[CANCER RESEARCH 62, 6831– 6836, December 1, 2002] Advances in Brief Cationic Charge Determines the Distribution of Liposomes between the Vascular and Extravascular Compartments of Tumors 1 Robert B. Campbell, 2 Dai Fukumura, Edward B. Brown, Laureen M. Mazzola, Yotaro Izumi, Rakesh K. Jain, Vladimir P. Torchilin, and Lance L. Munn 3 Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts 02114 [R. B. C., D. F., E. B. B., Y. I., R. K. J., L. L. M.], and Department of Pharmaceutical Sciences, Boston, Massachusetts 02115 [L. M. M., V. P. T.] Abstract Tumor vessels possess unique physiological features that might be exploited for improving drug delivery. In the present study, we investigate the possibility of modifying polyethylene glycol-ylated liposome cationic charge of polyethylene glycol coated liposomes to optimize delivery to tumor vessels using biodistribution studies and intravital microscopy. The majority of liposomes accumulated in the liver, and increasing charge resulted in lower retention in the spleen and blood. Although overall tumor uptake was not affected by charge in the biodistribution studies, intravital microscopy showed that increasing the charge content from 10 to 50 mol % doubled the accumulation of liposomes in tumor vessels, suggesting a change in intratumor distribution; no significant effect of charge on interstitial accumulation could be detected, possibly attributa- ble to spatial heterogeneity. Increased vascular accumulation of cationic liposomes was similar in two different tumor types and sites. Our results suggest that optimizing physicochemical properties of liposomes that ex- ploit physiological features of tumors and control the intratumor distri- bution of these drug carriers should improve vascular-specific delivery. Introduction Physiological barriers hinder the effective delivery of drugs to tumors (1, 2). To target cancer cells, a blood borne therapeutic agent must first cross the vasculature and then travel through the intersti- tium. Recent strategies have attempted to avoid these barriers by attacking the blood supply instead of the cancer cells, either by suppressing the formation of new vessels (antiangiogenic therapy) or by abolishing established vascular networks (antivascular therapy). This approach has the advantage that the delivery vehicle, once in the blood stream, should have direct access to the target endothelial cells. Recent studies have shown that cationic liposomes have a propensity for localizing in tumor vessels, but the mechanism behind this selec- tivity and the optimal formulation to maximize this effect have not been defined. We propose that optimizing physicochemical properties of liposome vehicles should improve their interactions with tumor vessels and, more generally, control their intratumor distribution. As a first step in identifying the optimal liposome formulation for im- proving accumulation in tumors, we investigated the influence of cationic charge on the distribution of liposomes in tumors using bio-distribution studies and intravital microscopy (3). We also per- formed these studies in two tumor types and two implantation sites to examine the role of host–tumor interactions. Materials and Methods Liposome Preparation. In general, liposomes were prepared as described previously (4). PEG 4 -modified cationic and electroneutral liposomes were prepared from DOPC, cholesterol, DOTAP, PEG-PE, and rhodamine-PE stock obtained from Avanti Polar Lipids (Alabaster, AL). Doxorubicin hydrochlo- ride was obtained from Sigma (St. Louis, MO). Lipids were stored at -70°C under argon. Solvents were obtained from Fisher Scientific (Pittsburgh, PA). The molar ratio of phospholipid to cationic lipids used to make liposomes varied as a function of total net charge. Typically, when preparing PEGylated electroneutral (DOPC:Chol:PEG-distearoylphosphatidylethanolamine:label) and cationic (DOPC:DOTAP:Chol:PEG-distearoylphosphatidylethanolamine: label) liposomes, the components responsible for lending the desired physic- ochemical feature were added at the expense of DOPC. The fluorescent label concentration (1 mol %) and PEG content (5 mol %) were similar for all liposomes. Percentage of charge was 0, 10, 25, or 50 mol %. Large multila- mellar vesicles were extruded 15 times through a 100-nm polycarbonate membrane using an Avanti Extruder (Avanti Polar Lipids) to produce smaller unilamellar vesicles. Liposome sizes were estimated with a Coulter N4 plus sub-micron particle sizer (Miami, FL).  potentials for PEGylated cationic liposomes were measured at 25°C in double distilled water using the -PLUS  potential analyzer (Brookhaven Instruments, Holtsville, NY). Animals and Tumors. SCID mice were bred and maintained in our de- fined flora gnotobiotic animal facility (Massachusetts General Hospital, Bos- ton, MA). The two tumor cell lines (LS174T and MCAIV) were maintained in Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum at 37°C in a humidified CO 2 atmosphere. Tumors grown s.c. in 8 –10- week-old SCID mice were resected aseptically, and all necrotic tissue was removed. The viable tumor was cut into 1-mm-size pieces. Mice were anes- thetized with a mixture of ketamine (90 mg/kg body weight) and xylazine (9 mg/kg body weight). Tumor pieces were implanted in the s.c. space in the cranial or dorsal window chambers described previously (5). Biodistribution Studies. Biodistribution studies were performed in tumor (LS174T human colon adenocarcinoma)-bearing SCID mice (25 grams). Tumor pieces were implanted s.c. in mice. Mice were used for experimental purposes when the tumor was between 8 and 10 mm in size. Twenty-four hours postinjection, the mice were anesthetized and sacrificed only after blood had been removed with a Pasteur pipet via retro-orbital sinus. Percentage of recovery of radioactivity was measured in liver, spleen, kidneys, lungs, blood, and tumor by a Beckman Gamma 5500B counter (Fullerton, CA), and data are expressed as a percentage of injected dose accumulated per organ. Weight of mouse blood was assumed to be 7.3% of the body weight (6). Intravital Fluorescence Microscopy. Anesthetized mice bearing dorsal and cranial windows were restrained on a custom-designed stage and observed with an intravital fluorescence microscope (Axioplan; Zeiss, Oberkochen, Germany). FITC-Dextran (10 mg/ml) was injected via tail vein to assess microvascular function before measuring interactions of liposomes with tumor Received 8/20/02; accepted 10/14/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant P01-CA80124 and by an NIH fellowship to R. B. C (T32-CA73479). 2 Current address: Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115. 3 To whom requests for reprints should be addressed, at Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114. Phone: (617) 726-4085; Fax: (617) 726-1962; E-mail: Lance@steele.mgh.harvard.edu. 4 The abbreviations used are: PEG, polyethylene glycol; ROI, regions of interest; FOV, field of view; SCID, severe combined immunodeficiency; PE, phosphatidylethanolamine; DSC, dorsal skin fold chamber; CRW, cranial window; DOPC, 1,2-dioleoyl-sn-glycero- 3-phosphatidylcholine; DOTAP, 1,2-diacyl-3-trimethylammonium propane. 6831 Research. on June 27, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from