Delivery of an anti-HIV-1 ribozyme into HIV-infected cells via cationic liposomes

Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA–RNA ribozyme targeted to the HIV-1 5 X LTR was investigated, using THP-1, THP-1 r HIV-1 or HeLa r LAV cells. Different fluorescence patterns were IIIB (cid:14) . observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE 1:1 complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1 r HIV-1 IIIB cells were more susceptible to killing by liposome–ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (cid:14) . (cid:14) . a 4-h treatment complexes of 5, 10 or 15 m M Lipofectin or DOTAP:DOPE 1:1 and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1 r HIV-1 cells in spite of the intracellular accumulation of the IIIB (cid:14) . ribozyme. A 24-h exposure of THP-1 r HIV-1 cells to 5 m M Lipofectin or DOTAP:DOPE 1:1 complexed with either IIIB the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 m M after a 24-h treatment. A 4-h treatment of HeLa r LAV cells with Lipofectin at 5, 10 or 15 m M was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4 q cells immediately after infection with HIV-1 at the same lipid concentrations and IIIB lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications. q 1998 Elsevier


Ž .
Ribozymes Rz are RNA molecules capable of catalytically cleaving specific phosphodiester bonds w x in complementary RNA molecules 1-4 .When delivered intracellularly, they can interfere with both pre-integration and post-integration events of the HIV replication cycle, by cleaving incoming viral RNA w x and transcribed mRNAs 5 .Delivery of Rz to cells Ž .has been attempted by i endogenous expression of Ž .the Rz-encoding gene or ii exogenous delivery of Ž .chemically-synthesized pre-formed Rz targeted to w x highly conserved regions of the virus 6 .
w x Chang et al. 7 have demonstrated that an anti-HIV-1 gag Rz can be expressed under the control of the constitutive human b-actin promoter in HeLa CD4 q cells.The stable expression of this anti-gag Rz in HeLa CD4 q cells reduces gag transcripts and w x p24 levels upon infection with HIV-1 1 .Since then, many of the genetic elements of the retroviral genome have been targeted including functional proteins, leader sequences and regulatory protein sequences w x 2,8 .A hairpin Rz designed to cleave HIV-1 RNA in the 5 X leader sequence suppresses virus expression in w x HeLa cells cotransfected with proviral DNAs 9,10 .Human T-cell lines, primary T cells and CD34 q hematopoietic stemrprogenitor cells transduced with retroviral vectors containing Rz were shown to be resistant to challenge with diverse strains of HIV-1, w x including uncloned clinical isolates 11-16 .
A number of problems need to be addressed before pre-formed Rz can be used successfully for therapy: Ž .
i stabilization of the Rz against serum and cellular nucleases without compromising its catalytic activity; Ž .Ž .ii efficient delivery to the target cells; iii intra-Ž .cellular localization of an active Rz; iv co-localization of the Rz with its mRNA target inside cells.In contrast to endogenous expression, exogenous deliv-Ž .ery permits the use of chemically modified Rz MRz which are more resistant to degradation by nucleases, w x while maintaining cleavage capability 17,18 .Chimeric DNA-RNA hammerhead Rz, with DNA in helices I and III, have an increased resistance to nucleases and a six-fold greater catalytic activity than w x an analogous all-RNA Rz 19 .
Several cationic liposome formulations have been w x developed to facilitate delivery of DNA 20-23 , w x mRNA 24 and antisense oligonucleotides into eu-w x karyotic cells 25-30 .In the present study we evaluated the intracellular delivery and anti-HIV effect of a fluorescein-labeled chimeric DNA-RNA Rz targeted to the HIV-1 5 X LTR, using four cationic liposomal preparations.Lipofectamine, Lipofectin, wŽ and liposomes composed of either DMRIE:DOPE a Ž .1:1 wrw mixture of 1,2-dimyristyloxypropyl-3-di-Ž .methyl-hydroxyethylammonium bromide DMRIE .Ž .xŽ and DOPE : dioleoylphosphatidylethanolamine 1:1, .w Ž Ž .wrw or DOTAP:DOPE N-1-2,3-dioleoyloxy pro-.xŽ pyl-N, N, N-trimethylammonium propane 1:1, .wrw , were complexed with the Rz and added to cultures of differentiated monocytic THP-1 cells and chronically infected THP-1rHIV-1 cells, as well IIIB as chronically and de novo infected HeLa CD4 q cells.Differentiated THP-1 and THP-1rHIV-1 IIIB cells were used in our study because they represent a convenient model of monocyte-derived macrophages w x w x 31 and chronically infected macrophages 32,33 , respectively.Because HeLa cells are often used as a model system for cationic liposome-mediated DNA delivery, chronically infected HeLarLAV cells and de novo infected HeLa CD4 q cells were also included in our study.Some of our results have been w x presented earlier in preliminary form 34,35 .

