Reduced dose-limiting toxicity of intraperitoneal mitoxantrone chemotherapy using cardiolipin-based anionic liposomes

POTENTIAL CLINICAL RELEVANCE Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 769 – 776

Original Article www.nanomedjournal.com

Reduced dose-limiting toxicity of intraperitoneal mitoxantrone chemotherapy using cardiolipin-based anionic liposomes

Rae Sung Chang, MSca,b,1 , Jiyeon Kim, MSca,1 , Han Young Lee, MSca , Su-Eun Han, MSca , Jinhee Na, MScc , Kwangmeyung Kim, PhDd , Ick Chan Kwon, PhDd , Young Bong Kim, PhDe , Yu-Kyoung Oh, PhDb,⁎ a

School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea b School of Pharmacy, Seoul National University, Seoul, South Korea c Korea Institute of Science and Technology, Seoul, South Korea d Biomedical Research Center, Korea Institute of Science and Technology, Seoul, South Korea e Department of Animal Biotechnology, Konkuk University, Seoul, South Korea Received 18 January 2010; accepted 12 May 2010

Abstract Intraperitoneal chemotherapy confers limited clinical benefit as a result of the dose-limiting toxicity of anticancer drugs. We aimed to develop optimized liposomes for mitoxantrone (MTO) administration that provide high encapsulation efficiency and increase the therapeutic index. Cationic MTO was loaded onto anionic liposomes by electrostatic surface complexation. The anticancer activity was evaluated in a peritoneal carcinomatosis model. The retention of MTO at the tumor site was monitored by molecular imaging. MTO loading efficiencies by electrostatic complexation were N95% for all anionic liposomes but b5% for neutral liposomes. Among anionic liposomes, cardiolipin liposomes (CLs) exhibited the strongest binding affinity for MTO, the highest anticancer activity, and the lowest toxicity. MTO delivered by CLs showed prolonged retention at tumor sites. Unlike free MTO showing significant cardiotoxicity, MTO administered in CLs provided negligible cardiotoxicity. CL-mediated delivery may increase the therapeutic index of MTO chemotherapy by prolonged retention and reduced cardiotoxicity. From the Clinical Editor: The authors report the development of optimized liposomes for intraperitoneal mitoxantrone delivery that provides high encapsulation efficiency and increases the therapeutic index. Cardiolipin liposomes exhibited the strongest binding affinity for mitoxantrone, along with the highest anti-cancer activity and lowest toxicity, including negligible cardiotoxicity. © 2010 Elsevier Inc. All rights reserved. Key words: Intraperitoneal chemotherapy; Mitoxanthrone; Cardiolipin liposomes; Dose-limiting toxicity; Prolonged retention

Intraperitoneal chemotherapy has been used for the treatment of peritoneal carcinomatosis from colorectal cancers,1 ovarian cancers,2 and other surface malignancies3 within the peritoneal cavity. However, intraperitoneal chemotherapy has been associated with high mortality, reflecting the dose-limiting toxicity of anticancer drugs.4 Currently, there is an urgent need for a new

This work has been financially supported by grants from the Ministry of Education, Science and Technology (2009-0081879), 2009 Health & Medical Technology R&D program (Grant No. A090945), and Bio-Green 21 program (Code No. 20100301-061-200-001-03-00), Rural Development Administration, South Korea. ⁎Corresponding author: School of Pharmacy, Seoul National University, Gwanak-gu, Seoul 151-742, South Korea. E-mail address: [email protected] (Y.-K. Oh). 1 These two authors contributed equally to this work.

modality of intraperitoneal chemotherapy capable of providing enhanced anticancer activity with reduced systemic toxicity.5 The anthracenedione antineoplastic agent, mitoxantrone (MTO), has been commonly used in the treatment of peritoneal carcinomatosis.6 However, MTO has been reported to provide only limited clinical benefit in intraperitoneal chemotherapy due to severe systemic toxicity, notably cardiotoxicity.7 Various delivery systems have been developed to reduce the doselimiting systemic toxicities of MTO, including MTO-eluting beads8 and solid lipid nanoparticles.9 The specific objective for each of these modalities is to increase the retention of MTO in the tumor tissues while reducing their distribution to the systemic circulation. In addition to these approaches, liposomes have been studied as a potential system for delivering several different anticancer drugs. Because liposomal formulations are biocompatible and biodegradable, and lend themselves to large-scale production, a

