred on complexes by poly(ethylene glycol) (PEG) grafting. Here, we examined the effect that degree of PEGylation has on physicochemical properties, cytotoxicity and transfection activity of lipoplexes containing the cytofectin 3ß-[N-(N', N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE), the asialoglycoprotein receptor (ASGP-R) targeted cholesteryl-ß-d-galactopyranoside (Chol-ß-Gal) ligand, and plasmid DNA in ASGP-R-negative (HEK293) and receptor-positive (HepG2) human cell lines. Lipoplexes were characterized by hydrodynamic sizing, electron microscopy, band shift, ethidium bromide (EtBr) intercalation and nuclease digestion assays. Cryo-TEM and DLS studies revealed that PEGylation generated smaller and more densely aggregated lipoplexes than their non-PEGylated counterparts. MTT and AB reduction studies showed that the lipoplexes elicited a dose-dependent cytotoxic effect in both cell lines, with cell viability remaining above 65% (MTT) and 50% (AB). The Ricinus communis (RCA120) agglutination test confirmed that the galactosyl residues on the targeted lipoplexes were well exposed and accessible. Transgene activity increased by 63% and 77% when HepG2 was confronted by the 2 and 5mole% PEGylated lipoplexes, respectively, compared to their non-PEGylated counterparts. Furthermore, Chol-T Chol-ß-Gal 5% PEG complexes were able to achieve a 164% increase in transfection level in the ASGP-R positive cell line (HepG2) compared to HEK293 (ASGP-R negative). Results strongly indicate that PEGylation potentiates the activity of ASGP-R-targeted lipoplexes, highlighting their gene delivery potential.