402 Bioconjugate Chem. 1992, 3, 402-407

Interferon Production of L929 and HeLa Cells Enhanced by Polyriboinosinic Acid-Polyribocytidylic Acid pH-Sensitive Liposomes

Pierre G. Milhaud,+ BBatrice Compagnon,* Alain Bienvenue,* and Jean R. Philippot*,*

Universit6 Montpellier 11, Sciences et Techniques du Languedoc, DBpartement Biologie SantB, URA-CNRS 1191 Gbnbtique Molbculaire, and URA-CNRS 530 Interactions Membranaires, 34095 Montpellier Cedex 5, France. Received May 8, 1992

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons cy and p (IFN) genes. With L929 and HeLacells 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 alow 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.

INTRODUCTION

The double-stranded RNA polyriboinosinic acid-poly- ribocytidylic acid (PolyIC) is an inducer of interferon (IFN) cy and /3 genes (Whatelet et al., 1987). Pretreating the cells with IFN (priming) (Stewart et al., 1971) enhances the response of L929 or HeLa cells to PolyIC powerfully (De Clercq, 1981).

On the other hand PolyIC induces a powerful toxicity against IFN-primed L929 cells (Stewart et al., 1972) the mechanism of which is not yet fully understood. However we have demonstrated that free PolyIC enters the cell through the acidic endocytic compartment before trig- gering that toxicity (Milhaud et al., 1987). Recently we demonstrated the induction of IFN by PolyIC loaded into liposomes which were targeted by antibodies to primed L929 cells (Milhaud et al., 1989). Surprisingly the IFN- PolyIC toxicity which goes with the endocytosis of PolyIC is highly increased by liposome-entrapped PolyIC. These results suggest that either a higher amount of intracel- lular PolyIC and/or a more appropriate intracellular delivery potentiate PolyIC efficacy.

Transfer of genetic material or synthetic nucleic acids interfering with gene expression (Leonetti et al., 1988) needs vectors to cross natural membranes, to escape deg- radative enzymes, and to reach their intracellular relevant targets. In several families of RNA viruses the delivery of the viral genome into the host cell cytoplasm is pH- dependent (Marsh & Helenius, 1989). At low pH, as encountered in the endocytic compartment, a viral protein triggers the fusion of the viral and endosomal membranes, thus allowing delivery of the viral genome into the cytoplasm. pH-sensitive liposomes have been devised which simulate viral behavior although the mechanisms are different (Connor et al., 1984; Ellens et al., 1984; Na- yar & Schroit, 1985). Dioleoylphosphatidylethanolamine

* Corresponding author: Dr. J. R. Philippot, Universit6 Mont- pellier 11, DBpartement Biologie Sant4, URA-CNRS 530 Inter- actions Membranaires, Case 107, Place E. Bataillon, 34095 Montpellier Cedex 5, France. Phone: (33)67143741. Fax: (33)- 67 144286.

t URA-CNRS 1191 GBn6tique MolBculaire. * URA-CNRS 530 Interactions Membranaires.

(DOPE) and oleic acid (OA) liposomes are stable a t neutral pH. The tendency to revert the HI1 phase occurs under conditions of acidic pH in which the OA component becomes protonated (Liu & Huang, 1989a). The lipid bi- layer of such liposomes is destabilized at low pH and the loaded material enters the cytoplasm by a still unknown mechanism. Whatever the vector, virus, or liposome, the material to be delivered enters the cell by the endocytic pathway and escapes lysosomal degradation at least partially. The efficiency of pH-sensitive liposomes has been demonstrated in vitro with the help of encapsulated drugs such as antitumor drugs (Collins et al., 1988; Con- nor & Huang, 1986) or entrapped toxin (Collins & Huang, 1987; Chu et al., 1990) or fluorescent markers of aqueous compartment (Connor & Huang, 1985) or membrane lip- ids (Duzgunes et al., 1987). Efficient gene transfers have been performed in vivo (Wang & Huang, 198713; Nayar & Schroit, 1989) and in vitro (Wang & Huang, 1987a; Wang & Huang, 1989).