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2010.05.003 Please cite this article as: R.S., Chang, et al, Reduced dose-limiting toxicity of intraperitoneal mitoxantrone chemotherapy using cardiolipin-based anionic liposomes. Nanomedicine: NBM 2010;6:769-776, doi:10.1016/j.nano.2010.05.003

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number of liposomal anticancer agents are on the market, with different chemical series in clinical and preclinical trials.10 The key to the clinical use of liposomal MTO is reduced systemic toxicity, which may ultimately depend on achieving high encapsulation efficiency and prolonged retention at tumor sites. Electrostatic complexation has been widely used as a means for loading cationic liposomes for delivery of anionic DNA11 and small interfering RNAs.12 Electrostatic complexation is a single-step procedure that is ideally suited for translation to clinical trials and is capable of high loading efficiencies. Although electrostatic complexation technology is applicable to loading the cationic MTO onto anionic liposomes, thus far it has not been used to prepare liposomal MTO. Moreover, the effects of different anionic lipids on the anticancer activity and systemic toxicities of liposomal MTO following intraperitoneal chemotherapy are incompletely understood. Here, we proposed that the strengths of electrostatic interaction between cationic MTO and anionic liposomes might be modulated by the type of charged anionic lipids used, and that enhancing this interaction might prolong MTO retention at the site of administration, thereby lowering systemic toxicity. We report that electrostatic complexation resulted in highly efficient loading of cationic MTO onto anionic liposomes, and show that the therapeutic index of intraperitoneal MTO was significantly improved by in vivo administration in optimized anionic liposomes.

Methods Preparation of MTO-liposomes complexes Liposomes were prepared using a multilamellar vesicle method with a slight modification.13 Anionic cholesteryl hemisuccinate (CHEMS), egg L-α-phosphatidyl-DL-glycerol (PG), or L -α-phosphatidylserine (PS)-based liposomes (CHEMS-L, PG-L, or PS-L) were prepared by dissolving each anionic lipid in chloroform with egg L-α-phosphatidylcholine (PC), cholesterol (Chol), and 1,2-diacyl-sn-glycero-3phosphoethanolamine-N-(methoxy [polyethylene glycol]-2000) (mPEG2000-DSPE) at a molar ratio of 5:5:5:0.5. In the case of cardiolipin (CA)-based liposomes (CA-L), the molar ratio of lipids was 2.5:5:5:0.5 (CA/PC/Chol/mPEG2000/DSPE). Neutral PC-based liposomes (PC-L) were prepared by mixing PC and Chol at a molar ratio of 10:5. After formation of thin lipid films of the lipid mixtures, the films were hydrated with 1 mL of 20 mM HEPES buffer (pH 7.4) by vortexing. The resulting multilamellar vesicles were extruded three times through 200-nm polycarbonate membrane filters (Millipore, Bedford, Massachusetts). Electrostatic complexation of cationic MTO onto the surfaces of anionic liposomes was accomplished by vortexing 500 μg of MTO with 1 mL of CHEMS-L, PG-L, or PS-L (15.5 μmol total lipids) or with 1 mL of CA-L (13 μmol total lipids) to yield surface complexation of MTO to CHEMS-L (MTO/CHEMS-L), to PG-L (MTO/PG-L), to PS-L (MTO/PS-L), and to CA-L (MTO/CA-L). For comparison purposes, MTO was also made to form complexes with anionic liposomes by adding 500 μg of MTO to 1 mL of neutral PC-L containing 15 μmol total lipids. The efficiencies of loading MTO onto each liposomal formulation were measured by passing the vortexed mixtures

