Sepharose-bound poly (I)-poly (C): interaction with cells and interferon production.

Poly(I).poly(C) covalently coupled to a matrix by one point fixation through its 3′ terminal stimulated both antiviral activity and interferon production in primary rabbit kidney (PRK) cells. This effect could not be accounted for by free polynucleotide released from the matrix into the medium. Penetration of the polynucleotide into the cells does not appear to be necessary for interferon production. A limited amount of matrix-bound poly(I).poly(C) was associated with the cells. Since it was sensitive to extraneous ribonuclease treatment, this poly(I).poly(C) was believed to be localized at the cell surface. Preliminary findings suggest that the binding of the polynucleotide to the cell is not directly proportional to the amount of interferon induced.     

Continuous production of homopolynucleotides by immobilized polynucleotide phosphorylase

A polynucleotide phosphorylase was immobilized with glutaraldehyde, via an aminopropyl spacer, on porous glass. The specific activity of the immobilized enzyme was effectively increased by the addition of an appropriate ribonucleoside diphosphate on immobilization.A homopolynucleotide could be synthesized continuously by passing a nucleoside diphosphate solution through the immobilized enzyme column. The chain length of the product depended upon the temperature and the flow rate. Polyinosinic acid, poly(I), was continuously synthesized with the immobilized enzyme for about one month without appreciable loss of activity.Polyinosinic acid-polycytidylic acid, poly(I)·poly(C), prepared from poly(I) and poly(C) synthesized with the immobilized polynucleotide phosphorylase, induced interferon-β (IFN-β) in human cultured cells as effectively as that prepared from homopolynucleotides synthesized with the free enzyme.     

Macrophage activation: synergism between hybridoma MAF and poly(I). Poly(C) delivered by liposomes

High concentrations of a murine T cell hybridoma culture supernatant containing macrophage-activating factor (MAF) rendered resident mouse peritoneal macrophages cytotoxic for P815 mastocytoma cells. The capacity of the hybridoma-derived MAF (MAFH) to induce tumoricidal activity increased 10(3) to 10(4)-fold when the lymphokine was encapsulated into liposomes. Combinations of MAFH and poly(I) X poly(C) acted synergistically to render macrophages potently cytotoxic. Subthreshold (nonactivating) concentrations of free or liposome-encapsulated MAFH increased the potency of free poly(I) X poly(C) and liposome encapsulated poly(I) X poly(C). Either as free agent or encapsulated in liposomes, single-stranded poly(I) or poly(C) did not activate macrophages in the presence or absence of MAFH. Double-stranded poly(I) X poly(C) was thus required for macrophage activation and synergism with MAFH.     

Intracellular digestion of saturated and unsaturated phospholipid liposomes by mucosal cells. Possible mechanism of transport of liposomally entrapped macromolecules across the isolated vascularly perfused rabbit ileum

The mechanism of intestinal absorption of liposomally entrapped [14C]inulin and 125I-labelled poly(vinylpyrrolidone) was studied using the isolated rabbit intestinal loop with intact perfused vasculature, a system more closely resembling an in vivo system than the everted sac technique. [14C]Inulin or 125I-poly(vinylpyrrolidone) was entrapped in liposomes prepared from unsaturated egg phosphatidylcholine and soya phosphatidylcholine, and saturated distearoylphosphatidylcholine (18:0), dipalmitoylphosphatidylcholine (16:0) and dimyrostoylphosphatidylcholine (14:0). Free and liposomally entrapped macromolecules were introduced in the ileum and the transport of liposomes and entrapped macromolecules into the venous effluent was monitored by measuring the presence of the aqueous marker 125I-poly(vinylpyrrolidone) or [14C]inulin, and lipid marker [3H]cholesterol. The results show that intact liposomes are not transported across intestine into the venous effluent, but they are taken up by mucosal cells and digested intracellularly, releasing the entrapped markers 125I-poly(vinylpyrrolidone) and [14C]inulin. These markers are then transported into the venous effluent as free molecules. The absorption of liposomally entrapped [14C]inulin into the venous effluent is biphasic, first slow for 30 min (i.e., a lag period of 30 min), followed by a rapid linear increase. The duration of the lag period and the rate of absorption of the entrapped [14C]inulin are dependent on the degree of saturation and the transition temperature of the phospholipids used to prepare liposomes. The possible explanation of the lag period based on the evidence presented here is that it is the time required for the liposomes to be taken up by mucosal cells and digested intracellularly. Intracellular digestion of liposomes prepared from saturated phospholipids is more rapid than from those prepared from unsaturated phospholipids, and the greater the fatty acid chain length of the saturated phospholipids the more rapid the intracellular degradation of liposomes.     