These previous observations prompted us to test the efficiency of pH-sensitive liposomes for PolyIC delivery. In the present study we show that PolyIC entrapped into pH-sensitive liposomes induced the secretion of IFN from L929 cells in the absence of priming and only triggered a weak toxicity. However in the absence or in the presence of priming, PolyIC entrapped into pH-sensitive liposomes only induced a weak antiviral activity in HeLa cells.

EXPERIMENTAL PROCEDURES

Cell Lines and Viruses. Murine L929 cells (American Type Culture Collection, Rockville, MD; Ref. CCL 1) were grown in MEM medium (Gibco, Cergy Pontoise, France) supplemented with 5% (v/v) fetal bovine serum (FBS) and antibiotics. HeLa cells were obtained from G. Huez (Universitb libre de Bruxelles, Belgium); they were grown in RPMI 1640 medium enriched with glutamine and supplemented with 5% (v/v) FBS and antibiotics. The human lymphoblastic T CEM cells were cultured in RPMI 1640 supplemented with 10% FBS (v/v) and antibiotics.

Vesicular stomatitis virus (VSV) and encephalomyo- carditis virus (EMCV) were grown and titrated on L929 cells.

Antibodies. Polyclonal rabbit antibodies to murine

0 1992 American Chemical Society

High Efficiency of Poly IC pH-Sensitive Llposomes

IFN-a-j3 were obtained from Lee Biomolecular Research (San Diego, CAI. The monoclonal murine antibodies (mAbs) to human IFN-a and to human IFN-j3 were obtained from Alpha Therapeutic Corp. (Los Angeles, CAI. The murine mAbs UM21.1 (IgG,2b), a generous gift from Dr. Kraaijeveld and Dr. Snippe (State University of Utrecht, the Netherlands), are neutralizing antibodies to EMCV (Vlaspolder et al., 1989). The goat peroxidase conjugated F(ab')z antibodies to whole mouse immuno- globulin G molecules were obtained from Immunotech (Marseille, France).

Interferon and Double-Stranded RNA. Murine IFN-a-fl (specific activity 6 X lo4 units/mg), a generous gift from Dr. G. Rossi (Instituto Superiore di Sanita, Roma), was diluted to 4 X lo5 units/mL in phosphate- buffered saline (PBS: 137 mM NaC1, 2.7 mM KC1, 1.5 mM KHzP04, 8.1 mM NazHPO4, pH 7.4) containing 2 mg/mL bovine serum albumin and then aliquoted and frozen at -70 "C until use. Recombinant human interferon a2a (HuIFN-a2a) was similarly treated and stored.

A high molecular weight complex of PolyIC (Pharma- cia, Uppsala, Sweden) (2 mg/mL) was sonicated (Bran- son, Danbury, CT, 20 W, 10 X 30 s) until a mean length of 500-600 base pairs was obtained, as verified by 2% (w/v) agarose gel electrophoresis (buffer, 89 mM Tris- HC1, 89 mM borate, 1 mM EDTA (pH 7.5), 0.5 pg/mL ethidium bromide).

PolyIC was labeled by [T-~~PIATP using polynucleotide kinase, according to standard protocols, to a final specific activity of 5 X lo5 Bqlpmol.

Preparation of pH-Sensitive Liposomes. In routine experimenta 20 pmol of dioleoylphosphatidylethanolamine (DOPE), cholesterol (Chol), and oleic acid (OA) in the molar ratio 2:2:1 were dried from solvents to a thin film under a stream of nitrogen. Materials to be encapsulated (0.7-1.4 mg of PolyIC) were introduced in a high ionic strength 150 mM 4-(2-hydroxyethyl)-l-piperazineethane- sulfonic acid (HEPES), 1 mM ethylenebis(oxyethy1enen- itri1o)tetraacetic acid (EGTA) and 150 mM NaC1, pH 8.01 and lipids were hydrated for 30 min at 37 "C. A 100-pmol portion of octyl glucoside in the corresponding buffer was added and the volume of the samples was adjusted to 0.5- 0.65 mL with the buffer. After vigorous shaking the detergent was removed by dialysis against 50 mL of buffer containing 2 g of Amberlite XAD2 (Philippot et al., 1983). Liposome preparations were treated for 30 min at 37 "C with RNase A (EC 3.1.4.22) (25-100 pg/mL) to digest un- encapsulated polynucleotides. Liposomes were collected on a 5%-20% (w/v) dextran (average M , = 162 000) gradient (Sigma) at 20 "C (45 min at 40 000 rpm). The yield of PolyIC encapsulated and the diameter of lipo- somes were measured for each sample. On a regular basis 2-4 pg of PolyIC/pmol of lipid was encapsulated, and the diameters were around 450-650 nm.