Ž
. linkages at the 3 terminal mol.wt.12,519.2, was targeted to the HIV-1 5 X LTR.The catalytic activity of the Rz was first tested in a cell-free system.A control Rz with a scrambled sequence in the binding Ž .arms was also designed and synthesized MRz .The sequences of these Rz are as follows: Rz: 5 X -ACACAACAcugaugaGTCCGTGAG -GACgaaaCGGGC)A)C-3 X MRz: 5 X -CAAACAACcugaugaGTCCGTGAG-GACgaaaACCGG)G)C-3 X where the ')' indicates a phosphorothioate internucleotide linkage, capital letters represent deoxyribonucleotides and lowercase letters represent ribonucleotides.
The mixed oligodeoxyribo-and ribonucleotides Ž were synthesized on an automated synthesizer Ap-.plied Biosystems 394 RNArDNA as described prew x viously 19 .

Cells and Õirus
THP-1 cells were obtained from the American Ž .For all experiments, the indicated amounts of lipid and Rz were complexed for 30 min at room temperature in RPMI or DME-HG medium without serum or antibiotics.Meanwhile the cells were washed twice with 1 ml medium, and 0.4 ml of medium was placed on each well.The lipid-Rz mixture was then added gently in a volume of 0.1 ml per well.The mixture remained on the cells for 4 h or 24 h at 378C in a 5% CO incubator.Cells treated with either medium or ( ) K. Konopka et al.r Biochimica et Biophysica Acta 13721998 55-68 58 Rz alone served as controls.Following treatment, cells were washed with PBS and viewed by phase contrast and fluorescence microscopy, using a Nikon Ž Diaphot microscope Nikon Instrument Group; . Melville, NY .Cell morphology was evaluated by inverted phase contrast microscopy at 250 = or 400 = magnification.Photographs of fluorescence and phase-contrast fields were made on Fuji color print Ž .film at ISO ASA 1600 or on Kodak black and white print film at ASA 400.The treated cells were fed Ž every 2-3 days with fresh medium RPMIr10 for THP-1 and HeLa CD4 q cells, or DMEr10 for .HeLarLAV cells and incubated for the indicated periods of time before the Alamar Blue assays and monitoring of the p24 level in supernatants.p24 Ž values represent the amount of virus produced in .ngrml between the time of medium change and the time of p24 determination.This time period is indicated in the figure legends.Results were compared to medium-treated controls.Statistical significance was evaluated by the unpaired Student's t-test, using Ž .StatView software BrainPower, Calabasas, CA .p values of F 0.05 were taken to indicate a significant difference.

HIV infection of HeLa CD4 q cells
The virus and infected cells were handled in a BL-3 facility.HeLa CD4 q cells were plated in 12well plates at 2 = 10 5 cellsrwell, and on the next day, at ; 50% confluency, they were exposed to HIV-1 for 2 h at 378C, at 5 = 10 3 TCID per IIIB 50 well.Following infection, the cells were washed to remove unbound virus and were treated with liposome-Rz complexes or medium alone.Control cells were treated similarly but not exposed to virus.The infection was monitored by determining viral p24 in culture supernatants by an antigen capture ELISA w x Ž assay 42 , using a Molecular Devices Menlo Park, .CA V microplate reader. max The supernatant of chronically infected H9rHTLV-IIIB cells was used as a source of the HIV-1 strain, HIV-1 .The culture supernatants IIIB were harvested at times of peak p24 production and stored at y808C.The p24 concentration of the stock was 1.4 mgrml as determined by ELISA.The tissue Ž .culture infectious dose, 50% endpoint TCID , was

Cell Õiability assay
The number of viable cells used for experiments was determined by Trypan Blue exclusion.Cell viability after treatment with free or liposome-complexed Rz was quantified by a modified Alamar Blue w x Ž .assay 44 .Briefly, 1.0 ml of 10% vrv Alamar Blue dye in the appropriate medium was added to each Ž well.After incubation for various times described .under figure legends at 378C, 200 ml of the supernatant was collected from each well and transferred to 96-well plates.The absorbance at 570 nm and 600 nm was measured with a microplate reader.Cell Ž viability as a percentage of mock-treated control .Ž cells was calculated according to the formula, A 570 .Ž .y A of test cells = 100r A y A of control 600 570 600 cells.After removal of the Alamar Bluermedium mixture, fresh growth medium was added, and cells were returned to the incubator.Thus, the Alamar Blue assay allows determination of viability over the culture period without the detachment of adherent cells.A good correlation was obtained between the w x Alamar Blue assay and Trypan Blue staining 39 .