of MTO and liposome solutions through a Sephadex G-25M column (PD-10 Columns, GE Healthcare, Amersham, Buckinghamshire, United Kingdom) to remove free MTO. The amounts of MTO bound to the liposomes were measured by absorbance at 662 nm. Measurement of particle sizes and zeta potentials A dynamic light-scattering method was used to measure liposome particle sizes, and laser-Doppler electrophoresis was used to determine zeta potentials of liposome solutions. Liposomes with or without MTO were diluted with 20 mM HEPES buffer, and analyzed using an ELS-8000 dynamic lightscattering instrument (ELS-8000; Photal, Osaka, Japan). The hydrodynamic diameters of the particles were determined using dynamic He-Ne laser (10 mW) light scattering. Zeta potentials were determined using laser Doppler microelectrophoresis at an angle of 22 degrees. Data were analyzed using a software package (ELS-8000 software). Measurement of binding affinity between MTO and anionic liposomes The strengths of electrostatic interaction of cationic MTO with various anionic liposomes were tested using a heparin competition assay as previously described.14-16 In this assay the relative binding forces between MTO and each anionic liposome formulation is inferred from the liberation of MTO from liposomes after the addition of heparin, visualized by the electrophoretic mobility of MTO on agarose gels. For heparin competition, 20 μL of MTO (500 μg/mL), free or in a liposomal formulation, were vortexed with 3 μL of heparin (20 mg/mL) and incubated for 20 minutes. After adding 7 μL of 50% glycerol, free MTO and MTO-liposomal complex formulations with or without heparin pretreatment were then loaded onto a 1% agarose gel and electrophoresed at 100 V for 10 minutes. MTO bands were visualized using a gel documentation system (Gel Doc XR system; Bio-Rad, Hercules, California), and band densities were quantified using image analysis software (Quantity One; Bio-Rad). The relative band density of MTO liberated from liposomal formulations was calculated from the following equation: Relative band density = (band density of liberated MTO in liposome samples treated with heparin – background density) / (band density of free MTO treated with heparin – background density). Cell line The L1210 murine leukemia cell line from American Type Culture Collection (Manassas, Virginia) was cultured in Dulbecco's Minimal Essential Medium supplemented with 10% heat-inactivated horse serum (Gibco BRL Life Technologies, Carlsbad, California), and 100 units/mL penicillin plus 100 μg/mL streptomycin at 37°C. The cells were maintained in the exponential growth phase by periodic passaging. Evaluation of in vitro cell viability The viability of cells was tested using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. L1210

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cells were seeded in 96-well plates at a density of 3 × 10 cells per well. After seeding the cells, free MTO, MTO/CA-L, or empty liposomes without MTO were added to each well. Untreated group was used as a control. After incubating for various times, 20 μL of an MTT solution (2.5 mg/mL) were added and incubated for 2 hours. The cells were then dissolved in 100 μL of 0.06 M HCl in 2-propanol. Absorbance at 570 nm was measured using a microplate reader (Sunrise Basic TECAN, Männedorf, Switzerland). Evaluation of in vivo anticancer activity Animal study plans were approved by the Institutional Review Board of Korea University, and all animal studies were performed in accordance with the guidelines for animal experiments of Korea University. The anticancer activity of free MTO and MTO in various liposomal formulations was tested in a mouse peritoneal tumor model. Five- to six-week-old female BDF1 mice (Orient Bio Inc., Seongnam, Gyeonggi-do, South Korea) were inoculated intraperitoneally with 2 × 105 L1210 murine leukemia cells on day 0. On days 1, 3, and 5, MTO in free or liposomal formulations was injected intraperitoneally. Five mice were used per each group, and the experiments were repeated three times. Mice injected with MTO formulations but not inoculated with L1210 cells served as controls. After MTO treatment, body weights were measured three times per week and survival was assessed daily. In vivo optical imaging of MTO The abdominal retention of MTO, injected intraperitoneally at a dose of 2.5 mg/kg to BDF1 mice, was studied using molecular imaging. The mice were positioned on the plate of the eXplore Optix system (Advanced Research Technologies Inc., Montreal, Quebec, Canada), and the abdominal region was scanned at 25 mW laser power and a count-time setting of 0.3 sec/point. A 670nm pulsed laser diode was used to excite MTO molecules, which possess an endogenous fluorophore with maximum excitation and emission wavelengths of 607 and 684 nm, respectively. Long-wavelength fluorescence emission (600–700 nm) was detected using a fast photomultiplier tube (Hamamatsu Photonics, Hamamatsu, Japan) and a time-correlated single-photon counting system (Becker and Hickl GmbH, Berlin, Germany). Cardiac histopathology Saline or MTO (2 mg/kg) in free or liposomal forms was intraperitoneally injected into female BDF1 mice (5–6 weeks old, 18–22 g) every day for 10 days. On day 11 the hearts were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5-μm-thick slices. The sections were stained with hematoxylin and eosin, and assayed by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) using a commercial apoptosis detection kit (fragEL DNA Fragmentation Detection Kit; Merck KGaA, Darmstadt, Germany). Figure 1. Chemical structures and schematic diagram of MTO-loaded liposomes. (A) Chemical structure of MTO and (B) lipid components. The neutral PC and the anionic lipids are shown. (C) Anionic PEGylated liposomes were loaded with cationic MTO by electrostatic complexation.