The stability of the pH-sensitive liposomes was assessed as follows. pH-sensitive liposomes containing 32P-labeled PolyIC were prepared and separated from nonincorpo- rated PolyIC. The day following their preparation the liposomes were incubated in different media for 30 min and then loaded on a Biogel 5-m column (Bio-Rad) equilibrated with the buffer used for their preparation. The leakage was expressed as the ratio of the radioactivity trailing as a second peak to the total radioactivity eluted from the gel. The liposomes were incubated in the preparation buffer or in MEM without serum at 37 "C: the leakage was 23 74 and 44 % , respectively (mean of two determinations, standard deviation = f0.6) in the first experiment; in the second independent experiment 38 % and 63 5% were obtained. These experiments underscored the destabilizing effect of MEM on pH-sensitive liposomes.

Bloconlugate Chem., Voi. 3, No. 5, 1992 403

As a rule we used the liposomes the day following their preparation.

Fluorescent pH-sensitive liposomes were prepared by replacing a few percent of DOPE by (4-nitrobenz-2-oxa- 1,3-diazolyl)phosphatidylethanolanine (NBD-PE): DOPE/

Lipid Mixing Assays. Vesicle membrane fusion results in the communication between two aqueous compartments initially separated by the two fusing membranes: it involves intermixing of aqueous contents and intermixing of membrane components. The last event can be followed using a fluorescence method. The high concentration (5 5% ) of NBD-PE (excitation, 460 nm; emission, 534 nm) present on the labeled pH-sensitive liposomes self-quenches its fluorescence. The fusion between these liposomes and cells dilutes the probe, resulting in an increase of NBD- PE fluorescence (Wilschut & Hoekstra, 1986). Kinetics were carried out a t room temperature in the cell of a spec- trofluorimeter.

Nonattached cells were used in this assay: 2.5 X 107 CEM cells in 1 mL (10 mM Tris, 150 mM NaC1) at pH 8 were mixed to 3 pL of fluorescent liposomes and then the pH of the medium was shifted to pH 5.5 with 10% HC1 (v/v) (zero time). At the plateau of the reaction, 10 pL of Triton XlOO (10%) was added and the fluorescence measured. Fluorescence changes were graphically esti- mated and expressed as percent of fluorescence variation between zero time and Triton XlOO addition. In control experiments buffer was used instead of acidic solution.

Induction of IFN and of IFN-PolyIC Toxicity. IFN production was obtained by modification of published protocols (Milhaud et al., 1989). L929 cells were seeded at lo5 cells/mL per well in 24-well tissue culture dishes and exposed to 800 U/mL IFN for 8 h. This step is defined as "priming" (Stewart et al., 1971). The cell monolayers (two wells per treatment) were washed and then various concentrations of free or liposome-encapsulated polynu- cleotides were added in serum-free medium and the dishes incubated for a further 2 h a t 37 "C. Cell monolayers were washed with MEM supplemented with 5% (v/v) FBS and incubated a t 37 "C for a further 18-20 h. The supernatant fluids were transferred to wells containing lo5 L929 fresh cells, incubated for 20 h, and then challenged with lo5 IU/mL VSV. The virus was titrated by end-point dilutions 24 h later.

Cell supernatants testing the antiviral activities induced by free or encapsulated PolyIC were incubated with antibodies to murine or human IFN-a-/3 (lo00 neutralizing units) for 2 h; they were then incubated with L929 or HeLa cells and the antiviral activities were estimated by chal- lenge with EMCV and the enzyme immunoassay as indicated below. The antiviral activities were completely neutralized, indicating that IFN-a and/or IFN-/3 were secreted by the cells treated with either free or liposome encapsulated PolyIC (data not shown).

Estimation of the PolyIC Toxicity. The toxicity was estimated on the primed and PolyIC-treated cells. As soon as a supernatant was transferred, 1 mL of diluted trypsin was added onto the cell monolayers, and the cells were counted with a Coulter counter (Coultronics). The results were expressed as the ratio of the number of the surviving cells after treatment with free or encapsulated PolyIC to the number of control cells (i.e. untreated or treated with empty liposomes, respectively).

