Nucleic Acids Research: VOLUME 40 ISSUE 16 2012

A method of ultramicroinjection of nucleic acids into cultured cells by means of liposomes is described. Messenger RNA, ribosomal RNA and transfer RNA were entrapped in large unilamellar liposomes and subsequently the liposomes were fused with cells. The uptake of RNA by the cells was stimulated 6 8 times by our method. Possible applications of microinjection of RNA by means, of liposomes are discussed. INTRODUCTION Experimental studies on the interaction of biologically active molecules with cells cultured ̂ n vitro are often hindered by the failure of cells to incorporate molecules added to the culture medium. Macromolecules have been introduced into cells by spontaneous uptake (1), by virus-induced fusion of cells with erythrocyte ghosts (2,3), and by microinjection (4,5). The last two techniques are the most efficient currently used. The first of these is quite simple and is applicable to almost all types of animal cells but has the disadvantage of introducing erythrocyte membrane, residual haemoglobin, viral envelopes and inactivated viral RNA, which can have undesirable effects on cells (6). The microinjection method, of introducing material directly into the cytoplasm, leads to little waste of the material, but has the disadvantage of being laborious, particularly when applied to normal-sized cells (4,5). Recent work has shown that cells _iii vivo, as well as ̂ n vitro, can incorporate large numbers of lipid vesicles (liposomes) without significant cytotoxicity (7,8). Entrapment of water-soluble materials inside such vesicles and subsequent fusion of the vesicles with cells offers a potential method for introducing non-permeable, biologically active molecules directly into the intracellular compartment, without the disadvantages of the previously described methods. In the particular case of introducing ribonucleic acids into cells, the main problem is their degradation by the ribonucleases which © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 1381 Nucleic Acids Research are present in the medium. Entrapment of RNAs in liposomes would protect them from ribonuclease activity and would greatly assist their introduction into the cells. We present here data of entrapment of ribonucleic acids of different sizes, namely reticulocyte ribosomal RNA (rRNA, 28s, 18s, 7s and 5s), globin messenger RNA (mRNA, 9s) and yeast transfer RNA (tRNA, 4s) in liposomes and their introduction into cells in culture. MATERIALS AND METHODS Preparation of liposomes containing RNAs. Large unilamellar liposomes were prepared by dissolving in chloroform 10 |/mol of beef brain phosphatidylserine (Lipid Products, Nr. Redhill, Surrey, England) and the solvent was removed by flask evaporation on a rotary evaporator at room temperature. The thin film thus obtained was suspended in NHTE buffer, pH 7.4 (0.1M NaCl, 2mM histidine, 2mM TES, 0.4mM EDTA, pH 7.4) (9). The suspension was vortexed and subsequently sonicated in a bath-type sonicator for 30 min at 30 C under nitrogen. 0.02 ranoles of Ca + was added and the mixture was incubated for 1 hr at 37°C after which the preparation was centrifuged at 2.500xg for 10 min. The pellet was suspended in 0.1 ml of a solution of RNA which had previously been dialysed 125 against NHTE buffer, pH 7.4. Trace amounts of [ I] RNA were added for quantitating RNA capture inside vesicles. The mixture was vortexed, 0.2 mmoles EDTA was added and the mixture was incubated for 30 min at 37 C. The liposomes were recovered by centrifugation (30,000xg, 20 min at 20 C) and washed with phosphate-buffered saline (PBS). Washed liposomes were suspended in 1 ml PBS, equilibrated for 30 min at room temperature and then passed through a Sepharose 4B column, equilibrated with the same buffer, to separate vesicles containing RNA from untrapped RNA. Interaction of liposomes with cells. Mouse L929 cells were grown in minimum essential medium (Eagle) containing 10% heat-inactivated calf serum, 100 U/ml penicillin and 100 ng/ml streptomycin, in 60 mm plastic petri dishes at 37 C in an atmosphere of 5% COin air. The monolayers were rinsed with PBS and either liposomes (100 6 1 ? S 125 nmol of lipid per 10 cells) containing [ I] RNA or free [ I] RNA, in 1 ml PBS were added. Subsequently the cells were incubated at 37 C for 60 min and then the unabsorbed material was removed by washing three times with PBS. The cells on the monolayer were harvested by scraping with a rubber policeman in PBS.