Improved methods for the delivery of liposome-sequestered RNA into eucaryotic cells

RNA sequestered by negatively charged liposomes becomes cell-associated following interaction between eucaryotic cells and the liposomes. This paper provides evidence that cell-associated RNA is internalized by the cells. In fact, (a) when Escherichia coli and mammalian RNA are entrapped within the same liposome population and delivered into cultured cells, one can observe degradation of the procaryotic but not the eucaryotic RNA. Such an event cannot happen extracellularly. (b) Scanning electron microscopy reveals no more than 10 liposomes adhering to each cell upon liposome-cell interaction under conditions in which the RNA entrapped by 140 liposomes becomes associated with each cell. The ability of liposomes prepared by (a) the cochleate process, (b) the reverse-phase evaporation technique, and (c) the ether infusion technique, to sequester and deliver RNA into cells was investigated. Reverse-phase evaporated liposomes were most efficient in sequestering RNA (20–40%), however, all types of liposomes delivered RNA with comparable efficiency. The rate of liposome-mediated RNA delivery into mammalian cells could be substantially improved when: (a) liposome-cell interaction was carried out at pH 6.5 (twofold increase over pH 7.5), (b) a basic protein (methylated albumin) was present (two- to threefold increase), (c) liposome-cell cultures were treated with polyethylene glycol 6000 (four- to eight-fold increase), and (d) DEAE-dextran was added during interaction of liposomes with cell monolayers (four- to eight-fold increase).     

Cellular uptake of ribonucleic acids entrapped into liposomes.

Ribonucleic acids were entrapped into phospholipid vesicles (liposomes). After incubation of the liposomes containing RNA (L- RNA), the RNA was introduced into the cells. The kinetics of L- RNA uptake by the cells in culture were studied. The uptake of L- RNA is linear over a broad vesicle concentration range depending on temperature, and at 37 degrees C uptake levels reach a plateau after 3 hours. Inhibitors of cellular energy metabolism have little effect on the uptake, and thus fusion, as the main mechanism of uptake, is proposed.     

Parameters affecting the liposome-mediated insertion of RNA into eucaryotic cells in vitro

The effect of various parameters on the liposome-mediated insertion of RNA into eucaryotic cells in vitro has been studied. Maximization of the insertion of liposome-encapsulated RNA into cells was approached at three levels: (1) alteration of liposome membrane composition, (2) alteration of the recipient cell membrane, and (3) manipulation of the conditions of liposome-cell cocultivation. (1) Changes in liposome membrane composition failed to affect the amount of RNA sequestered within liposomes but did alter the efficiency and mode of liposome uptake by human epithelial carcinoma cells, rabbit spleen lymphocytes, and carrot protoplasts. Addition of lysolecithin to the liposome membrane enhanced the cellular uptake of liposome-sequestered RNA by a “fusion” mechanism (uptake in the presence of cytochalasin B), while addition of cholesterol was inhibitory. (2) Uptake of liposome-sequestered RNA was enhanced when (a) cells were in the mitotic phase of the cell cycle; (b) cells were pretreated with cholesterol-free liposomes; and (c) cells were treated with Piracetam (2-oxo-1-pyrrolidine acetamide). The increased cellular uptake of liposomes appeared in most cases to be due to enhanced cell membrane fluidity. (3) Liposome uptake by cells was directly proportional to the time of liposome-cell cocultivation and to cell number. Increasing doses of liposomes resulted in a reduction of the percentage of RNA uptake, possibly due to a saturation phenomenon. When several of the investigated parameters were simultaneously maximized, as high as 20% of the liposome-sequestered RNA was inserted into human epithelial carcinoma cells.     