Virus Titration by End-Point Dilution. Dishes containing L929 cells and virus were frozen and thawed twice, and the VSV titers measured by end-point dilutions on L929 cells seeded in 96-well tissue culture dishes. The number of infectious units (IU/mL) was estimated by the maximum likelihood method (Milhaud et al., 1983). The

NBD-PE/Chol/OA, 1.75:0.25:2:1 (M/M).

404 Bloconjugate Chem., Vol. 3, No. 5, 1992

99% confidence limits for the number M or IU/mL, obtained with a dilution factor 10 are M X 0.3 < M < M X 3.38.

Virus Titration by Enzyme Immunoassay. Relevant cells (L929 or HeLa) in 24-well Linbro dishes were pretreated by serial dilutions of the supernatants con- taining the secreted IFN. A dose effect of murine IFN- a-8 (for L929 cells) or HuIFN-a2a (for HeLa cells) was included in the assay. After a 20-h incubation the dishes were infected and reincubated for 5-7 h. The virus yield was estimated by a direct enzyme immunoassay of EMCV according to a modification of a previously published protocol (Vlaspolder et al., 1989). The immunoassay measured virus yields which in turn measured IFN concentrations. Briefly the cells were fixed by ethanol/ acetone (1/1) and washed with PBS. mAbs UM 21.1 were added, incubated at 37 "C for 1 h, and then washed with PBS. Polyclonal antibody to whole mouse immunoglo- bulin, conjugated peroxydase, was added and incubated for 1 h at 37 "C and washed with PBS. The conjugated peroxidase was revealed with the help of 0.1 mM 2,2'- azinobis(3-ethylbenzthiazoline-6-sulfonate) (Boehringer) or 4 mM 1,2-phenylenediamine (Fluka) as chromogen in the presence of 0.003% H 2 0 2 as substrate. The colored reaction was read at 410 or 492 nm, respectively. Com- parison between the results a t 0.5 absorbance allowed an accurate titration of IFN.

Mllhaud et al.

RESULTS

Liposome Encapsulated PolyIC Resists RNase Treatment. Incubation of free PolyIC or that encapsu- lated into pH-sensitive liposomes with IFN-pretreated L929 cells induced a transferable antiviral activity to mouse cells.

To ascertain that the secreted antiviral activity could be only ascribed to the encapsulated PolyIC, a RNase treatment (50-100 pg/mL) of the liposomes was carried out as indicated in the methods section. We checked for the efficacy of this step. PolyIC was encapsulated into pH-sensitive liposomes whereas pH-sensitive empty lip- osomes were incubated with labeled PolyIC; both liposome preparations were RNase treated as indicated in the methods. The induced antiviral capabilities of these liposomes were tested. PolyIC encapsulated into lipo- somes induced an antiviral activity while empty liposomes whose bound PolyIC had been chopped up by RNase were devoid of any activity.

Independent experiments (n = 2) showed that pH- sensitive liposomes, which had been pretreated with RNase as indicated above in the methods, were destroyed by a second harsh RNase treatment (50-100 pg/mL). Table I shows that the activity of the encapsulated PolyIC was still shielded from a second weak RNase treatment (2 pg/ mL). These experiments emphasized that pH-sensitive liposomes provided PolyIC with protection.

PolyIC Encapsulated into pH-Sensitive Liposomes Induces IFN and a Weak Toxicity against Nonprimed L929 Cells. It has been reported that DEAE dextran is instrumental in enhancing the penetration of PolyIC into L929 cells (De Clercq, 1981) and triggers the induction of IFN in the absence of priming. Similarly pH-sensitive liposomes made it possible for the A chain of diphtheria toxin to penetrate and kill diphtheria-toxin-resistant L929 cells (Collins & Huang, 1987). We examined whether pH- sensitive liposomes could induce the production of IFN in the absence of priming.