A comparison of negatively and positively charged liposomes containing entrapped polynosinic · polycytidylic acid for interferon induction in mice

Intravenous injection of negatively charged liposomes containing entrapped poly(I) · poly(C) induced a vigorous interferon response in mice with serum titers of interferon reaching twenty times those observed with comparable dosages of free poly(I) · poly(C). The response did not persist over an extended time period as was observed earlier for enhanced interferon production stimulated by positively charged liposomes containing the inducer. Both negatively and positively charged liposomes containing [¹⁴C]poly(I) · poly(C) were taken up chiefly by the liver when given intravenously. Negatively charged particles were concentrated somewhat preferentially by the spleen (7–9% of the dose compared to 4–6%). Less radioactivity was found in liver and spleen when negatively charged particles were given intraperitoneally than was the case when positively charged particles were injected by this route. Free [¹⁴C]poly(I) · poly(C) was extensively metabolized to low molecular weight materials within four hours of injection, while encapsulation of the polymer provided protection against in vivo degradation. When both preferential localization and protection were considered, from three to five times as much high molecular weight [¹⁴C]poly(I) · poly(C) was recovered from liver at four hours after intravenous injection when the compound was given in encapsulated form compared to the free polymer. Similarly, for spleen, seven times and three times as much polymeric [¹⁴C]poly(I) · poly(C) was recovered following injection of negatively charged liposomes and positively charged liposomes respectively compared to free [¹⁴C]poly(I) · poly(C). At 48 h after an intravenous injection of positively charged liposomes, as much as four percent of the dose remained in high molecular weight form in the liver and one percent in the spleen. Following intraperitoneal injections, polymeric [¹⁴C]poly(I) · poly(C) recovered from the liver never exceeded 4.3% of the dose, showing that most of the radioactivity in the liver consisted of metabolites. These results suggest that elevated and prolonged production of interferon in animals treated with encapsulated inducer results from a combination of factors including preferential tissue location and protection of the inducer from hydrolytic cleavage.     

Receptor-targeted liposomal delivery of boron-containing cholesterol mimics for boron neutron capture therapy (BNCT)

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.     

Stability and absorption of liposomes with incorporated insulin in the small intestine

Lecithin-cholesterol liposomes with the incorporated insulin were studied in vitro for their stability to the action of some factors of gastrointestinal tract--hydrochloric acid, gastric juice, pepsin, trypsin, bile. A considerable destructive action of bile on liposomes is established. Modification of the lipid composition of liposomes makes it possible to increase their stability to the bile action. Suction of liposomes from the isolated sites of rat small intestine is studied. Insulin from liposomes is established to be sucked most intensive from the initial parts of small intestine. In this case it penetrates into the blood bed not in a free state but in the composition of liposomes.     

Characteristics of the carbohydrate digestive transport carrier in rats exposed to the effect of a temperature factor in early postnatal ontogeny

Using accumulating preparations of the small intestine mucose, studies have been made of the initial stages of glucose uptake from solutions of glucose, maltose and starch in rat puppies at the age of 17, 24, 31 and 38 days, which were subjected from the 10th to the 17th day of postnatal life to daily heating (up to 40-41 degrees C) or cooling (up to 6-7 degrees C) for 2 hours. It was found that in heat-adapted animals, the rate of accumulation of "free" glucose remained the same as in control animals, whereas the rate of uptake of the glucose in the form of maltose or starch was significantly decreased. Cold adaptation promoted stable stimulation of carbohydrate hydrolysis and transport functions of the intestine.     

Induction of cell death in rat small intestine by ischemia reperfusion: differential roles of Fas/Fas ligand and Bcl-2/Bax systems depending upon cell types

Although ischemia reperfusion (I/R) induces apoptotic damage of mammalian small intestine, the molecular mechanism is largely unknown. We investigated the appearance of apoptosis at various time-points (0–24 h) of reperfusion after 1-h ischemia and the expression of various apoptosis-related proteins, such as Bcl-2, Bax, Fas, Fas ligand (FasL), activated caspase-3, and cytochrome c, immunohistochemically in rat small intestine. As assessed by TUNEL and electron microscopy, apoptotic cells were increased at 3 h of reperfusion in all intestinal parts (villous epithelium, crypt epithelium, and stroma of intestine). Moreover, the TUNEL-positive cells in the stroma were later identified as T cells. The expression of Fas and FasL as well as activated caspase-3 was markedly increased at 3 h of reperfusion in the stroma. In the villous epithelium, a transient decrease in Bcl-2 expression was found while in the crypt epithelium, Fas expression was induced. Finally, intraperitoneal injection of leupeptin (an SH-protease inhibitor) after I/R resulted in a significant inhibition of the induction of apoptosis in the stroma and crypt epithelium. Our results indicate that the triggering molecules of apoptosis in the I/R rat small intestine may vary depending on cell type and that the use of a broad-spectrum protease inhibitor may reduce intestinal damage.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Induction of the replicative synthesis of DNA by SV40 virus T antigen introduced into cells using liposomes

