Structural Requirements for Cationic Lipid Mediated Phosphorothioate Oligonucleotides Delivery to Cells in Culture

Abstract

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (C_14:0) and dioleyl (C_18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C_14:0) was more effective than dipalmityl (C_16:0) lipid, which was more effective than distearyl (C_18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56°C for the distearyl lipid, 42°C for the dipalmityl lipid and 24°C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.     

脂质体转染bcl-2反义寡核苷酸G3139在HL-60细胞中的动力学模式

目的　应用阳离子脂质体介导或直接转染时 ,建立HL 6 0细胞摄入由 5 FITC标记的bcl 2反义寡核苷酸G3139的动力学模式及观察G 3139在细胞内的分布。方法　流式细胞仪测定细胞内的相对平均荧光强度 ,荧光显微镜观察细胞内的荧光分布。结果　①脂质体介导转染法显著提高HL 6 0细胞对G 3139的摄入 ,当DOSPER/G 3139(μg/μg)为 2 2 / 1时 ,细胞内相对平均荧光强度可达G 3139直接作用时的 40倍 ,而且HL 6 0细胞对G 3139的摄入跟G3139的浓度和作用时间有关。②细胞内的G 3139可向胞外转运 ,在DOSPER介导转染后 ,胞内荧光强度衰减缓慢 ,t1/ 2 约为 4h ,而G 3139直接作用后 ,t1/ 2 约为 0 5h ;③DOSPER介导转染 6h后 ,细胞内荧光物质主要以明亮的点状聚集在细胞核和部分细胞浆的细胞器中 ,而G 3139直接作用时 ,荧光物质则弥漫分布于胞浆中。结论　DOSPER可以增加G 3139的细胞摄入并改变其胞内分布 ,可能是阳离子脂质体增加bcl 2反义寡核苷酸生物活性的重要原因。     

On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides.

The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.     

Studies on uptake, sub-cellular trafficking and efflux of antisense oligodeoxynucleotides in glioma cells using self-assembling cationic lipoplexes as delivery systems

The cellular uptake of antisense oligodeoxynucleotides (ODNs) may be enhanced by the use of carriers such as cationic liposomes or lipoplexes, but little is known about the intracellular fate and subcellular trafficking of these systems in target cells. In this study, we report on the cellular uptake and biodistribution of ODNs in the presence and absence of optimised self-assembled cationic lipoplexes using the C6 glioma cell line as an in vitro model. Biotin or radiolabelled 15-mer phosphorothioate (PS) ODNs were synthesised and their cellular uptake and subcellular biodistribution characterised in the presence and absence of an optimised cationic lipoplex delivery system using studies ranging from cellular association, cellular efflux and transmission electron microscopy (TEM). Ultrastructural studies clearly showed PS ODNs in the absence of liposomal delivery to be sequestered within endosomal and lysosomal vesicular bodies indicative of endocytic uptake. ODNs were also visible, to a lesser extent, in the nucleus and cytoplasm. By employing DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidospermine tetra trifluoroacetic acid) and DOPE (dioleoylphosphatidylethanolamine) complex in a 3 : 1 ratio, as a delivery system for ODNs at a optimal lipid/DNA charge ratio of 1 : 1, the level of ODN cellular association was significantly increased by approximately 10-12 fold with a concomitant change in subcellular distribution of PS ODN. TEM studies indicated enhanced penetration of ODN within the cytosol and the cell nucleus with reduced presence in vesicular compartments. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into 'deeper' cellular compartments, consistent with endosomal release. Optimised cationic lipoplexes improved cellular delivery of ODNs by enhancing cell association, uptake and by favourably modulating the intracellular trafficking and distribution of ODNs into non-vesicular compartments including the cytosol and nucleus.     

