Enhanced Delivery to Target Cells by Heat-Sensitive Immunoliposomes (Liposome Targeting/Controlled Release) SEAN M
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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6117-6121, August 1986 Medical Sciences Enhanced delivery to target cells by heat-sensitive immunoliposomes (liposome targeting/controlled release) SEAN M. SULLIVAN* AND LEAF HUANGt Department of Biochemistry, University of Tennessee, Knoxville, TN 37996-0840 Communicated by John D. Baldeschwieler, April 18, 1986 ABSTRACT Heat-sensitive immunoliposomes are capable somes maintains target cell specificity with respect to bind- of releasing the entrapped content at the target cell surface ing. Once bound to the target cell, these immunoliposomes upon a brief heating to the phase transition temperature of the are able to release their entrapped contents upon heating to liposome membrane. In this study we have examined the the phase transition temperature of the liposomal lipid. The delivery efficiency of drugs entrapped in heat-sensitive im- advantage arises from the formation of a large localized munoliposomes. Immunoliposomes composed of dipalmitoyl concentration of drug at the target cell surface, which serves phosphatidylcholine with entrapped [3H]uridine were incubat- to enhance drug uptake over that of drug released into the ed with target cells at 40C. The cell-4iposome mixture was then bulk medium. heated to 410C and the uptake of [3H]uridine into the intra- In the initial characterization of these immunoliposomes, cellular pool of phosphorylated uridine-containing molecules the fluorescent dye carboxyfluorescein was used to monitor was measured. The immunoliposomes showed maximal release the release properties (19). This dye was not transported by of the uridine at 41'C, the phase transition temperature of the cells (20) and therefore, the delivery efficiency could not dipalmitoyl phosphatidylcholine liposomes. The largest accu- be evaluated. In this report we examine delivery efficiency, mulation of [3H]uridine in the target cells also took place at using uridine uptake as a model system for delivery of 41PC. The initial level of uptake of [3H]uridine released from cytotoxic nucleosides. Nucleoside transport was chosen for immunoliposomes by heating was greatly enhanced over that three reasons. First, nucleoside transport is rapid and there- observed for free [3H]uridine and [3H]uridine released from fore able to take advantage of a high localized concentration liposomes without attached antibody. The nucleoside uptake created at the cell surface. Second, nucleoside metabolism inhibitors nitrothiobenzylinosine, dipyridamole, and unlabeled has been well characterized with respect to kinetic parame- uridine were able to inhibit uptake of [3H]uridine released from ters and availability ofinhibitors. Third, information obtained immunoliposomes. This supports the hypothesis that the en- from uridine delivery can be directly applied to the delivery hanced uptake is due to a heat-induced release of [3H]uridine of chemotherapeutic nucleoside analogues such as 1-,B-D- at the cell surface followed by transport and phosphorylation arabinofuranosylcytosine, which has similar uptake charac- of [3H]uridine by the target cells. These results indicate the teristics (21). We report here the enhanced uptake of uridine feasibility of using the heat-sensitive immunoliposomes as a released from immunoliposomes bound to the target cells. target-specific drug delivery system. MATERIALS AND METHODS Liposomes have been used as carriers for a wide variety of biologically active materials (1-5). Attachment of monoclo- Reagents. Isolation, iodination, and derivatization of nal antibodies to the liposome surface has resulted in specific mouse monoclonal anti-H2Kk IgG with the palmitic acid ester binding of the liposomes to cells expressing a cell surface ofN-hydroxysuccinimide have previously been described (8, antigen (for reviews see refs. 6 and 7). Upon binding, the 19, 22). Monoclonal P3 IgG was purified from ascites fluid of liposomes are internalized and delivered to the lysosomes mice bearing intraperitoneal P3-X63-Ag8 cells. This IgG has (8-11). The delivered drug must be able to escape this unknown binding specificity and does not bind with RDM4 degrading organelle into the cytosol to exert its therapeutic cells. It was used as a control IgG. Dipalmitoyl phospha- effect. Although this type of liposome delivery system has tidylcholine (Pam2-PtdCho) was purchased from Avanti Polar been effective in a few cases, it is inefficient for most of the Lipids and stored in CHC13 under N2 at -20°C. Phosphate commonly used drugs. To increase the delivery efficiency, was determined by the method ofBartlet (23). [3H]Hexadecyl site-directed liposomes with special functions have been