The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides

Macromolecules such as DNA and RNA can be entrapped within liposomes associated with gangliosides by reverse-phase evaporation. When these liposomes are incubated with HVJ2 (Sendai virus), they deliver their contents into cultured cells efficiently. More than 95% cells of a Ltk- cell line (thymidine kinase-deficient cells) transiently expressed thymidine kinase activity by thymidine kinase gene transfer using HVJ liposomes with gangliosides. Stable transformants could be obtained efficiently from various cell lines by use of HVJ liposomes containing the neoR gene. The neo+ transformants were obtained at frequencies of about 0.2-1.0, 0.06-0.25, and 0.06-0.1% in monolayers of L, CHO-Kl, and HeLa-S3 cells, respectively. Moreover, in Ehrlich ascites tumor cells which grow in suspension, the frequency was more than 0.01%. On introduction of plasmid pTK4 into Ltk- cells, about 0.5-1.0% TK+ transformants were obtained. Cosmid DNA containing the neoR gene (about 45 kbp) was also introduced into L cells by this method and neo+ transformants were obtained at a frequency of 0.1%. When rat liver mRNA was introduced into L cells by HVJ liposomes with gangliosides, immunoprecipitation studies showed that the L cells secreted rat albumin and some other proteins into the cultured medium. Moreover, using erythrocyte membrane vesicles containing IgM that had been incubated with HVJ empty liposomes with gangliosides, the IgM could be introduced into all the L cells.     

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

Liposomes as vehicles for cellular incorporation of biologically active macromolecules

Lipid vesicles (liposomes) have recently been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using large unilamellar vesicles composed of phosphatidylserine [LUV(PS)] we were able to encapsulate poliovirus and purified poliovirus ribonucleic acid (RNA) and show that it can be delivered efficiently to cells in an infectious form. LUV-entrapped poliovirus RNA produced infectious titers 100-fold higher than comparable RNA preparations delivered to cells by other techniques. We have made a quantitative analysis of the uptake and infectivity of the vesicle-encapsulated RNA by using various ratios of RNA copies per vesicle and by determining the percentage uptake of labelled lipid and RNA by HeLa cells.     

Interferon production of L929 and HeLa cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.     

Flow microfluorometric monitoring of the interaction of lipid vesicles with cells

Summary Flow microfluorometric techniques have been applied to experiments concerned with the penetration of cells by lipid vesicles. The high sensitivity of laser flow systems enables to measure the weak fluorescence emitted by individual liposomes tagged with perylene, inserted into their multilamellar layer. The total fluorescence of cells which have incorporated such perylene-loaded liposomes could be measured and well separated from that of unbound liposomes. Significant differences in the incorporation rates of cationic and anionic liposomes were shown by means of time-course analyses of cellular fluorescence spectra. The advantages of the rapid data analysis by flow fluorescence techniques is discussed in comparison with conventional radio-isotopic methods.     

Flow cytofluorometric investigation of the uptake by hepatocytes and spleen cells of targeted and untargeted liposomes injected intravenously into mice

Untargeted liposomes (composition: PC-PS-cholesterol) and targeted liposomes (composition: PC-PS-cholesterol-lactosylceramide) having encapsulated concentration-quenched carboxyfluorescein were injected intravenously into mice. 1 h after injection, the mice livers were perfused, excised and the hepatocytes were separated from nonparenchymal cells and analysed in a fluorescence-activated cell sorter analyzer. The result was that hepatocytes took up significantly more liposomes when lactosylceramide was inserted in the liposome bilayers, which was in good agreement with observations made on the in vivo uptake of liposome-encapsulated insulin gene (Soriano, P. et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 7128–7133). Cytofluorimetric analysis of the spleen cells showed that approx. 10% of the splenic lymphocytes take up high amounts of lactosylceramide liposomes, whereas most of the phospholipid liposomes are taken up by the phagocytic cells. The flow cytofluorimetric analysis shows, moreover, the internalization of the liposomes by the target cells and allows a quantitation of this uptake. Thus, in vivo targeting of the liposomes to specific liver and splenic cells, by means of glycolipid insertion in the liposome bilayer, is shown to take place with delivery of the liposomal aqueous space marker to these cells.     

Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region.