Statistics Statistical analysis was performed using a Student t-test or an ANOVA with a post hoc Student-Newman-Keuls test. SigmaStat

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software (version 3.5, Systat Software) was used for all analyses, and a P value less than 0.05 was considered significant.

Results Physicochemical properties of MTO in complex with liposomes MTO possesses two positively charged amine groups, conferring a cationic property that was exploited to electrostatically induce MTO to form complexes with the surfaces of anionic liposomes (Figure 1, A). To electrostatically load MTO onto liposomes, we prepared various liposomes using anionic lipids with a single negatively charged group (CHEMS, PG, and PS) or two negatively charged groups (CA) (Figure 1, B). The molar percentage of anionic lipids with a single negatively charged group was 32.3% for each liposome. Because CA contains two negatively charged groups, the molar percentage of CA in the CA-L was adjusted to 19.2% to achieve a similar charge density per liposome. Neutral liposomes containing PC were prepared for comparison with anionic liposomes. The PEGylated lipid, mPEG2000-DSPE, was incorporated into all liposomes to enhance in vivo stability. Both anionic and PEGylated liposomes may bind to cationic MTO in the resulting MTO-liposomes complexes. The scheme for complexation of cationic MTO onto anionic liposomes is presented in Figure 1, C. The loading efficiencies of MTO onto liposomes were determined by the surface charges of the liposomes, not by the type of anionic lipids. All anionic liposomal formulations demonstrated loading efficiencies greater than 95.0% (Figure 2, A), and there were no significant differences in loading efficiency among the anionic liposomes. In contrast, the loading efficiency of neutral PC-L liposomes was 3.8%. As shown in Figure 2, B, the mean sizes of the different anionic liposomes were in the range of 184 to 202 nm. Electrostatic complexation of MTO onto anionic liposomes did not affect the sizes of anionic liposomes except that of CHEMS-L, which was reduced from 201.5 ± 5.5 nm to 163.1 ± 2.8 nm upon complexation with MTO. In contrast to the general lack of effect of MTO complexation on particle size, the complexation of positive MTO increased the zeta potential values of negatively charged liposomes (Figure 2, C). After complexation with MTO, the zeta potentials of PG-L, PS-L, CA-L, and CHEMS-L increased to –32.5 ± 0.2, –31.9 ± 6.1, –25.7 ± 0.6, and –14.2 ± 1.1 mV, respectively. Effect of anionic lipids on electrostatic interaction with MTO To determine whether the strengths of electrostatic interaction between cationic MTO and anionic liposomes differed depending on lipid type, we used an electrophoresis-based, heparin competition assay. In this assay, in the absence of heparin, free positively charged MTO moves toward the anode upon electrophoresis in an agarose gel, whereas in the presence of heparin, free MTO forms complexes with negatively charged heparin and moves toward the cathode (Figure 3, A). In contrast, MTO bound to anionic liposomes does not move in the gel as a result of the size of liposomes in complex. In the presence of

