Lipid multilayer microarrays for in vitro liposomal drug delivery and screening.

Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed. Small-molecule arrays and microfluidics are promising approaches. Here we introduce a simple method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The multilayer patterns are of sub-cellular dimensions and controllable thickness and were formed by dip-pen nanolithography. The patterns successfully delivered a rhodamine-tagged lipid and drugs only to the cells directly over them, indicating successful encapsulation and no cross-contamination to cells grown next to the patterns. We also demonstrated multilayer thickness-dependant uptake of the lipids from spots with sub-cellular lateral dimensions, and therefore the possibility of delivering different dosages from different areas of the array. The efficacies of two drugs were assayed on the same surface, and we were able to deliver dosages comparable to those of solution based delivery (up to the equivalent of 30 µg/mL). We expect our method to be a promising first step toward producing a single high-throughput liposome-based screening microarray plate that can be used in the same way as a standard well plate.

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast.

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast.

Combination chemotherapy of human ovarian xenografts with intraperitoneal liposome-incorporated valinomycin and cis-diamminedichloroplatinum(II).

Intraperitoneal administration of liposomal valinomycin (MLV-VM) with cis-diamminedichloroplatinum(II) (cDDP) had significant antitumor activity against murine P388 leukemia and inhibited the growth of OVCAR-3 tumors in a nude mouse model of human ovarian cancer. This tumor is a teratoma originating in the ovary with pathogenesis and metastatic properties similar to those of human ovarian cancer. Drug was given to the mice once every 5 days for 4 doses beginning 1 day after i.p. implantation of 10(7) or 5 x 10(7) OVCAR-3 tumor cells. For P388 leukemia, drug was given i.p. once or on days 1 and 5 after tumor inoculation. Despite the use of low doses of MLV-VM, the antitumor activity of the combination [increase in life span (%T/C), 289%-294%] represents a 4-log cell kill over the additive effect of the two drugs, indicating a synergistic interaction between MLV-VM and cDDP. Likewise, low doses of the drug combination produced a synergistic interaction on human ovarian OVCAR-3 tumors, and tumor-free, long-term survivors were obtained. Combined therapy of liposome-incorporated valinomycin and cisplatin was well tolerated and produced no overlapping nephrotoxicity, although a decrease in liver enzyme markers (alkaline phosphatase and/or alkaline aminotransferase) with MLV-VM was observed. These results appear to suggest that MLV-VM with cDDP may have considerable potential for the treatment of ovarian cancer disseminated within the peritoneal cavity, although the frequency and sequence of drug administration may need to be improved.

In vitro interaction of liposomal valinomycin and platinum analogs: cytotoxic and cytokinetic effects.

Cisplatin is the most active agent in the chemotherapy of ovarian cancer and this activity can be enhanced by liposomal valinomycin (MLV-VM) in vitro. To test whether MLV-VM is capable of augmenting the cytotoxic and cytokinetic effects of other platinum analogs, drug combinations of MLV-VM and platinum drugs were tested against two human ovarian cancer cell lines (OVCAR-3 and CaOV-3) and on Chinese hamster ovary (CHO) cells in vitro. MLV-VM enhanced the sensitivity to cisplatin, ormaplatin and carboplatin on human ovarian carcinoma cells that show various degrees of drug sensitivity. This interaction was shown to be truly synergistic by median-effect analysis up to 90% cell kill. The combination index at 50% cell kill (Cl50) was also used to quantitate the extent of drug synergy. In the OVCAR-3 cell line, for example, the Cl50s were 0.62, 0.85 and 0.8 for cisplatin, ormaplatin and carboplatin, respectively. DNA histograms obtained by flow cytometry showed that CHO cells treated with cisplatin alone accumulated in the S-G2 segment, with a partial G2 block. The addition of 2 microM VM with cisplatin, significantly enhanced the accumulation of cells at the G2/M phase. Our results further demonstrate that in vitro treatment with VM, cisplatin and/or combination is associated with an increase in protein kinase C (PKC) activity. These findings suggest that accumulation of cells at G2/M phases and modulation of PKC activity could be among the basis for the cytotoxic synergism observed between cisplatin and VM.

[The effect of membrane-active compounds on the radiation-modifying effect of glucose in x-irradiated ascitic tumor cells]. / Vliianie membranoaktivnykh soedinenii na radiomodifitsiruiushchii éffekt gliukozy pri rentgenovskom obluchenii kletok astsitnoi opukholi.