As a preliminary experiment we titrated by ELISA the antiviral activity induced by free PolyIC from nonprimed and IFN-primed cells. As little as 0.02 pg/mL of free

Table I. RNase Resistance of PolyIC-Encapsulated Preparationse

liposome preparationb untreated RNase treated free PolyIC 100 0

lip. pHS-0 0 0

lip. pHNS-0 0 0

lip. pHS-IC 99.4 98.5

lip. pHNS-IC 97.6 93.8

Free and encapsulated PolyIC (2 pg/mL) were incubated in MEM in the absence or in the presence of RNase A (2 gg/mL) at 37 "C. This was a second RNase treatment in addition to the initial one performed during liposome preparation. After 1-h incubation mixtures were checked for antiviral activity: virus growth was titrated by end- point dilution. The control viral growth was 1.3 X lo7 IU/mL and in the presence of PolyIC was 1.3 X lo4 IU/mL. The results are expressed as the percents of viral reductions (1.3 X lo7 - Yi1.3 X l o 7 - 1.3 X lo4 IUlmL). The influence of RNase on virus growth was corrected accordingly. lip. pHS and lip. pHNS stand for pH-sensitive liposome and pH-insensitive liposome, respectively. -IC indicates that PolyIC was encapsulated, -0 indicates that the liposomes were empty.

Table 11. Induction of IFN and Toxicity to L929 Cells of Free PolyIC and PolyIC Encapsulated into pH-Sensitive Liposomese

values at PolyIC concn, pglmL

free encapsulated cell treatment 0.2 2 0.2 2

untreated cells <2 <2 12 512 (0.98 f 0.04) (0.97 f 0.04) (0.92 f 0.04) (0.77 f 0.03)

(0.40 f 0.02) (0.02 f 0.01) (0.77 f 0.03) (0.18 f 0.03)

"Results are expressed as units of IFN/mL. The toxicity is expressed as the fraction of surviving cells as detailed in Experimental Procedures and is in parentheses.

primed cells 68 137 152 448

PolyIC produced a detectable antiviral activity on primed cells and the secreted antiviral activity reached the equivalent of 185 units of IFN at 1 pg/mL. On the other hand 10 pg/mL of PolyIC were required to obtain a detectable antiviral activity with unprimed cells and no more than the equivalent of 40 units of IFN/mL could be harvested after a treatment with 50-400 pg/mL of free PolyIC. These results confirmed that IFN production by unprimed L929 cells required a 100-fold higher concen- tration of PolyIC than primed cells. Moreover the induction is weak and cannot be improved by a further increase of the dose of PolyIC (data not shown).

Comparable experiments ( n = 3) were carried out with PolyIC encapsulated into pH-sensitive liposomes. The pH-sensitive liposomes induced an antiviral activity with primed and unprimed L929 cells (Table 11). Further experiments (n = 2) showed that pH-insensitive liposomes failed to trigger any IFN production in nonprimed cells whereas they induced an antiviral activity in primed cells (data not shown).

Table I1 indicates in parentheses the relative toxicity of PolyIC loaded into pH-sensitive liposomes which was induced to nontreated or in IFN-pretreated L929 cells. At 2 pg/mL PolyIC the toxicity to primed cells was 3-4-fold higher than on nonprimed cells. pH-insensitive liposomes did not induce any toxicity to nonpretreated L929 cells.

Comparison between pH-Sensitive and Insensitive Liposomes as Vectors for IFN-PolyIC Toxicity and IFN Induction on Primed L929 Cells. We compared the IFN-inducing activity of free PolyIC to that of PolyIC encapsulated into pH-sensitive or pH-insensitive lipo- somes. Figure 1 indicates that free PolyIC and PolyIC loaded into pH-sensitive or pH-insensitive liposomes essentially displayed the same IFN-inducing activities.

High Efficiency of Poly IC pH-Sensitive Liposomes Bioconjugate Chem., Vol. 3, No. 5, 1992 405

Table IV. IFN Production by Free and Encapsulated PolyIC in Nontreated and Primed HeLa Cells

IFN production at 2 Ng/mL PolyIC

91 I t I

3: 2 ,002 .02 . 2 2

Equivalent Poly IC (Fgiml)

Figure 1. IFN activity induced by free and encapsulated PolyIC. Increasing doses of free PolyIC and PolyIC encapsulated into pH-sensitive and pH-insensitive liposomes were checked for antiviral activity. Results were plotted as log (IU/mL) (standard error = h0.205): free PolyIC (O) , pH-sensitive liposomes containing PolyIC (@), pH-insensitive liposomes containing PolyIC (A), empty pH-sensitive liposomes (O), empty pH- insensitive liposomes (A).