The role of virus SV40 T-antigen in the induction of cell DNA synthesis during its incorporation into cell liposomes was studied, using monolamellar liposomes obtained by phase reversal with incorporated highly purified T-antigen. Immunofluorescence studies revealed that T-antigen effectively penetrates inside the cells and after 10 hours is accumulated in the nuclei, where its level remains unchanged for 24 hours. Injections of purified T-antigen into the renal cells of serum-starved CV1 monkeys resulted in an almost 10-fold increase in the number of DNA-synthesizing cells 18 hours after the exposure. The same effect was observed during stimulation of a 10% serum culture. Removal of T-antigen from the preparation by specific immunoadsorption eliminated this effect. Centrifugation of cells grown in the presence of bromodeoxyuridine in a CsCl gradient was used to demonstrate the replicative type of cell DNA synthesis during T-antigen induction.     

Temperature sensitization of liposomes by use of N-isopropylacrylamide copolymers with varying transition endotherms

Three kinds of copolymers of N-isopropylacrylamide (NIPAM) with the same conformational transition temperature and varying transition endotherms were synthesized with N-acryloylpyrrolidine (APr), N,N-dimethylacrylamide (DMAM), and N-isopropylmethacrylamide (NIPMAM) as the comonomers. Two dodecyl groups were incorporated into the termini of these copolymers as an anchor for the fixation to a liposomal membrane. Egg yolk phosphatidylcholine liposomes having these copolymers were prepared and their temperature-sensitive contents release and association properties were investigated. While these copolymer exhibited a conformational transition at ca. 40 degrees C, DeltaH for the transition increased in the order of poly(APr-co-NIPAM) < poly(DMAM-co-NIPAM) < poly(NIPMAM-co-NIPAM). The liposomes containing poly(NIPMAM-co-NIPAM) showed a drastic release enhancement of entrapped calcein above the transition temperature, whereas the liposomes with poly(DMAM-co-NIPAM) and those with poly(APr-co-NIPAM) exhibited moderate and slight enhancement of calcein release above that temperature, respectively. On the contrary, the liposomes containing poly(APr-co-NIPAM) showed significant aggregation above the transition temperature, but the aggregation was hardly observed for the liposomes having poly(NIPMAM-co-NIPAM), indicating that poly(APr-co-NIPAM) more efficiently made the liposome surface hydrophobic. Thus, we concluded that the copolymer with a large DeltaH is suitable for obtaining functional liposomes with a temperature-sensitive contents release property, whereas the copolymer with a small DeltaH is appropriate for preparing functional liposomes with a temperature-sensitive surface property.     

Polynucleotides. LVII. Synthesis and properties of poly (2''-chloro-2''-deoxyinosinic acid).

Poly (2'-chloro-2'-deoxyinosinic acid) [poly(Icl)] was synthesized from Icl 5'-DP by polymerization with polynucleotide phosphorylase. UV absorption properties of poly(Icl) are very similar to those of poly(I). Poly(Icl) adopted a multi-stranded ordered form in the presence of 0.95M Na ion. The Tm value of this form was 36 degrees, which resembles that of poly(I) quadruple-stranded form at high salt. CD spectra also suggested presence of these two forms. Upon mixing with poly(C), poly-(Icl) forms a double-stranded 1 : 1 complex, which had very similar Tm-log[Na+] relationship to that of poly(I) . poly(C). Thus it was concluded that the chlorine substitution at 2'-position of the polynucleotide had the similar effect to OH on physical properties of polynucleotides.     

Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii.

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.     

Polyinosinic-polycytidylic acid accelerates intestinal stem cell proliferation via modulating Myc expression

It is well known that exposure of double-stranded RNA (dsRNA) to intestine immediately induces villus damage with severe diarrhea, which is mediated by toll-like receptor 3 signaling activation. However, the role of intestinal stem cells (ISCs) remains obscure during the pathology. In the present study, polyinosinic-polycytidylic acid (poly[I:C]), mimicking viral dsRNA, was used to establish intestinal damage model. Mice were acutely and chronically exposed to poly(I:C), and ISCs in jejunum were analyzed. The results showed that the height of villus was shorter 48 hr after acute poly(I:C) exposure compared with that of controls, while chronic poly(I:C) treatment increased both villus height and crypt depth in jejunum compared with control animals. The numbers of ISCs in jejunum were significantly increased after acute and chronic poly(I:C) exposure. Poly (I:C)-stimulated ISCs have stronger capacities to differentiate into intestine endocrine cells. Mechanistically, poly(I:C) treatment increased expression of Stat1 and Axin2 in the intestinal crypt, which was along with increased expression of Myc, Bcl2, and ISC proliferation. These findings suggest that dsRNA exposure could induce ISC proliferation to ameliorate dsRNA-induced intestinal injury.     