脂质体Geneshuttle20对STAT6反义核酸转染小鼠脾淋巴细胞影响的研究

目的探讨阳离子脂质体Geneshuttle20对STAT6反义核酸在小鼠脾淋巴细胞摄入、分布的影响作用。方法采用阳离子脂质体介导STAT6反义核酸转染小鼠脾淋巴细胞,应用流式细胞仪、荧光显微镜分别观察反义核酸的细胞摄入和胞内分布。结果反义核苷酸与脂质体(W/W)为2∶4时,细胞摄入率可以达到67.7%,较单独加入反义核酸提高转染效率6倍,细胞内平均荧光强度最强,细胞核染色较深。结论Geneshuttle20提高了细胞对反义寡核苷酸的摄取,促使其进入细胞核内发挥作用。     

Cationic Cholesterol Promotes Gene Transfection Using the Nuclear Localization Signal in Protamine

Purpose. The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin). Methods. Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37°C. The cells then were washed and cultured for another 40 h in the growth medium at 37°C before luciferase assay. Results. The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine. Conclusion. Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.     

阳离子脂质体增加bcl—2反义寡脱氧核苷酸G3139在HL—60细胞中的摄取及活性

目的:探讨阳离于脂质体DOSPER对bcl-2反义寡脱氧核苷酸G3139在HL-60细胞中的摄取和活性的影响。方法:流式细胞仪测定细胞结合的平均荧光强度和Bcl-2蛋白阳性细胞的百分数;荧光显微镜观察细胞内FTTC荧光素标记G3139的分布;RT-RCR测定bcl-2 mRNA的表达。结果:①DOSPER增加细胞对G3139的摄取,当DOSPER/G3139(μg:μg)为2:1时,细胞对G3139的摄取在2h左右达到高峰,为G3139直接作用时的20倍,细胞核和细胞浆中的G3139为明亮的点状聚集;而G3139直接作用时,G3139则弥漫分布于细胞浆中;②细胞内的G3139可向胞外转运。DOSPER介导转染G3139 4h后,细胞内的G3139符合方程C(t)=68.2e~(-0.60t) 31.8e~(-0.02t)(初值为100％),t_(1/2)约为1.1h,而G3139直接作用后,细胞内的G3139符合方程C(t)=64.8e~(-2.27t) 35.2e~(-0.04t),t_(1/2)约为18min;③在DOSPER介导转染下,G3139在浓度1μmol·L~(-1)时,特异性减少bcl-2 mRNA的表达,同时使Bcl-2蛋白阳性率从97％±4％下降到70.6％±2.1％。结论:DOSPER增加细胞对G3139的摄取和改变G3139在细胞内的分布可能是其增强G3139活性的重要机制。     

Intracellular Distribution and Mechanism of Delivery of Oligonucleotides Mediated by Cationic Lipids

Purpose. To study the parameters influencing the intracellular trafficking of oligonucleotides delivered by cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) lipids and to elucidate the mechanism of uptake. Methods. We have studied the intracellular localization of fluorescently labeled oligonucleotide (F-ODN) delivered by DOTAP using confocal microscopy and measured inhibition of luciferase synthesis. The delivery mechanism of ODN/DOTAP complexes was investigated using inhibitors of the endocytosis pathway. Results. F-ODN delivered by DOTAP liposomes redistribute from punctate cytoplasmic regions into the nucleus. The nuclear uptake of F-ODN depends on: charge ratio (+/–), time of incubation, temperature and presence of serum. A positively charged complex is required for enhanced uptake. The association of neutral lipids with DOTAP reduced the optimum charge ratio without altering the delivery efficiency. DOTAP lipids increased >100 fold the antisense activity of a specific anti-luciferase ODN. Inhibitors of the endocytosis pathway show that the majority of F-ODN are introduced through an endocytic pathway mainly involving uncoated vesicles. Nuclear accumulation of oligonucleotides can be decreased by inhibitors of actin microfilaments, energy metabolism and proteins implicated in the fusion of endosomes. Nuclear uptake is independent of acidification of the endosomal vesicles and unaffected by inhibitors of microtubules. Conclusions. Oligonucleotides are delivered by cationic lipids into the cytoplasm at an early stage of the endocytotic pathway which leads to a marked increase in antisense activity and oligonucleotide nuclear uptake.     