A study was made of the influence of membrane-active agents with different mechanisms of action (quercetin, amiloride, valinomycin, and digitonin), that modify the transmembrane transfer of inorganic ions, on a modifying effect of a glucose loading in X-irradiated Ehrlich ascites tumor cells. The combination of digitonin with glucose increased the damaging effect of radiation on tumor cells by 1.8-2.2 times as compared to glucose alone. Merely insignificant changes in the radiation-modifying effect of glucose were observed when it was combined with other membrane-active agents.

Valinomycin-induced modulation of adriamycin resistance and cationic probe distribution in MCF-7 cell lines.

In vitro restoration of adriamycin sensitivity in a resistant human breast tumor cell line was obtained by continuous exposure to nanomolar nontoxic valinomycin concentrations. Seven-day treatment with nanomolar valinomycin concentrations caused a slight increase of the signal of the cationic fluorescent cyanine probe DiOC5(3) but did not appreciably affect adriamycin incorporation in the cells. A marked increase of the DiOC5(3) signal was obtained in the presence of micromolar valinomycin concentrations, which were incompatible with the in vitro cellular growth.

Synergistic cytotoxic actions of cisplatin and liposomal valinomycin on human ovarian carcinoma cells.

We have previously shown that the toxicity of valinomycin (VM), a membrane-active agent with antineoplastic activity, can be dramatically reduced with no loss of the antitumor efficacy of the drug by incorporating it into liposomes. In the present study, we investigated the interaction between cis-diamminedichloroplatinum(II) (CDDP) and VM in terms of in vitro cytotoxicity to human ovarian tumor cells. Using the MTT assay and analyzing the data using the median-effect principle, we showed that synergistic cytotoxic interactions exist between CDDP and VM in their liposomal form. The degree of cytotoxic synergism was influenced by the duration of drug exposure and the dose ratio. The cellular accumulation of platinum by ovarian cells at 37 degrees C was slightly higher after exposure to VM as compared with controls; however, it is not clear that this accounts for the cytotoxic synergism. These results suggest that the combination of liposomal VM and CDDP may have merit as a form of localized drug delivery for the treatment of ovarian cancer disseminated within the peritoneal space.

Modulation of doxorubicin resistance by valinomycin (NSC 122023) and liposomal valinomycin in Chinese hamster ovary cells.

Recently, we have reported that the toxicity of the membrane-active agent valinomycin (VM) can be reduced with maintenance and/or enhancement of its antitumor activity by incorporation in liposomes (S. S. Daoud and Juliano, Cancer Res., 46:5518-5525, 1986). Since the underlying defect(s) in multidrug resistance reside mainly in the cell membrane, it seems reasonable to attempt to overcome multidrug resistance with membrane-active drugs. Here, we report on the in vitro restoration of Adriamycin (ADR) sensitivity in a resistant Chinese hamster ovary cell line (CHRC5) by treatment with nontoxic doses of valinomycin or of liposomal valinomycin. During a 1-h drug exposure, the sensitivity of CHRC5 to ADR was enhanced 21- to 28-fold when 20 or 40 nM VM was present, doses which are not toxic to CHRC5 cells. At the same time, modest synergistic toxicity could be seen in the parent drug-sensitive cell line (AUX B1). At 100 nM VM, the sensitivity of CHRC5 to ADR was restored to almost that of the sensitive AUX B1 cells. The effects of liposomal VM on ADR sensitivity were similar to the effects produced by free VM. At nontoxic doses and with continuous exposure of the drug, valinomycin was highly active in restoring ADR sensitivity in CHRC5 cells. In cells treated for 72 h, valinomycin enhanced the sensitivity to ADR 208- to 250-fold in CHRC5 and 3- to 5-fold in AUX B1 cells. Measurements of ADR uptake and efflux indicate that, unlike other multidrug resistance modifiers, valinomycin exerts its actions in modulating ADR resistance by mechanism(s) other than increasing intracellular accumulation of Adriamycin. The possible mechanisms of the restoration of ADR sensitivity by valinomycin are discussed.

In vitro effect of liposome-incorporated valinomycin on growth and macromolecular synthesis of normal and ras-transformed 3T3 cells.