DeliÕery of liposome-Rz complexes to uninfected, differentiated THP-1 cells
Differentiated THP-1 cells were exposed to the liposome-Rz complexes for 4 or 24 h in RPMI medium with 8% FBS.The cells were exposed to Lipofectamine at 3 or 8 mM, and to Lipofectin and DMRIE at 15 or 40 mM, complexed with Rz, at a Ž .lipid:Rz wrw ratio of 1.3:1.Under these condi-Ž .tions, the charge ratio qry for Lipofectamine was 1.7, while for Lipofectin and DMRIE this ratio was about 0.3.Thus, a net positive charge was not absolutely required for the complex to be taken up.Different fluorescence patterns were observed with the three liposomal preparations.With Lipofectamine, intense cell-associated fluorescence was found.Treatment with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE, an Ž intense but sporadic fluorescence was observed Figs.

Ž
. mgrml 0.96 to 3.2 mM , showed no significant Ž .cellular accumulation data not shown .The cells were further incubated and fed every 2-3 days with RPMIr10 medium before cell viability was quantified on days 12 or 13 post-treatment.The only cytotoxic treatment was that with 8 mM Lipofectamine Ž .which caused disintegration of the cells Fig. 3 .

Ž
. charge ratio qry for the 8:1 complex was 1.8 while for the 4:1 complex this ratio was 0.9.Following a 4 or 24-h treatment in RPMI medium with 8% FBS, micrographs were taken.After a 4-h treatment, p24 production was determined on days 3, 5, 7, 9 and 14, and cell viability was measured on day 14.After a 24-h treatment, p24 production was determined on days 2, 4 and 6, and cell viability was measured on day 6.A concentration-dependent, cell-associated dif-fuse fluorescence was observed after a 4-h treatment.However, neither Lipofectin-nor DOTAP-Rz complexes reduced cell viability or affected p24 produc-Ž .tion after a 4-h treatment data not shown .
Ž The p24 levels were reduced by 25.4% 0.005 -p F .Ž .0.01 and 19.1% 0.025 -p F 0.05 on day 4 and 6, respectively.Thus, on day 2 virus production was reduced to a greater extent by the liposome-complexed Rz than by a three to six-fold higher concentration of free Rz.This concentration of free Rz corresponded to the Rz added with lipid:Rz complex at 15 mM lipid at the ratio 4:1.Therefore, liposomemediated delivery of the Rz enhanced its anti-HIV effect, as determined by reduction of p24 levels on day 2.However, treatment with the Lipofectin:Rz Ž .
Ž .8:1 and DOTAP:Rz 8:1 and 4:1 complexes could not prevent virus production from increasing over time.On days 4 and 6, the level of p24 antigen produced within the previous 48-h period was similar in the treated and control wells.Under the same conditions, both liposomal preparations at 10 and 15 mM were highly cytotoxic, causing substantial disin-Ž .tegration of the cells data not shown and a decrease Ž . in p24 levels Fig. 6 .
To determine whether the modest inhibitory effect observed at 5 mM lipid concentration was sequencespecific, differentiated THP-1rHIV-1 cells were IIIB exposed to 5 mM Lipofectin or DOTAP complexed Ž with the functional Rz, or with control MRz Table .1 .When cells were treated for 24-h with free Rz at Ž .5.5 mgrml 0.44 mM , the p24 level was reduced by Ž .30.7% compared to untreated controls p F 0.005 .Under the same conditions MRz was not inhibitory.However, both Rz and MRz were equally inhibitory when they were complexed with cationic liposomes.For example, Lipofectin complexed with Rz or MRz at a ratio of 8:1 reduced the p24 levels by 30% Ž .p F 0.005 .Under the same conditions DOTAP complexed with Rz or MRz reduced the p24 levels Ž .Ž .by 34.5% p F 0.0005 or 28.5% p F 0.005 , respectively.
Ž .Interestingly, the charge ratios qry for the 8:1 and 4:1 complexes are 1.8 and 0.9, respectively.Thus, it appears that the functional delivery of Rz in the 4:1 complex is not dependent on the presence of a net positive charge on the complex.However, it is  .Ž . of 8:1 or 4:1 0.93 and 1.9 mg Rzrml 0.074 and 0.15 mM , respectively , or the Rz alone at 5.5 mgrml 0.44 mM , for 24 h.The p24 production within the previous 48-h period was measured on day 2. Data represent the mean " standard deviation of p24 determinations Ž . in duplicate, in supernatants of duplicate wells n s 4 , except in controls which were measured in three wells.b Untreated controls.
possible that positively charged regions of the complexes mediate their attachment to the cells.