Bovine serum albumin modified the intracellular distribution and improved the antiviral activity of an oligonucleotide

In the present work, we describe the ability of bovine serum albumin (BSA) to improve the cytoplasmatic delivery of a phosphodiester oligonucleotide (PO), whose phosphorotioate analogue is ISIS 2922 (Vitravene). The changes in intensity and lambda(max) in fluorescence spectroscopy and the increase in fluorescence anisotropy upon complex formation between the oligonucleotide and the protein (PO-BSA) were used to determine the dissociation constant, Km=6.3 x 10(-8) M. The stoichiometry of the complex is mainly 1:1, although 2 PO per BSA have been detected at high PO/BSA ratios. The complexation did not protect PO against enzymatic degradation by snake venom phosphodiesterase (0.1 mg/mL). Fluorescence microscopy revealed that PO-BSA complexes showed a decreased uptake and modified pattern of intracellular distribution with a rapid nuclear accumulation (rather than vesicular localization in the cytoplasm observed for free oligonucleotide). The effect was only observed over a certain threshold of BSA concentration (higher than found in media supplemented with 10% serum). By this way, the interaction with albumin increased the antiviral activity of the oligonucleotide, tested by a plaque reduction assay in MRC-5 fibroblasts infected with human cytomegalovirus (strain RC 256) at a MOI of 0.0035. At 10 microM PO concentration, free PO decreased virus plaques to 85% of the control, while PO-BSA complexes reduced to 60%, in the same order than ISIS 2922. The phosphorotioate analogue complexed by BSA (ISIS 2922-BSA) showed the highest activity with 45% of the control plaques.     

Effect of cationic liposomes on intracellular trafficking and efficacy of antisense oligonucleotides in mouse peritoneal macrophages

We have investigated the intracellular fate and antisense effect of oligonucleotide/cationic liposome complexes using phosphorothioate oligonucleotides (S-Oligo) targeted to inducible nitric oxide synthase in mouse peritoneal macrophages. Confocal laser microscopic analysis revealed that, after application of fluorescein isothiocyanate (FITC)-labeled S-Oligo alone, the intracellular localization of fluorescence exhibited a punctate pattern in the cytoplasm, suggesting that the oligonucleotides were mainly confined to the endosomal and/or lysosomal compartments. In the case of complexation with Lipofectin and DMRIE-C liposomes, cellular uptake of FITC-S-Oligo was not greatly enhanced and the fluorescence localization in the cells was similar to that of FITC-S-Oligo alone. LipofectAMINE slightly enhanced cellular uptake of FITC-S-Oligo; however, the intracellular localization profile of FITC-S-Oligo remained largely unchanged. The antisense effect was slightly enhanced by LipofectAMINE under only very limited experimental conditions. It was concluded that cationic liposomes are not a potential carrier for S-Oligo in peritoneal macrophages because of their inability to promote the release of S-Oligo from the endosomal compartments to the cytosol over a non-toxic concentration range.     

Disposition of non-viral gene delivery systems and oligonucleotides

At the 1998 annual meeting of the American Association of Pharmaceutical Scientists (AAPS), held in San Francisco, there was a roundtable discussion meeting entitled 'Disposition of Non-viral Gene Delivery Systems and Oligonucleotides'. Four speakers presented the advances made by their research groups in this area. The non-viral delivery systems utilized lipid, peptide, and polymeric carriers. The disposition of naked plasmid DNA was compared with complexed and condensed DNA. Although the disposition kinetics were more favorable with cationic systems, the expression levels obtained from naked DNA were superior. Interpolyelectrolyte complexes (IPECs) containing pluronic units are efficient for transfection. With the FDA approval of the first antisense oligonucleotide for therapeutic applications, there is growing interest in the development of antisense oligonucleotides. ISIS Pharmaceuticals Inc (Carlsbad, CA, USA) is evaluating various routes of delivery for oligonucleotides, including oral, rectal, inhalation, intravitreal and dermal routes.     

Cationic submicron emulsions overcome multidrug resistance in SGC7901/VCR cells

The over-expression of P-glycoprotein (P-gp) is associated with the development of multi-drug resistance (MDR) in cancer cells. In this study, we examined whether cationic submicron emulsions (CSEs) can efficiently deliver hydroxycamptothecin (HCPT) into MDR cells (SGC7901/VCR cells) via electrostatic-mediated endocytosis, thus overcoming MDR. We prepared HCPT-CSEs and rhodamine-123-CSEs (RH-123-CSEs), and examined the in vitro cytotoxic activity of HCPT-CSEs and the intracellular accumulation of HCPT and RH-123 in SGC7901/VCR cells. The HCPT-CSEs significantly increased the intracellular accumulation of HCPT (8.2-fold higher than HCPT-injection) and enhanced cytotoxic activity of HCPT (2.7-fold higher than HCPT-injection with verapamil). The fluorescence microscopic and flow cytometric detection on RH-123 supported the intracellular accumulation effect of CSEs. These results indicate CSEs may enhance drug-CSEs internalization followed by releasing their contents into the cytoplasm (near nuclear), thus lowering P-gp-mediated drug efflux. Furthermore, these in vitro results suggest that CSEs are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumor cells.     