Interaction of metal ions with polynucleotides and related compounds. IX. Unwinding and rewinding of poly(A + U) and poly(I + C) by copper(II) ions

It is demonstrated that, poly(A + U) and poly(I + C) are both formed under low ionic strength conditions. Continuous variation studies indicate the formation of copper(II) complexes of poly A, poly C, and poly I, but not of poly U. Copper(II) in a 1:1 ratio to polynucleotide prevents the formation of poly(A + U) and brings about the dissociation of the poly (A + U) complex produced in the absence of the metal. Poly (I + C) is similarly dissociated by copper(II) ions. The addition of sufficient electrolyte reverses the copper(II) induced dissociation of poly(I + C). The effect of copper(II) on ordered synthetic polynucleotides is thus very similar to its effect on DNA.     

Experimental thermodynamics of the helix--random coil transition. 3. Determination of the transition enthalpies of the helical complexes poly(I+C) and poly I in solution

The enthalpies of the helix-coil transitions of the ordered polynucleotide systems of poly(inosinic acid)–poly(cytidylic acid) [poly(I + C)], (helical duplex), and of poly (inosinic acid) [poly(I + I + I)], (proposed secondary structure: a triple-stranded helical complex), were determined by using an adiabatic twin-vessel differential calorimeter. Measuring the temperature course of the heat capacity of the aqueous polymer solutions, the enthalpy values for the dissociation of the helical duplex poly (I + C) and the three-stranded helical complex poly(I + 1 + 1), respectively, were obtained by evaluating the additional heat capacity involved in the conformational change of the polynucleotide system in the transition range. The ΔH values of the helix-coil transition of poly (I + C) resulting from the analysis of the calorimetric measurements vary between the limits 6.5 ± 0.4 kcal/mole (I + C) and 8.4 ± 0.4 kcal/mole (I + C). depending on the variation of the cation concentration ranging from 0.063 mole cations kg H₂O to 1.003 mole cations/kg H₂O. The calorimetric investigation of an aqueous poly I solution (cation concentration 1.0 mole/kg H₂O) yielded the enthalpy value ΔH = 1.9 ± 0.4 kcal/mole (I), a result which has been interpreted qualitatively following current models of inter- and intramolecular forces of biologically significant macromolecules. Additional information on the transition behavior of poly(I+ C)Was obtained by ultraviolet and infrared absorption measurements.     

Poly(2-methylthio-7-deazainosinic acid)--hydrophobic stabilization of polynucleotide secondary structure by the 2-methylthio group.

Poly(2-methylthio-7-deazainosinic acid) [poly(ms2c7I)] was enzymatically synthesized by polymerization of 2-methylthio-7-deazainosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer shows much higher thermal stability than its parent polynucleotides poly(7-deazainosinic acid) [poly(c7I)] and poly(I). Its sigmoidal melting curve and pronounced hypochromicity imply a rigid, ordered structure. Poly(ms2c7I), like poly(2-methylthio-inosinic acid) [poly(ms2I)], does not form a complex with poly(C) because of the bulky 2-methylthio substituent. On the other hand, two poly(ms2c7I) strands form very rigid triple strands with poly(A). Different from poly(I) and poly(c7I) the homopolymer poly(ms2c7I) is very stable against cleavage by nuclease S1 and ribonuclease T2 as expected from its rigid secondary structure.     

TNF-α Producing Innate Lymphoid Cells (ILCs) Are Increased in Active Celiac Disease and Contribute to Promote Intestinal Atrophy in Mice

Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of this study was to characterize the tissue distribution of ILCs in celiac disease (CD), a gluten-driven enteropathy, and analyze their role in gut tissue damage. ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs) isolated from duodenal biopsies of CD patients and healthy controls (CTR) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with peptidoglycan, poly I:C, or CpG, the agonists of TLR2, TLR3, or TLR9 respectively, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Although the percentage of total ILCs did not differ between CD patients and CTR, ILCs producing TNF-α and IFN-γ were more abundant in CD mucosa compared to controls. ILCs expressed TLR2, TLR3 and TLR9 but neither TLR7 nor TLR4. Stimulation of LPMC with poly I:C but not PGN or CpG increased TNF-α and IFN-γ in ILCs. RAG1-deficient mice given poly I:C exhibited increased frequency of TNF-α but not IFN-γ/IL17A-producing ILCs in the gut and depletion of ILCs prevented the poly I:C-driven intestinal damage. Our data indicate that CD-related inflammation is marked by accumulation of ILCs producing TNF-α and IFN-γ in the mucosa. Moreover, ILCs express TLR3 and are functionally able to respond to poly I:C with increased synthesis of TNF-α thus contributing to small intestinal atrophy.     

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.