Previous studies revealed that antisense oligodeoxynucleotides to specific regions of the human immunodeficiency virus-1 (HIV-1) are potent inhibitors of replication of HIV-1 in vitro (Zamecnik, P. C., Goodchild, J., Taguchi, Y., and Sarin, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4143-4146). We now report that antisense RNA, synthesized in vitro using T7 and SP6 RNA polymerase, displayed an anti-HIV-1 effect in the HTLV-IIIB/H9 system in vitro. Treatment of HIV-1-infected H9 cells with viral env region antisense RNA encapsulated in liposomes targeted by antibodies specific for the T cell receptor molecule CD3 almost completely inhibited HIV-1 production. The viral env segment covered a part of exon II of HIV-1 tat gene. No anti-HIV activity could be detected with similarly targeted liposome-encapsulated sense env RNA or with pol RNA synthesized in either the sense or antisense orientations, or with env region antisense RNA free in solution, or encapsulated in liposomes in the absence of the targeting antibody. A semiquantitative evaluation revealed that 4000-7000 RNA molecules became cell-bound in targeted liposomes; the half-life of the intracellularly present hybridizable antisense env RNA was approximately 12 h. Western blots showed that antisense env RNA suppressed tat gene expression by approximately 90% and gp160 production by 100%. These data were confirmed by immunoprecipitation studies. Northern blots (using an env probe) demonstrated the existence of all major HIV RNA species (9.3-, 4.3-, and 2.0-kb mRNA) in HIV-infected cells treated with antisense env RNA although at a reduced level. We conclude that the antisense env RNA inhibited viral protein production at the translational level.     

Flow cytofluorometric investigation of the uptake by hepatocytes and spleen cells of targeted and untargeted liposomes injected intravenously into mice

Untargeted liposomes (composition: PC-PS-cholesterol) and targeted liposomes (composition: PC-PS-cholesterol-lactosylceramide) having encapsulated concentration-quenched carboxyfluorescein were injected intravenously into mice. 1 h after injection, the mice livers were perfused, excised and the hepatocytes were separated from nonparenchymal cells and analysed in a fluorescence-activated cell sorter analyzer. The result was that hepatocytes took up significantly more liposomes when lactosylceramide was inserted in the liposome bilayers, which was in good agreement with observations made on the in vivo uptake of liposome-encapsulated insulin gene (Soriano, P. et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 7128-7133). Cytofluorimetric analysis of the spleen cells showed that approx. 10% of the splenic lymphocytes take up high amounts of lactosylceramide liposomes, whereas most of the phospholipid liposomes are taken up by the phagocytic cells. The flow cytofluorimetric analysis shows, moreover, the internalization of the liposomes by the target cells and allows a quantitation of this uptake. Thus, in vivo targeting of the liposomes to specific liver and splenic cells, by means of glycolipid insertion in the liposome bilayer, is shown to take place with delivery of the liposomal aqueous space marker to these cells.     

Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins

We have studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. We describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by [3H]thymidine incorporation. We show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein.     

Liposome-mediated RNA transfection should be used with caution

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.     

Systemic delivery of complexes of melanoma RNA with mannosylated liposomes activates highly efficient murine melanoma-specific cytotoxic T cells in vivo

The efficiency of the antitumor immune response triggered by dendritic cell (DC)-based vaccines depends predominantly on the efficiency of delivering tumor antigen-coding nucleic acids into DCs. Mannosylated liposomes were used to deliver tumor total RNA into DCs both ex vivo and in vivo, and the cytotoxic T-lymphocyte (CTL) antitumor response was assayed. The liposomes contained the mannosylated lipid conjugate 3-[6-(α-D-mannopyranosyloxy)hexyl]amino-4-{6-[rac-2,3-di(tetradecyloxy)prop-1-yl oxycarbonylamino]hexyl}aminocyclobut-3-en-1,2-dione), the polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), and the zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) at a molar ratio of 1: 3: 6 and were used as a transfection agent. Total RNA isolated from B16-F10 mouse melanoma cells served as a source of tumor antigens. Systemic administration of mannosylated liposomes–tumor RNA complexes into circulation of melanoma- bearing mice induced an efficient CTL response, which reduced the melanoma cell index in vitro with the same efficiency (by a factor of 2.8) as CTLs activated via an inoculation of DCs loaded with complexes of the same composition ex vivo. Complexes of tumor RNA with control liposomes, which lacked the mannosylated lipid conjugate, or DCs transfected with these complexes ex vivo were less efficient and reduced the melanoma cell count by a factor of only 1.6–1.8.     