Stability of polycationic complexes of an antisense oligonucleotide in rat small intestine homogenates.

Presystemic degradation in the gastrointestinal tract is one of the major problems contributing to the poor oral absorption of antisense oligonucleotides. Complexes between the antisense phosphorothioate oligodeoxynucleotide ISIS 2302 and the polycationic carriers protamine sulfate grade X, protamine chloride grade V, protamine phosphate grade X, poly-L-lysine hydrobromide (PLL), spermidine phosphate salt, spermine diphosphate salt, and Protasan G113 and CL113 were formulated in order to increase stability against intestinal nucleolytic degradation. Specific conductivity measurements were carried out to determine the charge ratio of the complex systems. Nuclease stability assays were performed in a rat small intestine homogenate model, which displayed significant exo- and endonuclease activity. Full-length oligonucleotide and metabolites were analyzed by capillary gel electrophoresis with UV detection at 260 nm. Most of the complexes of ISIS 2302 and the polycationic materials, except PLL-based systems, showed a better protection against enzymatic metabolism than free oligonucleotide. Protamine sulfate and protamine chloride considerably enhanced the nuclease stability of the phosphorothioate antisense oligonucleotide. The association of oligonucleotides with several polycationic substances proved to be an alternative to chemical modification in order to stabilize oligonucleotides in the gastrointestinal tract against nucleolytic degradation.     

Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells

Selective gene inhibition by antisense oligodeoxynucleotide (AS-ODN) or by small interference RNA (siRNA) therapeutics promises the treatment of diseases that cannot be cured by conventional drugs. However, antisense therapy is hindered due to poor stability in physiological fluids and limited intracellular uptake. To address these problems, a ligand targeted and sterically stabilized nanoparticle formulation has been developed in our lab. Human lung cancer cells often overexpress the sigma receptor and, thus, can be targeted with a specific ligand such as anisamide. AS-ODN or siRNA against human survivin was mixed with a carrier DNA, calf thymus DNA, before complexing with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes consisting of DOTAP and cholesterol (1:1, molar ratio) to obtain LPD (liposome-polycation-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with DSPE-PEG-anisamide, a PEGylated ligand lipid developed earlier in our lab, by the postinsertion method. Nontargeted nanoparticles coated with DSPE-PEG were also prepared as a control. Antisense activities of nanoparticles were determined by survivin mRNA down-regulation, survivin protein down-regulation, ability to trigger apoptosis in tumor cells, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to anticancer drugs. We found that tumor cell delivery and antisense activity of PEGylated nanoparticles were sequence dependent and rely on the presence of anisamide ligand. The uptake of oligonucleotide in targeted, PEGylated nanoparticles could be competed by excess free ligand. Our results suggest that the ligand targeted and sterically stabilized nanoparticles can provide a selective delivery of AS-ODN and siRNA into lung cancer cells for therapy.     

Oral bioavailability and multiple dose tolerability of an antisense oligonucleotide tablet formulated with sodium caprate

In vivo study was performed to determine the tolerability and pharmacokinetics of ISIS 104838, a phosphorothioate antisense oligonucleotide targetting human tumour necrosis factor alpha (TNF-alpha) mRNA, following multi-dose administration via intravenous and oral routes. Oral tablet formulations of ISIS 104838 were pre-formulated with the permeation enhancer, sodium caprate, in an enteric-coated solid dosage form. The average plasma bioavailability of ISIS 104838 was 1.4% relative to IV. The tissue distribution profile was similar following both routes of administration, with highest concentrations observed in the kidney followed by the liver, lymph nodes and spleen. Plasma bioavailability underestimated the tissue accumulation of ISIS 104838 observed 1 day after the last dose. Mean systemic tissue bioavailability ranged from 2.0 to 4.3%, relative to IV tissues, and was dependent on tissue type. No marked differences were noted in the pharmacokinetic parameters following multi-dosing either via intravenous or oral routes. All formulations administered were well tolerated. This paper reports the first evaluation of solid oral dosage forms comprising sodium caprate and an antisense oligonucleotide. Furthermore, this study demonstrates the oral delivery of ISIS 104838 from solid oral dose formulations, with the achievement of comparable tissue concentrations of the oligonucleotide to that of the intravenous treatment.     