Table 111. IFN-PolyIC Toxicity Induced by Free and Encapsulated PolyIC.

toxicity at PolyIC concn, pg/mL liposome preparation 0.022 0.20 2.00 lip. pHS-IC 0.85 f 0.03 0.59 f 0.03 0.20 f 0.01

(Diameter 640 nm) lip. pHNS-IC 0.98 f 0.04 0.89 f 0.04 0.59 f 0.03

(Diameter 492 nm) free PolyIC 0.99 f 0.04 0.88 f 0.04 0.50 f 0.02

IFN pretreated L929 cells were incubated for 2 h with free or encapsulated PolyIC or empty liposomes. The cultures were washed with fresh medium, incubated for 18 h, and counted with a Coulter counter. Results are expressed as fractions of surviving cells as detailed in the Experimental Procedures. The error values were computed to allow for the errors on treated and untreated wells at 95 % confidence limits. For abbreviations see legend of Table I.

The empty liposomes did not induce any transferable antiviral activity.

The IFN-PolyIC toxicity induced by free and encap- sulated PolyIC is presented in Table 111. Two independent experiments confirmed the higher toxicity of the PolyIC delivered by pH-sensitive liposomes as compared to pH- insensitive liposomes, and to free PolyIC. We had observed that small-sized liposomes loaded with PolyIC were more toxic in these experimental conditions independent of their lipid composition (data not shown). The higher toxicity of the pH-sensitive liposomes was not due to smaller size. Indeed the diameters of the pH-sensitive and the pH- insensitive liposomes wee respectively 640 and 492 nm in that particular experiment. The empty liposomes did not display any toxicity. These results suggested a more efficient delivery of PolyIC from the pH-sensitive lipo- somes.

IFN Induction by Free and Encapsulated PolyIC into Nontreated and Primed HeLa Cells. HeLa cells are refractory to IFN induction by PolyIC unless a complex regimen is carried out including IFN priming and super- induction with PolyIC, cycloheximide, and actinomycin D (De Clercq, 1981). So we addressed the same questions for HeLa cells as we did for L929 cells previously. The HeLa strain we used was weakly induced (4 units IFN) by free PolyIC at 10 pg/mL after priming with HuIFN-a2a. A better induction was obtained when the primed cells were treated with PolyIC and cycloheximide; a weak toxicity accompanied the IFN production (data not shown).

cell treatment free PolyIC lip. pHS-IC lip. pHNS-IC control 0 >0.5 0 priming 0 1.6 0 Antiviral activity were tested by ELISA; results are expressed

as units of IFN/mL. For abbreviations see the legend of Table I.

I

p H 5 5 m pH 8.0

- I , , ,

0 20 43 to &I 1 m 1 a 1 4 0

Time (sec) Figure 2. Mixing between liposome and cell membrane lipids. 2.5 X 107 CEM cells in 1 mL of buffer, pH 8, were set in the cell of a spectrofluorimeter under continuous magnetic stirring at room temperature. Then 3 pL of fluorescent liposomes were added and the suspension was allowed to equilibrate. At zero time the pH of the medium was shifted to pH 5.5 with 0.1 N HCl (0% fluorescence). The fluorescence emission of NBD-PE was monitored all along the kinetics for 200 s, then 10 NL of 10% Triton XlOO was added to determine 100% fluorescence emission. In control experiments pH of the cell suspension stayed un- changed at pH 8.0.

Comparable experiments (n = 2) were performed with encapsulated PolyIC (2 pg/mL) on nonprimed and primed HeLa cells. A weak antiviral activity was only observed with pH-sensitive liposomes on nonprimed and primed cells: in both cases the antiviral activity was reproducibly weak but clear (around 1 unit IFN/mL) (Table IV). The immunoassay titrated less than 0.5 units of HuIFN/mL. In the same experiments no antiviral activity was detected for PolyIC loaded into pH-insensitive liposomes nor for empty liposomes.