DeliÕery of liposome-Rz complexes to chronically infected HeLa r LAV cells
Chronically infected HeLarLAV cells were exposed to Lipofectin at 5, 10 and 15 mM, complexed Ž .with Rz, at a lipid:Rz wrw ratio of 8:1 or 4:1 in DME medium with 8% FBS.Micrographs were taken following a 4-h treatment at 378C.The cells were further incubated and fed every day with fresh DMEr10 medium; p24 production was measured on days 1, 2 and 3.Although a concentration-dependent, cell-associated diffuse fluorescence was observed, the treatment did not affect p24 production, nor did it Ž .cause cytotoxicity data not shown .

DeliÕery of liposome-Rz complexes to de noÕoinfected HeLa CD4 q cells
One explanation for the lack of inhibition of p24 production in HeLarLAV cells by liposome-Rz complexes is that the efficiency of delivery was too low to affect significantly the level of p24 antigen in culture supernatants of cells producing high quantities of virus.Therefore, we next examined if p24 production could be reduced by Lipofectin-Rz complexes in de novo infected HeLa CD4 q cells, producing much lower levels of virus.
HeLa CD4 q cells were infected with HIV-1 at IIIB 5 = 10 3 TCID per well, as described in Section 2.

50
Immediately after infection the cells were exposed to Lipofectin at 5, 10 and 15 mM, complexed with Rz, Ž .at a lipid:Rz wrw ratio of 4:1 for 4 h in RPMI medium with 8% FBS.On 1 and 2 days post-treatment, the culture medium was replaced with fresh medium, and 3 days later the culture supernatants were analyzed for viral p24.Under these conditions treatment of de novo infected HeLa CD4 q cells with the Lipofectin-Rz complexes was cytotoxic while the Ž .Rz alone was not toxic Fig. 7 .A similar cytotoxic effect was observed when uninfected HeLa CD4 q Fig. 7. Effect of LF complexed with Rz on p24 production in de novo infected HeLa CD4 q cells.The cells were exposed to the Ž .LF-Rz complexes at a lipid:Rz ratio wrw of 4:1, or to the Rz Ž .Ž alone at 5.5 mgrml 0.44 mM , for 4 h see Section 2 for .details .The p24 antigen production within the previous 72-h period was measured on day 5 and was expressed as percent of Ž .the control 100%s 0.56"0.11ng p24rml .Cell viability was Ž measured on day 5 post-infection incubation with Alamar Blue .for 90 min at 378C and was expressed as percent of the control.Data represent the mean"standard deviation obtained from duplicate wells or quadruplicate control wells.( ) K. Konopka et al.r Biochimica et Biophysica Acta 13721998 55-68 65 cells were incubated with the Lipofectin-Rz com-Ž .plexes data not shown .