Figure 2. Loading efficiencies and physicochemical properties of MTO in complex with liposomes. (A) Efficiencies of loading MTO onto various liposomes were determined after removal of unbound MTO by gel filtration. (B) Sizes of liposomes were measured using a light-scattering method. (C) Zeta potentials of liposomes were determined using an electro-Doppler method. Asterisks denote significantly different from the group without MTO (P b 0.05).

heparin, free positively charged MTO forms MTO-heparin complexes that migrate to the cathode upon electrophoresis under the influence of associated heparin. Using this assay, we found that MTO in complex with anionic liposomes showed partial movement upon addition of heparin, reflecting that portion of MTO that was released from liposomes and subsequently bound heparin (Figure 3, A). Notably, the MTO/

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CA-L formulation showed the least electrophoretic movement of MTO after addition of heparin, indicating the greatest retention of MTO. The relative band density of mobile MTO liberated from the liposome complexes by heparin competition was the highest for CHEMS-L (88.3% ± 8.0%), followed by PG-L (79.1% ± 2.4%), PS-L (54.1% ± 2.1%), and CA-L (20.7% ± 11.1%) (Figure 3, B). In vitro cell viability after treatment with liposomes with or without MTO MTO in complex with CA-L exerted anticancer activity in vitro similar to that of free MTO but with different kinetics. During the first 12 hours, free MTO showed significantly higher anticancer activity than did liposomal MTO (Figure 3, C). The percentage of surviving L1210 cells was 42.4% ± 2.0% after free-MTO treatment and 77.8% ± 2.6% after treatment of MTO in complex with CA-L. At 24 hours, the differences in L1210 cell survival between free MTO- and liposomal MTO-treated groups became smaller, and by 48 hours of treatment, the cell survival percentages were similar for free and liposomal MTO. Empty liposomes alone did not significantly affect the viability of cells. The groups treated with empty liposomes showed viability similar to the untreated control group (Figure 3, D). In vivo systemic toxicity of liposomal MTO As a peritoneal carcinomatosis model, we used BDF1 mice intraperitoneally inoculated with L1210 murine leukemia cells, which have been used to induce tumors in the peritoneal cavity.17 The systemic toxicity of MTO differed significantly in vivo depending on the type of anionic lipids used in the liposomal formulations (Figure 4, A) and paralleled the relative electrostatic interaction strengths observed in heparin competition assays. As expected, survival of peritoneal L1210 tumor-bearing mice was lowest in the free MTO–treated group, which showed 0% survival on day 7 after peritoneal administration. All liposomal MTO-treated groups showed more prolonged survival of peritoneal tumor-bearing mice than did those treated with free MTO. At 60 days postadministration, the survival was highest with the MTO/CA-L–treated group (80% survival), followed by MTO/PS-L–, MTO/PG-L–, and MTO/CHEMS-L–treated groups, which had 60-day survival rates of 40%, 20%, and 0%, respectively. Most liposomal MTO formulations did not significantly alter the body weights of mice compared to the control (no tumor) group, which exhibited a modest, but insignificant, upward trend Figure 3. Heparin competition assay for release of MTO from liposome complexes and in vitro cell viability after treatment of free and anionic liposomal MTO. After incubation of liposomal MTO with or without heparin, the mixtures were electrophoresed on a 1% agarose gel, and MTO bands were visualized using a gel documentation system (A), and relative band densities were determined (B). Data in B are expressed as mean ± SD (n = 3). Asterisk denotes significantly lower than other groups (P b 0.05). L1210 cells were treated with free MTO or MTO/CA-L (C), or empty liposomes without MTO (D). MTO-untreated cells were used as a control. The viability of cells after 24 hours' treatment of MTO formulations was measured by MTT assays. Asterisk denotes significantly higher than free MTO-treated group (P b 0.05).