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.     

Oligonucleotides encapsulated in pH sensitive liposomes are efficient toward Friend retrovirus.

Retroviruses present multiple RNA targets for antisense oligonucleotides. An oligodesoxyribonucleotide (15 mer) complementary to the region of the initiation codon AUG of the env gene mRNA of Friend retrovirus was an inhibitor of the translation of Env protein in vitro. No effect was observed on cells infected with Friend retrovirus. We observed that these oligomers were rapidly degraded in cellular medium. After encapsulation into liposomes, they inhibited the spreading of the virus for chronic or de novo infection. We have compared the efficiency of two compositions of liposomes: pH sensitive and non pH sensitive formulations. Oligomers encapsulated in pH sensitive liposomes were more active that those encapsulated in non pH sensitive liposomes. pH sensitive liposomes could allow to avoid degradation of oligomers by lysosomes.     

Antibody targeted liposomes containing poly(rI).poly(rC) exert a specific antiviral and toxic effect on cells primed with interferons alpha/beta or gamma

Double-stranded RNA can stimulate interferon production and mediate an antiproliferative effect on certain cell types. We evaluated the possibility of specifically targeting to cells in vitro the RNA duplex poly(rI).poly(rC) in pharmacologically active form after its encapsulation in small, unilamellar liposomes, to which was covalently coupled protein A. These liposomes became bound to and were endocytosed by murine L929 cells in the presence of protein A-binding monoclonal antibodies specific for an expressed cell surface protein, the H-2K molecule. When L929 cells were preincubated in the presence of low doses of interferon alpha/beta or gamma, they could be activated to produce interferon following exposure to either free poly(rI).poly(rC), or specifically bound liposomes poly(rI).poly(rC), but not the same liposomes in the presence of non-cell binding control antibodies. Specifically bound liposome-encapsulated poly(rI).poly(rC) was toxic to L929 cells at dose levels at least three logs lower than free poly(rI).poly(rC). This toxicity was also dependent on pre-treatment with interferon. These results indicate that liposome-encapsulated poly(rI).poly(rC) can survive endocytosis and can be released in active form to specific cell populations, at concentrations much lower than that required for pharmacologic effects of the same molecule in free form. They suggest that introduction into cells of other nucleic acids might benefit from the antibody-targeted liposome technology described here.     

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).     

Liposome-mediated delivery of tobacco mosaic virus RNA into petunia protoplast

Liposome-mediated delivery of TMV RNA into petunia protoplasts and resulting virus antigen production has been used as an assay for determining incubation conditions which favor increased uptake of vesicle contents by plant cells. Vesicle phospholipid composition, incubation buffer divalent metal ion concentration, the type and concentration of polyalcohol used to stimulate vesicle uptake and the RNA content of the liposome preparation were determined to be critical factors influencing the efficiency of delivery. Manipulation of these parameters resulted in a 50-fold improvement in virus antigen production over that obtained with conditions previously used for liposome-protoplast incubations (Proc Natl Acad Sci 79: 1859–1863, 1982). Virus antigen production could be detected following incubation of protoplasts with <0.5 ng of encapsulated TMV RNA, while at higher concentrations of added liposomes, >80% of the protoplasts could be infected. Comparisons with other techniques used to introduce nucleic acids into plant protoplasts indicated that liposome-mediated delivery was 10-to 1 000-fold more efficient than these other methods. The general use of liposomes to introduce RNA and DNA molecules into plant protoplasts is discussed.     

Expression of a cascading genetic network within liposomes

Liposomes have long been used as possible compartments for artificial cells, and it has been shown that liposomes can sustain various types of biochemical reactions. To elevate the degree of molecular complexity of the system in liposomes, we have constructed a two-stage genetic network encapsulated in liposomes. This two-stage genetic network was constructed with the plasmid pTH, in which the protein product of the first stage (T7 RNA polymerase) is required to drive the protein synthesis of the second stage (GFP). We show that the two-stage genetic network constructed in a cell-free expression system is functional within liposomes.