Valinomycin is a depsipeptide antibiotic that selectively translocates potassium ion across biologic membranes. This drug has been reported to display antitumor effects, but its use has been limited by its extreme toxicity. However, its incorporation into lipid vesicles (liposomes) has resulted in a reduction in toxicity and in the enhancement of the drug's therapeutic index. As a preliminary investigation of the mechanistic basis for this enhancement, the in vitro response of normal 3T3 and ras-transformed cells to free (VM) and liposomal valinomycin (VM-MLV) was examined. The incorporation of [3H]-leucine and [methyl-3H]-thymidine was used to assess macromolecular synthesis, and the MTT vital dye assay was used to measure cell survival and growth. Pretreatment of exponentially growing NIH/3T3 cells with 20 nM VM for 1 h decreased [3H]-leucine and [methyl-3H]-thymidine incorporation by 90% and 80%, respectively. However, Ha-ras 3T3 cells showed resistance to VM treatment with inhibitory doses in the range of 200 nM. At equimolar VM concentrations, VM-MLV was found to be less inhibitory than VM for protein and DNA synthesis. Specifically, marked protective activity was apparent with normal 3T3 cells. In this report we also demonstrate that VM selectively killed normal cells compared with ras-transformed cells grown in vitro. However, VM-MLV displayed a modest cytotoxic selectivity (3- to 4-fold) to ras-transformed cells. Our data suggests that first, there is good correlation between growth inhibition and inhibition of DNA and protein synthesis by VM, and second, VM-MLV exhibits a modest, selective toxicity to the ras-transformed 3T3 cell line as compared with nontransformed 3T3 cells, whereas free VM has the opposite selectivity.

Liposome mediated dissipation of valinomycin-imposed potassium potential across erythrocytes membrane.

Influence of liposomes made of phosphatidylcholine (PC) on the valinomycin-imposed potassium potential across erythrocyte membrane was examined by measuring the fluorescence change of the potential-sensitive cyanine dye. We concluded that the liposomes modulate ion selectivity of the membrane embedded valinomycin, on the basis of the following lines of evidence. (i) The valinomycin-imposed potassium potential across erythrocyte membrane (interior negative) was dissipated in the presence of PC-liposomes. (ii) When PC-liposomes were added to the cell suspension before the valinomycin, a membrane potential could not be imposed. (iii) Liposomes containing only the PC of saturated fatty acids were inactive in the potential dissipation, whereas the liposomes containing PC of unsaturated fatty acids were fully active. (iv) Liposome-mediated dissipation of the imposed-membrane potential was similarly observed in the resealed erythrocyte ghosts. (v) The liposomes did not show a detectable effect on the gramicidin-mediated proton potential. (vi) The effect of liposome was somewhat analogous to the nigericin-mediated dissipation of the valinomycin-imposed potassium potential.

Reduced toxicity and enhanced antitumor effects in mice of the ionophoric drug valinomycin when incorporated in liposomes.

Valinomycin (NSC 122023) is a cyclic depsipeptide antibiotic with potassium selective ionophoric activity. This drug has been reported to display antitumor effects but its utilization has been limited by its extreme toxicity. Here we report that the incorporation of valinomycin into multilamellar liposomes composed of dimyristoyl phosphatidyl choline:cholesterol:phosphatidyl serine (10:4:1 M ratio) results in a profound reduction in toxicity with maintainence of antitumor efficacy. Thus the median lethal dose (LD50) for i.p. administered valinomycin (VM) in C57BL/6 X DBA/2 mice is 1.7 mg/kg whereas the LD50 for liposome incorporated valinomycin (MVL-VM) is in excess of 50 mg/kg. In like manner, the LD50 for i.v. administered VM is 0.18 mg/kg where the LD50 for MLV-VM preparations passed through a 0.6-micron filter is greater than 10 mg/kg. The antitumor efficacies of i.p. administered VM or MLV-VM against i.p. P388 mouse leukemia were similar in multiple dose formats using doses below the maximal tolerated dose for VM. However, since MLV-VM was substantially less toxic than VM, the liposomal drug also produced significant (170% median survival time of treated mice/median survival time of untreated control) antitumor effects when administered as a single dose at levels above the maximal tolerated dose for free VM; single doses of free VM at the maximal tolerated dose were ineffective in this context. In experiments with i.v. inoculated P388 leukemia, MLV-VM but not free VM, displayed antitumor activity (144% median survival time of treated mice/median survival time of untreated control) when administered i.v. at equitoxic doses. Thus the use of a lipid vesicle drug carrier system permits a reduction in the toxicity of valinomycin with maintainence or enhancement of antitumor activity against i.p. or i.v. P388 leukemia.