cholestanyl ether was used as a lipid marker (24). Uridine was developed. purchased from Sigma. [3H]Uridine was purchased from pH-sensitive liposomes are one type of special-function ICN. S-(4-Nitrobenzyl)-6-thioinosine was purchased from liposomes that are capable of fusing with the endosomal Aldrich. Dipyridamole [2,6-bis(diethanolamino)-4,8-dipiperi- membrane dinopyrimido[5,4-d]pyrimidine] was obtained from Boeh- upon acidification of the endocytic vacuole, ringer Mannheim. Deoxycholate was purchased from Cal- thereby releasing its entrapped contents into the cytoplasm biochem and recrystallized twice. (12-14). However, this mechanism, although quite novel and Preparation of Immunoliposomes with Entrapped Uridine. very successful, is dependent upon endocytosis of the lipo- Immunoliposomes were prepared as previously described somes by the target cells. Efficient delivery of immunolipo- (19). Briefly, small unilamellar liposomes composed ofPam2- some-encapsulated drugs to cells that do not actively PtdCho were prepared and allowed to fuse at 40C for 3-30 endocytose requires an alternative approach. Heat-sensitive days. The large liposomes were separated from smaller ones liposomes have been shown to potentially fulfill this need by using a 35-ml preparative 5-20% (15-18). Attachment of antibody to the heat-sensitive lipo- continuous sucrose Abbreviation: Pam2-PtdCho, dipalmitoyl phosphatidylcholine. The publication costs of this article were defrayed in part by page charge *Present address: Division of Chemistry and Chemical Engineering, payment. This article must therefore be hereby marked "advertisement" California Institute of Technology, Pasadena, CA 91125. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 6117 Downloaded by guest on September 28, 2021 6118 Medical Sciences: Sullivan and Huang Proc. Natl. Acad. Sci. USA 83 (1986) density gradient centrifuged at 80,000 x g for 18 hr. A 27 mM incubation at 18'C was continued for another 10 min before Pam2-PtdCho liposome suspension was prepared in Ca2+- heating. and Mg2+-free phosphate-buffered saline containing 50 mM [3H]uridine, 1 mM EGTA, and 0.02% NaN3. The [3H]uridine RESULTS specific activity was 2-4 x 105 cpm/nmol. The suspension was placed in a temperature-regulated chamber set at 41TC. Phosphorylation of uridine occurs only intracellularly and is A 15-mg/ml solution of palmitoyl-specific antibody in phos- therefore a good criterion for examining the efficiency of phate-buffered saline containing 3.8 mM deoxycholate, pH uridine delivery by the immunoliposomes. Uridine transport 8.0, was injected into the liposome suspension at a rate of0.26 and metabolism are collectively referred to as "uptake." /.l/min by using an infusion pump. The suspension was Specifically, the uptake process involves diffusion of extra- annealed at 43TC for 20 min and equilibrated to room cellular nucleosides across the plasma membrane via a temperature over a period of 20 min. The suspension was transport system, followed by the intracellular kinase- dialyzed overnight against phosphate-buffered saline con- catalyzed phosphorylation of transported uridine. An assay taining 1 mM EGTA and 0.02% NaN3 to remove untrapped was developed that measured all phosphorylated uridine [3H]uridine and residual deoxycholate. Unincorporated metabolites, including UMP, UDP, UTP, UDP-glucose, and palmitoyl antibody was removed by the same centrifugation RNA. Phosphorylated uridine metabolites were collected on procedure used to fractionate the stock Pam2-PtdCho lipo- an anion-exchange filter and unphosphorylated uridine was somes. The uridine/antibody peak was pooled and dialyzed washed away. This assay and paper chromatographic anal- to remove sucrose. "Bare liposomes" (without attached ysis of perchloric acid-soluble material (26) yielded compa- palmitoyl antibody) were prepared in the same fashion as the rable results (data not shown). immunoliposomes except that only phosphate-buffered Temperature-Dependent Release of Uridine from Im- saline/3.8 mM deoxycholate was injected into the liposome munoliposomes. Heat-sensitive immunoliposomes were pre- suspension. Bare liposomes had the same trapping efficiency pared containing 50 mM [3H]uridine. Incubation with i07 as immunoliposomes. Prior to cell incubation, im- RDM4 cells yielded 15-20% ofpalmitoyl antibody bound and munoliposomes and bare liposomes were dialyzed against 5-8% of [3H]uridine bound. A 50-fold excess offree antibody culture media without serum to remove NaN3. was able to inhibit the palmitoyl antibody binding and Determination of Uridine Uptake. RDM4 cells were cul- [3H]uridine binding (data not shown). This demonstrated the tured in RPMI 1640 medium supplemented with 1 mM sodium cell-associated uridine to be the result of immunoliposome pyruvate and 10% fetal bovine serum. For incubation