Ž .
Our results demonstrate that i treatment with liposome-Rz complexes results in cell-associated flu-Ž .orescence in a concentration-dependent manner; ii different cellular fluorescence patterns are observed Ž with the four cationic liposomal preparations Lipo-.Ž .fectamine, Lipofectin, DMRIE and DOTAP ; iii treatment of THP-1rHIV-1 cells with both func-IIIB tionally active or MRz complexed with 5 mM Lipofectin or DOTAP for a 24-h period causes a reduction Ž . in HIV production; iv differentiated chronically infected THP-1rHIV-1 cells are more susceptible to IIIB killing by liposome-Rz complexes than uninfected Ž .THP-1 cells; v CD4-expressing HeLa cells are more susceptible to killing by liposome-Rz complexes than chronically infected HeLarLAV cells.
Compared to the wide variety of methods used to deliver DNA, progress in introducing RNA molecules into cells has been slow, probably because of the rapid degradation of RNA by both exo-and endow x ribonucleases 24 .Only a few studies have reported results obtained with exogenous delivery of Rz.In one study polybrene-treated H9 cells were incubated with 1 or 100 mM anti-HIV-1 gag Rz and subsew x quently infected with HIV-1 45 .The formation of syncytia was assessed 7 days after infection.The Rz inhibited the formation of syncytia by 9% and 67%, at 1 and 100 mM, respectively, when compared to untreated controls.These results suggest that the exogenous administration of anti-HIV-1 Rz may be effective in blocking viral infection in H9 cells.However, the pre-formed Rz RNAs were short-lived, and 5 h after addition no intact Rz RNAs could be detected inside the cells.In a subsequent study, the Ž .anti-HIV-1 gag Rz 142 nucleotides in length was encapsulated in liposomes composed of 50 mol% dipalmitoylphosphatidylglycerol.This procedure enhanced the uptake and protected the RNA molecules w x from degradation over a 48-h period in H9 cells 2 .Similarly, encapsulation of an all-RNA Rz in liposomes containing dimyristoylphosphatidylglycerol Ž .DMPG , phosphatidylethanolamine and cholesterol, or cardiolipin, phosphatidylcholine and cholesterol increased its resistance to serum ribonucleases for at least 24 h and enhanced the accumulation of Rz in HepG2 cells to up to 1.5% of the added dose after 24 w x h 30 .
Cationic liposomes as an exogenous delivery system for Rz have likewise been described in only a few studies.In one study chimeric DNA-RNA Rz complexed with Lipofectin were shown to be more stable in H9 cells over a 48-h period than their w x all-RNA counterparts 19 .Lipofectin was also used to deliver hammerhead Rz targeted to human urokinase receptor mRNA, into human osteosarcoma cells w x 46 .While free Rz were degraded immediately in medium, Rz complexed with Lipofectin were stable for up to 22 h and were efficiently transported into the cytoplasm.Another cationic liposome formula-Ž .tion, Transfectam lipopolyamine , was used to deliver a chimeric hammerhead Rz directed against Ž .bcr-abl mRNA, to a cell line EM-2 derived from a w x patient with chronic myelogenous leukemia 47 .The Rz decreased the level of bcr-abl mRNA and inhibited expression of the bcr-abl gene product, as well as cell growth.A multi-unit Rz that targets bcr-abl mRNA was delivered into murine myoloblasts trans-Ž .formed with the bcr-abl gene 32D cells , using w x either Lipofectin or folic acid-polylysine 48 .Both delivery systems protected Rz from degradation in serum-free-medium up to 24 h and enhanced Rz uptake; however, the folate receptor-mediated uptake was up to 10-fold more efficient than that mediated by Lipofectin.Another cationic lipid, DOTAP, was used to deliver hammerhead Rz targeted to the MDR-1 mRNA, into human pleural mesothelioma cells w x 49 .A 42-mer chimeric DNA-RNA hammerhead Rz targeted to a leukocyte-type 12-lipoxygenase mRNA, was delivered into porcine aortic vascular smooth muscle cells after complexation with Transfectam w x 50 .The Rz almost completely inhibited specific mRNA expression at 4 mM, but appeared to be ineffective at 0.5 mM.We should note that the use of Rz concentrations in the range 0.5-4 mM in our experiments would have been toxic to the cells.
Our results indicate that cationic liposome-Rz complexes are readily taken up by differentiated THP-1rHIV-1 cells.However, in spite of the IIIB intracellular accumulation of the Rz, its effect on virus production is confined to a very limited set of non-cytotoxic conditions.The lack of specific inhibi- Konopka et al.r Biochimica et Biophysica Acta 13721998 55-68 66 tion by the liposome-complexed functional Rz vs. the MRz may be explained by a non-specific oligonucleotide effect.In contrast, the free MRz did not inhibit p24 production when compared with the free Ž .functional Rz Table 1 .Since the functionally active chimeric Rz have been effective against cellular tarw x gets 19 , the lack of major efficacy against HIV is most likely due to the inability of the Rz to enter the cytoplasm in sufficient quantities to inhibit virus production.It is also not clear what fraction of the Rz is free to interact with substrate, and what fraction is bound to cellular proteins or remain in the endocytotic pathway.It is also possible that cationic lipids augment the production of nucleases which degrade the Rz.Our results suggest that cationic liposome-Rz complexes are sequestered in intracellular membrane-bound compartments, such as endosomes, similar to the fate of cationic liposome-DNA comw x plexes 51 .Such sequestering, together with the low efficiency of cytoplasmic delivery could diminish the potential effectiveness of the Rz, especially in chronically infected cells producing high quantities of virus.Although we have utilized four cationic liposome formulations and two Rz:lipid ratios in our studies, it is possible that a more extensive matrix of Rz and lipid concentrations may enhance the cytoplasmic delivery of exogeneous Rz.
It will be of interest to investigate whether endosome disrupting peptides, such as those used to enhance the expression of transferrinrpolylysine-comw x plexed DNA 52 , or transferrin-cationic liposome w x complexes 53 will facilitate the delivery of Rz into the cytoplasm.We have shown that peptides that interact with and destabilize membranes at mildly acidic pH enhance gene delivery by cationic lipow x somes 54 .Likewise, the intracellular delivery of Rz in pH-sensitive liposomes, which are thought to destabilize the endosome membrane at the mildly w x acidic pH of the endosome lumen 55,56 , may result in enhanced cytoplasmic delivery.Preliminary experiments in our laboratory have shown that the Rz used in this study is effective in inhibiting HIV production by macrophages when delivered in pH-sensitive lipo-Ž somes N. Duzgunes ¸, E. Pretzer and S. Simoes, un-¨¨.
published data .The recent development of sterically stabilized pH-sensitive liposomes with a prolonged w x circulation half-life 57 may render feasible the in vivo delivery of Rz to target cells and tissues.
Differentiated THP-1rHIV-1 cells were more IIIB sensitive to cytotoxic effects of liposome-Rz complexes than differentiated THP-1 cells.Interestingly, the formation of complexes with Rz significantly changes the toxic effect of liposomes alone.For example, a 4-h treatment of differentiated THP-1 cells with the Lipofectamine-Rz complex at 8 mM Ž .lipid caused a decrease in cell viability Fig. 3 , while 8 mM Lipofectamine alone was not toxic even after a w x q 24-h treatment 39 .HeLa CD4 cells were more susceptible to cytotoxic effects of liposome-Rz complexes than uninfected THP-1 cells, chronically infected THP-1rHIV-1 cells or HeLarLAV cells.