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Figure 4. Changes in body weight and survival of tumor-bearing mice after peritoneal treatment with free or liposomal MTO. L1210 tumor-bearing mice were injected intraperitoneally with saline, free MTO, or liposomal MTO. Injections were administered on days 1, 3, and 5 after inoculation with tumors. Mice in the control group were not inoculated with L1210 cells and received no treatment. (A) Survival of mice was monitored daily and (B) body weights were measured three times per week.

in body weight (∼3 g) over 60 days (Figure 4, B). The lone exception was the MTO/CHEMS-L–treated group, which tended to reduce body weight. Only the saline-treated group of L1210-inoculated mice exhibited a significant increase in body weight, which became evident after 20 days postadministration. Saline-treated mice developed swollen abdomens filled with ascitic L1210 tumors and died within 36 days. However, all the groups treated with MTO did not develop tumor in the peritoneal cavity during their life spans. Prolonged abdominal retention of MTO administered using CA-L The kinetics of MTO retention at the injection site differed significantly between free and CA-L/MTO. Near-infrared optical molecular imaging of MTO revealed that free MTO was retained in the abdominal cavity 1 hour after injection (Figure 5, A). However, by 6 hours postinjection, the MTO signal in the abdominal cavity had decreased to nearly background levels. In contrast, MTO administered using CA-L showed more pro-

Figure 5. Retention of MTO at the tumor sites. (A) Near-infrared fluorescence molecular imaging was used to monitor the retention of MTO in the abdominal cavity following injection of free MTO or MTO/CA-L. Images were collected at various times and normalized to the same scale. (B) Total photon counts in the abdominal regions of mice were measured after intraperitoneal treatment with free MTO or MTO/CA-L. Asterisk denotes significantly higher than free MTO-treated group (P b 0.05).

longed retention in the abdominal cavity, exhibiting significantly higher MTO-derived photon counts compared with free MTO for up to 12 hours after intraperitoneal injection (Figure 5, B). Reduced cardiac toxicity of MTO administered using CA-L The administration of MTO/CA-L induced lower cardiotoxicity compared with injection of free MTO. Mice treated with free MTO (Figure 6, B) showed an increase in apoptotic myocardia and heterogeneous nuclear location compared with saline-treated mice (Figure 6, A). In contrast, cardiac tissues of mice treated with the MTO/CA-L (Figure 6, C) exhibited histology similar to that of saline-treated mice. Consistent with the histology data, the frequency of apoptotic bodies and vacuole formation was higher in free MTO–treated mice (Figure 6, E) than in saline(Figure 6, D) or MTO/CA-L–treated groups (Figure 6, F).

Discussion In this study we demonstrated that the therapeutic index of MTO could be enhanced by the optimized liposomal delivery.

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Figure 6. Histological analysis and apoptotic assessment of cardiac tissues following MTO treatment. Heart sections from mice treated with saline (A, D), free MTO (B, E), or MTO/CA-L (C, F) were stained with hematoxylin and eosin (A–C) and analyzed by TUNEL assay to measure apoptosis (D–F). Black arrows indicate apoptotic bodies. White arrows indicate vacuole formation. Scale bar = 50 μm.

Specifically, we report that anionic CA-L allowed the convenient electrostatic surface loading of MTO at high efficiency, prolonged retention of MTO in the peritoneal cavity after injection, provided the greatest protection against peritoneal tumor-induced mortality in mice, and reduced MTO cardiotoxicity. The efficiencies of loading MTO onto anionic liposomes achieved here using electrostatic complexation were greater than 95%. We calculated the loading efficiencies of MTO by its fluorophore properties. For the analysis of the MTO fluorophore absorbance, all liposomal MTO formulations were dissolved in methanol. Because MTO was bound to the liposome electrostatically, but not covalently, it is unlikely that MTO was still bound to the disassembled lipid components of the liposomes in the methanol medium. Indeed, we scanned the excitation properties of MTO in free and liposomal form in methanol and observed no distinct differences in the excitation wavelengths. MTO has previously been encapsulated in neutral liposomes by remote-loading methods. However, this method suffers from relatively complicated preparation steps, which would increase the cost of manufacturing for clinical applications. Optimizing a remote-loading process for liposomal formulation of chemotherapeutic agent is thus likely to be relatively time-consuming and involve a substantial effort.18 Because electrostatic MTOloading technology has a high efficiency comparable to that of remote-loading technology, this single-step surface complexation process may reduce manufacturing time and prove to be more convenient and cost-effective for clinical applications. The differences in the strengths of electrostatic interaction between MTO and the various anionic liposomes might be explained by the pKa of the individual anionic lipids. The pKa of the succinate group in CHEMS is 5.6, whereas that of the phosphate in anionic lipids is 2. Among the anionic lipids, CA has two phosphate groups per molecule. This difference in pKa of the anionic groups in each anionic lipid might differentially influence the ionic strength of the negative charges at neutral pH and may result in different rates of release of free MTO molecules from the liposomes by heparin. Moreover, we used the