Lipid Mixing between Liposomes and Cell Mem- branes. Liposomes do not spontaneously fuse with other liposomes or cells unless conditions are established to promote this event. It is a feature of pH-sensitive lipo- somes to undergo lipid mixing when they are exposed to an acidic environment (Collins et al., 1989). In order to evaluate the fusiogenic ability of the pH-sensitive lipo- somes used throughout these studies we kinetically monitored the intensity of the fluorescence of NBD-PE embedded on the liposome membrane upon mixing of lip- ids between liposome and cell membranes. In this assay (Figure 2) fusion occurred at the plasma membrane level as the acidification was directly performed in the incu- bation medium. A shift to acidic medium induced an increase of fluorescence in suspensions containing fusi- ogenic liposomes mixed with cells. Data from Figure 2 are the average of two assays. The phenomenon was completed in 2-3 min. Under control conditions fluo- rescent liposomes did not provoke any significative flu- orescent change.

Electron microscopy and photon correlation spectros- copy indicated that the mean diameter of the liposomes was several-fold enlarged a few minutes after medium acidification (data not shown). These observations were consistent with a fusion process induced by the pH- sensitive liposomes.

400 Bloconjugate Chem., Vol. 3, No. 5, 1992

DISCUSSION

Characteristics of the pH-Sensitive Liposomes. Induction of IFN in the presence or in the absence of priming is a good model to demonstrate the efficiency of pH-sensitive liposomes in spite of their instability. The resistance of DOPE/OA liposomes has been improved by cholesterol so we adopted the composition DOPE/OA/ CHOL, 4:2:4 (M/M), in accordance with published results (Liu & Huang, 1989b) without further investigation. However we have indicated in the methods section that pH-sensitive liposomes are leaky in the presence of MEM and cannot withstand a second harsh treatment with RNase A. The influence of the entrapped PolyIC on the stability of the liposomes cannot be excluded.

The liposome preparations were not extruded. Under standard conditions the mean diameter of the liposomes did not vary significantly unless the buffer composition was modified. L929 cells exhibit a high phagocytic ability so that large variations of liposome characteristics are required to obtain different biological responses.

Fusion of pH-Sensitive Liposomes. pH-sensitive lip- osomes are efficient vectors to introduce molecules into animal cells (Connor & Huang, 1985; Collins et al., 1988; Connor & Huang, 1986; Collins & Huang, 1987; Chu et al., 1990; Wang& Huang, 1987b; Nayar & Schroit, 1989; Wang & Huang, 1989). Among the possible mechanisms our results favor the fusion process; indeed the lipid exper- iments were performed with liposomes exhibiting larger diameters than CEM cell-coated pits (Carriere et al., 1989). First, the exposure of pH-sensitive liposomes to low pH at constant osmotic pressure led to a diameter increase. It was verified by electron microscopy that pH-sensitive liposomes did not aggregate upon medium acidification and remain paucilamellar. Moreover, mixing cellular membrane lipids and NBD-PE-associated liposomes in- duced fluorescence dequenching at low pH (Figure 2); such dequenching was observed with pH-sensitive liposomes only. Other indirect evidence using nonpermeant fluo- rescent dyes such as calcein have been published (Connor & Huang, 1985). We also observed a leakage of calcein from pH-sensitive liposomes by lowering the pH of the medium. These results reinforce but do not prove conclusively that fusion operates a t the plasmic membrane level a t low pH. However our results are not a t variance with a fusion process a t the endosome membrane following endocytosis, since L929 cells are able to internalize large diameter liposomes (Machy et al., 1987). Indeed, plasmatic or intracellular membrane-liposome fusion might occur through different mechanisms. Other mechanisms have been suggested to explain the delivery into the endosome and the escape from this compartment (Ellens et al., 1984).

Cytoplasmic Delivery. Induction of IFN along this work was carried out with encapsulated PolyIC. The lip- osomes were efficiently treated with RNase, which indi- cated that an important part of the liposome population shielded PolyIC; however, because of the leakage induced by MEM, a piggyback cell delivery cannot be ruled out. Indeed cell transfections with DNA bound at the external surface of pH-sensitive liposomes have already been reported (Wang & Huang, 1987b).