IIIB
These data are consistent with previous observations that both the efficiency of cationic lipid-mediated transfection and the toxicity of lipids or lipid-DNA com plexes, vary for different cell types w x 20, 24,26,51,58,59 .
In summary our results indicate that the effective delivery of functional Rz into cells by cationic liposomes is an inefficient process and needs extensive improvement before the technique can be used successfully in ex vivo and in vivo applications.Viral targets such as HIV, especially in chronically infected cells, may not be as well suited for exogenous Rz delivery as are intracellular targets present in smaller molar amounts.It is possible that delivery of gene constructs for continuous endogenous expression of the Rz is a more promising strategy.

Fig. 3 .
Fig. 3. Effect of LFA, LF or DMRIE complexed with Rz on the viability of differentiated, uninfected THP-1 cells.PMA-treated THP-1 cells, 5 days post-differentiation, were exposed to the Ž .liposome-Rz complexes LFA: Lipofectamine, LF: Lipofectin Ž .Ž or the Rz alone 40 mgrmls3.2mM for 4 or 24 h see Section. 2 for details .Cell viability was measured on day 13 for the 4-h treatment, and on day 12 for the 24-h treatment, by incubating the cells with Alamar Blue overnight at 378C.The viability is expressed as percent of the control.Data represent the mean" standard deviation obtained from duplicate wells.
et al.r Biochimica et Biophysica Acta 1372 1998 55-68 63 Ž .Ž .Fig. 6.Effect of LF A or DOTAP B complexed with Rz on p24 production in differentiated THP-1rHIVliposome-Rz complexes, at lipid:Rz ratios wrw Ž . of 8:1 or 4:1, or the Rz alone at 5.5 mgrml 0.44 mM , for 24 h Ž .see Section 2 for details .The p24 antigen production within the previous 48-h period was measured on days 2 and 4. Data represent the mean"standard deviation of p24 determinations in Ž .duplicate, in supernatants of duplicate wells ns 4 except in Ž .controls which were measured in five wells.Asterisks ) indicate that the values were significantly different from the untreated controls.LF and DOTAP at 10 and 15 mM were cytotoxic.