A Fluorescent hPept1 Transporter Substrate for Uptake Screening

PURPOSE: To synthesize fluorescent analogues of hPept1 substrates, FITC-Val-OCH3, Lys-FITC-OH, and Lys-FITC-OCH3, and to characterize their hPept1 transporter-mediated uptake. METHODS: FITC analogues of amino acids were synthesized using established synthetic procedures, and the extent of their [3H]Gly-Sar uptake inhibition in HeLa/hPept1 cells was determined. The uptake of Lys-FITC-OCH3 was evaluated in HeLa, HeLa/hPept1, and Caco-2 cells in the presence and absence of Gly-Sar using a fluorescence microscopy-based assay. The uptake and transport of the Lys-FITC analogues were also determined in Caco-2 cells using HPLC assays. RESULTS: In HeLa/hPept1 cells, [3H]Gly-Sar uptake was significantly inhibited by Lys-FITC-OCH3 (74%) but not by FITC-Val-OCH3 (22%). The uptake of Lys-FITC-OCH3 (100 microM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, Lys-FITC-OCH3 (100 microM) uptake in HeLa/hPept1 and Caco-2 cells was reduced by 77% and 80%, respectively, in the presence of 1 mM Gly-Sar. Dipeptides and cephalexin significantly reduced Lys-FITC-OCH3 uptake in Caco-2 cells. The apical permeability of Lys-FITC-OCH3 (1.5 x 10(6) cm/s) in Caco-2 cells was significantly lowered in the presence of Gly-Sar. Fluorescence micrographs revealed that this analogue was localized in the cytoplasm and in the nucleus. CONCLUSIONS: The combined results indicate that Lys-FITC-OCH3 is recognized and transported by hPept1 in HeLa/hPept1 and by peptide transporters in Caco-2 cells. The results also suggest that Lys-FITC-OCH3 might be a useful fluorescent substrate for rapid assessment of peptide transporter activity in cells of interest.     

PAMAM Dendrimers as Delivery Agents for Antisense Oligonucleotides

Purpose. To investigate the potential use of PAMAM dendrimers for the delivery of antisense oligonucleotides into cells under conditions that mimic the in vivo environment. Methods. We used HeLa cells stably transfected with plasmid pLuc/ 705 which has a luciferase gene interrupted by a human -globin intron mutated at nucleotide 705, thus causing incorrect splicing. An antisense oligonucleotide overlapping the 705 splice site, when delivered effectively, corrects splicing and allows luciferase expression. The ability of dendrimers to deliver oligonucleotides to HeLa Luc/705 cells was evaluated in the absence or presence of serum. Results. PAMAM dendrimers formed stable complexes with oligonucleotides that had modest cytotoxicity and showed substantial delivery activity. The dose of the oligonucleotide, the charge ratio of oligonucleotide to dendrimer, and the size (generation) of the dendrimers were all critical variables for the antisense effect. The physical properties of dendrimer/oligonucleotide complexes were further investigated using sedimentation and gel electrophoresis methods. Effective oligonucleo-tide/generation 5 dendrimer complexes were macromolecular rather than particulate in nature, and were not sedimented at 100,000 RPM. Compared to other types of delivery agents, PAMAM dendrimers were more effective in delivering oligonucleotides into the nucleus of cells in the presence of serum proteins. Conclusions. Our results suggest that PAMAM dendrimers form non-particulate delivery complexes that function in the presence of serum proteins and thus may be suited for in vivo therapeutic applications.     

Efficient telomerase inhibition in human non-small cell lung cancer cells by liposomal delivery of 2'-O-methyl-RNA

The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase.     

Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions

Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes. Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing. Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo. This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL. The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense action of S-oligos. We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.