That at least part of the load of the pH-sensitive lipo- some is directly delivered into the cytoplasm has been demonstrated by means of fluorescent probes or drugs whose site of action are cytoplasmic as diphtheria toxin (Connor & Huang, 1985; Collins et al., 1988; Connor & Huang, 1986; Collins & Huang, 1987; Chu et al., 1990; Nayar & Schroit, 1989). The killing of diphtheria-toxin- resistant L cells by pH-sensitive liposomes loaded with diphtheria toxin A chain exemplifies such a cytoplasmic

Milhaud et el.

delivery whereas comparable pH-insensitive liposomes failed to generate any toxicity (Collins & Huang, 1987).

Whether the improved biological activities of PolyIC encapsulated into pH-sensitive liposome stems from an appropriate or an increased cytoplasmic delivery cannot be formally demonstrated. Indeed neither IFN induction nor cytotoxicity triggered by PolyIC on IFN-treated cells is fully understood. Arguments which favor cytoplasmic targets for PolyIC have however been proposed. For example PolyIC inhibits translation by causing the in- activation of eIF-2 (eukaryotic initiation factor 2) and binds to this protein tightly. The PolyIC/eIF2 complex intro- duced into cells reduces the toxicity and increases IFN production, suggesting that eIF-2 is involved (Harary et al., 1990). The IFN-PolyIC toxicity would stem from the removal of eIF-2 from the translation machinery. This hypothesis does not explain the lytic event but implicates a cytoplasmic delivery.

Induction of IFN in the Absence of Priming. The present study shows that with L929 cells PolyIC loaded into pH-sensitive liposomes triggered a good induction of IFN (around 500 units of IFN/mL for 2 pg/mL PolyIC) in the absence of priming (Table 11) while large doses of free PolyIC (up to 200 pg/mL) were unable to induce more than 40 units IFN/mL. As for HeLa cells we show that PolyIC loaded into pH-sensitive liposomes succeeded in inducing a reproducible but weak antiviral activity a t doses where free PolyIC induced no IFN at all (Table IV). In both cases the efficiency of the PolyIC entrapped into pH-sensitive liposomes thus appeared several orders of magnitude better than that of free PolyIC or PolyIC loaded into pH-insensitive liposomes.

The induction obtained with L929 cells was far better than with HeLa cells; the difference might be explained both by the high endocytic capacity of L929 cells, which favors liposome internalization, and by cell-specific factors involved in the induction process (De Maeyer & De Maeyer, 1988). Using more appropriate pH-sensitive lipids, re- ducing liposome size for fitting with endocytic vesicles, and targeting with efficient antibodies should improve the overall results.

Whether the first role of priming is to induce the eIF-2 kinase which PolyIC will bind to (Harary et al., 1990) or fragilize the cells, rendering the access to cytoplasm easier (Milhaud et al., 19871, requires deeper investigations. Priming exerts important effects on IFN induction al- though its mechanism remains unclear, likely dependent on cell lines and IFN genes (Dron et al., 1990); however it is not compulsory to induce IFN. With L929 cells priming can be replaced by DEAE dextran which is reported to enhance PolyIC and polynucleotide intra- cellular penetration (De Clercq, 1981; Sompayrac & Danna, 1981). Likewise, microinjection of PolyIC with micropi- pets into HeLa cells has been reported to bring about IFN induction in the absence of any priming (Silhol et al., 1986).

We previously have shown that encapsulation of PolyIC into antibody-targeted liposomes protected the drug from nucleolytic attack and changed drastically its pharma- cological characteristics. Here we show that encapsulation of PolyIC into pH-sensitive liposomes increases by several orders of magnitude its inducing capabilities. Such improvements might be beneficial if PolyIC or Ampligen, its analog, should be used as an antitumor (Chapekar & Glazer, 1985; Hubbell, 1986) or as an anti-HIVagent (Mon- tefiori & Mitchell, 1987; Carter et al., 1987).

ACKNOWLEDGMENT We thank B. Lebleu for advice and reading the manu-

script. This work was supported by grants from Centre National de la Recherche Scientifique, Agence Nationale

High Efficiency of Poly IC pH-Senskive Liposomes

de Recherches sur le SIDA, and Association pour le Developpement de la Recherche sur le Cancer to B. Lebleu (URA CNRS 1191) and independently to J. R. Philippot (URA CNRS 530).

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Registry No. PolyIC, 24939-03-5.

2985-2989.

U.S.A. 84, 7851-7855.

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