PEGylated lipid in the liposomes at the molar ratios ranging from 3.2% to 3.8% and measured zeta potential values of all liposome formulations. Although PEG layers on liposome surface can prevent the charge detection, our observation indicates that the use of PEGylated lipids less than 4 mol% may allow the detection of zeta potentials. We used the L1210 cell line to develop intraperitoneal tumor models. L1210, a murine leukemia cell line, has been used to develop mice tumor model by intraperitoneal inoculation.17,19,20 Similarly, we observed that intraperitoneal inoculation of L1210 cells has provided high reproducibility of developing tumors in the intraperitoneal sites of mice. Although the leukemia cell line may have different biological properties from human peritoneal carcinomatosis, we aimed to test the antitumor effects of intraperitoneally administered MTO liposomal formulations on the intraperitoneal tumor cells. In this study we intended to test the reduction of systemic toxicities of free MTO by use of various liposomal formulations. Actually, none of the groups treated with MTO developed tumor in the peritoneal cavity during their life spans. Moreover, the empty liposome formulations did not significantly affect the viability of cells. Thus, empty liposomes have been excluded in the in vivo test for evaluating the reduction of systemic toxicities of free MTO. In this study we hypothesized that the systemic toxicity of free MTO could be reduced by sustained-release MTO formulations. To evaluate the hypothesis we should eliminate the possible deaths of mice due to the overgrowth of established peritoneal tumors. Thus, we treated mice with various MTO formulations before development of intraperitoneal tumors. We observed that all the MTO formulations prevented the development of tumors in the peritoneal cavity but differed in the survival of mice. This observation indicates that the systemic toxicity of MTO could be governed by tailoring the liposomal formulations. Although none of the MTO-treated groups developed intraperitoneal tumors during their survival, free MTO–, MTO/ CHEMS-L–, and MTO/PG-L–treated groups showed even

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shorter survivals than the saline-treated group. Saline-treated mice died within 36 days due to the development of tumor. Except the saline-treated mice, none of the groups with free or liposomal MTO developed tumor in the peritoneal cavity. Instead, the mice treated with MTO died from the systemic toxicity of high-dose MTO. Because the deaths of mice due to the systemic MTO toxicity could be more acute as compared with those due to tumor growth, the mice treated with free MTO might have shown the shorter survival than the saline-treated group. Especially, the shorter survival of mice treated with MTO/ CHEMS-L or MTO/PG-L might be further explained by the lower binding affinities of MTO onto CHEMS-L or PG-L, resulting in the faster release of free MTO. Our results indicate that the stability of liposomes in vivo may vary depending on the compositions, and that the stability of MTO/CA-L may be the highest. The heparin competition assay reveals that MTO molecules associated with CA-L were hardly dissociated from CA-L even after mixing with heparin. Moreover, the biodistribution study of MTO using the optical imaging technique supports that MTO/CA-L showed prolonged retention as compared with free MTO in vivo. Taken together, the heparin competition and biodistribution studies provide evidence that MTO/CA-L may retain the stability in vivo. We have developed a simple method for preparing liposomal formulations of MTO by electrostatic surface loading of MTO onto anionic liposomes. Four MTO–anionic liposomes formulations were characterized physicochemically and screened in vitro and in vivo. We observed that MTO/CA-L showed the highest anticancer efficacy and the lowest toxicity among all tested liposomal MTO formulations and might be applicable for intraperitoneal chemotherapy.

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