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 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.

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.

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.

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.

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.

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.

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.

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.

Novel serum-tolerant lipoplexes target the folate receptor efficiently.

Gene transfer using non-viral vectors is a promising approach for the safe delivery of nucleic acid therapeutics. In this study, we investigate a lipid-based system for targeted gene delivery to malignant cells overexpressing the folate receptor (FR). Cationic liposomes were formulated with and without the targeting ligand folate conjugated to distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG2000), the novel cytofectin 3ß[N(N(1),N(1)-dimethlaminopropylsuccinamidoethane)-carbamoyl]cholesterol (SGO4), which contains a 13atom, 15Å spacer element, and the helper lipid, dioleoylphosphatidylethanolamine (DOPE). Physicochemical parameters of the liposomes and lipoplexes were obtained by zeta sizing, zeta potential measurement and cryo-TEM. DNA-binding and protection capabilities of liposomes were confirmed by gel retardation assays, EtBr intercalation and nuclease protection assays. The complexes were assessed in an in vitro system for their effect on cell viability using the MTT assay, and gene transfection activity using the luciferase assay in three cell lines; HEK293 (FR-negative), HeLa (FR(+)-positive), KB (FR(++)-positive). Low cytotoxicities were observed in all cell lines, while transgene activity promoted by folate-tagged lipoplexes in FR-positive lines was tenfold greater than that by untargeted constructs and cell entry by folate complexes was demonstrably by FR mediation. These liposome formulations have the design capacity for in vivo application and may therefore be promising candidates for further development.

PEGylated and non-PEGylated siRNA lipoplexes formulated with cholesteryl cytofectins promote efficient luciferase knockdown in HeLa tat luc cells.

Delivery of small interfering RNAs (siRNAs) remains a major challenge in their development for therapeutic applications, and cationic liposomes are being actively investigated for this purpose. Six liposome formulations containing the cytofectins 3ß[N-(N ',N '-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3ß[N-(N ',N ',-dimethylaminopropylsuccinamidohydrazido)-carbamoyl] cholesterol (MS09) and varying amounts of distearoylphosphatidylethanolamine poly(ethylene glycol)2000, were prepared. Lipoplexes formed with siRNA were characterized by gel retardation analysis and cryo-electron microscopy. All lipoplexes exhibited low cytotoxicity in the HeLa tat luc cell line while Chol-T lipoplexes, containing anti-luciferase siRNA, achieved 93.4% knockdown of the luciferase transcript at 30 nM siRNA. Poly(ethylene glycol)2000 incorporation only marginally reduced knockdown efficiency.

Glycosylated liposomes with proton sponge capacity: novel hepatocyte- specific gene carriers.

Interest in hepatocyte-directed liposomal gene delivery is driven, in part, by the lack of effective treatment for several liver-associated disorders. To impart a hepatocyte targeting capability on DNA-lipoplexes, and to promote early release of cargo DNA from endosomes, novel glycosylated and imidazolylated cholesteryl derivatives have been synthesized and evaluated in vitro. Thus cholesteryl-3ß-N-[(lactobionyl) amino] carbamate (Chol-LAC) and cholesteryl-3ß-N- [(urocanyl) amino] carbamate (Chol-UAC) have been formulated with the cytofectin cholesteryl-3ß-[(N',N'- dimethylaminopropyl) carbamate (Chol-T) and the neutral co-lipid dioleoylphosphatidyl ethanolamine (DOPE). Liposomes, which displayed a buffering capability at endosomal pH, effectively bound DNA at a N/P ratio of 0.8:1 and offered partial protection against serum nuclease digestion. The MTT cell viability assay showed that lipoplexes were well tolerated by human hepatoma cells (HepG2), which were efficiently transfected almost exclusively by asialoglycoprotein receptor (ASGP-R)-mediation, as demonstrated in competition assays. In the ASGP-R-negative human kidney cell line (HEK293) transfection levels were considerably lower (P < 0.001). Therefore the combination of Chol-LAC and Chol- UAC in cationic liposomal formulations may provide a platform for the development of useful hepatotropic gene delivery systems.

Novel targeted liposomes deliver sirna to hepatocellular carcinoma cells in vitro.

Liposomes form a major class of non-viral vectors for short interfering RNA delivery, however tissue and cell-specific targeting are additional requirements in the design of short interfering RNA delivery systems with a therapeutic potential. Selective delivery of liposomes to hepatocytes may be achieved by directing complexes to the asialoglycoprotein receptor, which is expressed on hepatocytes, and which displays high affinity for the ß-d-galactopyranosyl moiety. We aimed to show that the d-galactopyranosyl ring in direct ß-glycosidic link to cholesterol, when formulated into liposomes with 3ß[N-(N',N'-dimethylaminopropane) carbamoyl] cholesterol (Chol-T) or its quaternary trimethylammonium analogue (Chol-Q), may promote targeted delivery of cytotoxic short interfering RNA to the human hepatoma cell line HepG2 via the asialoglycoprotein receptor. Liposome-short interfering RNA interactions were characterized by electron microscopy, dye displacement, gel retardation and nuclease assays. Stable short interfering RNA-protective lipoplexes were formed at N/P ratios in the range 5:1-7:1. Targeted lipoplex 4 achieved high transfection efficiencies at 50 nm short interfering RNA (70%) and <10% in a competition assay, whilst untargeted complexes reached low levels at the same concentration (<25%). Transfection efficiencies of all lipoplexes in the asialoglycoprotein receptor-negative cell line HEK293 under the same conditions were low. Lipoplexes containing cholesteryl-ß-d-galactopyranoside may therefore form the basis for the development of useful hepatotropic short interfering RNA delivery vectors.

Novel neo glycolipid: formulation into pegylated cationic liposomes and targeting of DNA lipoplexes to the hepatocyte-derived cell line HepG2.

Liver parenchymal cells are an important target for the treatment of several metabolic and viral disorders. Corrective gene delivery for this purpose is an avenue that is receiving increasing attention. In the present study, we report a novel neo glycolipid that may be formulated into cationic liposomes with or without poly(ethylene glycol) decoration. Lipoplexes formed with plasmid DNA are nuclease resistant and are targeted to the human hepatoblastoma cell line HepG2 by selective asialoglycoprotein receptor mediation. Transfection levels achieved by lipoplexes containing the targeting ligand cholesteryl-3ß-N-(4-aminophenyl-ß-D-galactopyranosyl) carbamate were sixfold greater than those obtained with similar but untargeted lipoplexes.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Studies on the inhibition of Moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds.

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 '-phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5'-AMP inhibited the enzyme partially using poly (rA).oligo d(pT)(16-18) as template-primer with [(3)H]dTTP. In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m, and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above template-primer system. The compounds all inhibited the reverse transcriptase with IC(50) values of approximately 60-80 microM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the nucleotide 8-(6-aminohexyl) amino-adenosine-5'-phosphate, the inhibitory activity of this compound increased significantly (IC(50) = 5 microM).

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.