Vol. 38, No. 3461 Biol. Pharm. Bull. 38, 461–469 (2015)
Regular Article
Liposomal Pemetrexed: Formulation, Characterization and in Vitro Cytotoxicity Studies for Effective Management of Malignant Pleural Mesothelioma Noha Essam Eldin,a,b Hanan Mohamed Elnahas,a Mahmoud Abd-Elghany Mahdy,a and Tatsuhiro Ishida*,b a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University; Zagazig 44 519, Egypt: and b Department of Pharmacokinetics and Biopharmaceutics, Institute of Health Biosciences, The University of Tokushima; Tokushima 770–8505, Japan. Received November 9, 2014; accepted December 8, 2014 Pemetrexed (PMX) is a newly developed multi-targeted anti-folate with promising clinical activity in many solid tumors including malignant pleural mesothelioma (MPM). However, PMX does not show sufficient anti-tumor activity in vivo when used alone either due to inefficient delivery of adequate concentrations to tumor tissue or dose-limiting side effects. In order to overcome these problems and to achieve potent anti-tumor activity, PMX was encapsulated into a liposomal delivery system. In the present study, various formulations of liposomal PMX were prepared. The effect of formulation parameters on the encapsulation efficiency of PMX within liposomes was evaluated. In addition, the influence of drug release rate on the in vitro cytotoxicity was investigated. Encapsulation of PMX within liposomes was remarkably increased by the incorporation of cholesterol within liposomal membranes and by increasing the total lipid concentration. Encapsulation efficiency was found to be unaffected by the type of phospholipid used or the inclusion of a cation lipid, DC-6-14. Interestingly, encapsulation of PMX within “fluid” liposomes was found to allow efficient release of PMX from liposomes resulting in a potent in vitro cytotoxicity against MPM MSTO-211H cell line. On the other hand, entrapment of PMX within “solid” liposomes substantially hindered PMX release from liposomes, and thus PMX failed to exert any in vitro cytotoxicity. These results suggest that encapsulation of PMX within “fluid” liposomes might represent a novel strategy to enhance the therapeutic efficacy of PMX while minimizing the side effect encountered by the non selective delivery of free PMX to various body tissues. Key words
liposome; malignant pleural mesothelioma; polyethylene glycol; pemetrexed
Pemetrexed (PMX) is a new-generation anti-folate, approved for the treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer, currently being evaluated for the treatment of a variety of other solid tumors.1,2) Unlike other classical anti-metabolites, such as methotrexate, which selectively target a single enzyme critical in purine and pyrimidine biosynthetic pathways, PMX has multi-targeted activity on the enzymes such as thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT), which participate in de novo purine/pyrimidine synthesis.3,4) This multiple enzyme–inhibitory properties of PMX create a combinatorial effect wherein inhibition of three enzymes at multiple sites gives an advantage in overcoming acquired or intrinsic resistance associated with over-expression or mutation of any one of the enzyme.5) However, like other chemotherapeutic agents, its cytotoxic efficacy is limited, in part, by the inadequate delivery to the target tissue and/or dose-limiting side effects.6,7) Currently, significant progress has been achieved by targeted delivery of cytotoxic drugs to tumor tissue, which could effectively minimize the adverse effects of chemotherapy,8,9) while maximizing its therapeutic efficacy via attaining adequate concentrations of the chemotherapeutic agent within the tumor tissue.10,11) Liposomes are one of the promising drug delivery systems used in cancer therapy, and a number of reviews have been published on the advancement of liposomal delivery as the
enabling technology for anti-cancer drug delivery.12,13) One of the major advantages of liposomal delivery is the ability of liposomes to alter the pharmacokinetics and biodistribution of the encapsulated agent.14,15) Liposomes of sizes ranging from 50 to 150 nm are able to capitalize on the discontinuities in the tumor vascular endothelium and extravagate more readily as compared to normal healthy endothelium.16,17) Combining with an impaired lymphatic system in tumor tissues, liposomal systems allow increased preferential accumulation of the encapsulated agent in tumor site with a concomitant decrease in the extent and types of non-specific toxicities. These advantages of liposomal delivery have culminated in two approved liposomal formulations of doxorubicin in the treatment of metastatic breast cancer, with the dose-limiting cardiotoxicity much reduced and tolerability greatly improved.18,19) Malignant pleural MPM is a locally invasive and rapidly fatal malignancy with a poor prognosis. The incidence of MPM is expected to peak at the coming decades especially in developing countries where the use of asbestos-containing materials is still very common.20) PMX, as a single agent or in combination with other chemotherapeutic agents such as platinum analogs or PEGylated liposomal doxorubicin,21,22) has been applied clinically as a first-line treatment of a wide variety of solid tumors, including MPM. However, the overall prognosis of patients with MPM remains very poor with response rates of approximately 40%.23) The objective of this study, therefore, is to evaluate the
To whom correspondence should be addressed. e-mail: [email protected] * © 2015 The Pharmaceutical Society of Japan
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potential of liposome as a delivery system for PMX. The physicochemical properties, stability and in vitro release studies were investigated in detail. The in vitro cytotoxicity of the formulated liposomal PMX was also evaluated using a human MPM cell line.
MATERIALS AND METHODS Materials Pemetrexed disodium (PMX; Alimta®), a freely water soluble crystalline powder with a molecular weight of 471.37 g/mol, was purchased from Eli Lilly (Indianapolis, IN, U.S.A.). Dioleoyl phosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoyl phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-n[methoxy(polyethylene glycol)-2000] (mPEG2000 -DSPE) were generously donated by NOF (Tokyo, Japan). Cholesterol (CHOL) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). A cationic lipid, O,O′-ditetradecanoylN-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14) was purchased from Sogo Pharmaceutical (Tokyo, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesque (Kyoto, Japan). All other reagents were of analytical grade. Tumor Cell Lines A human malignant pleural MPM cell line, MSTO-211H, was purchased from the American Type Culture Collection (Manassas, VA, U.S.A.) and was maintained in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum (Japan Bioserum, Hiroshima, Japan) and gentamicin (10 µg/mL). Cells were incubated under standard culture conditions (20% O2, 5% CO2, 37°C). Preparation of Liposomes All liposomal formulations were composed of the bilayer-forming phospholipid phosphatidylcholine, with fatty acyl chains of various lengths and degrees of saturation (HSPC, POPC/DOPE or DOPC/DOPE), in combination with or without cholesterol and/or a cationic lipid, DC-6-14. DOPE was added to act as a membrane fusion promoter.24) All formulations contained 5 mol% (relative to phospholipid) of mPEG2000 -DSPE. The detailed composition of different liposomal formulations was summarized in Table 1. Liposomes were prepared using the reverse phase-evaporation method as described previously.25) Briefly, lipids (50 mmol) were dissolved in 6 mL of chloroform–diethyl ether (1 : 2 v/v) and then 2 mL of PMX solution (25 mg/mL) in phosphate buffer saline (pH 7.4) was dropped into the lipid mixture to form a water/oil (W/O) emulsion. For preparation of “empty” Table 1.
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(no drug-containing) PEG-coated liposomes, phosphate buffer saline (pH 7.4) was added instead of PMX solution. The volume ratio of the aqueous to the organic phase was maintained at 1 : 3. The emulsion was sonicated for 15 min and then the organic phase was removed to form liposomes by evaporation in a rotary evaporator at 40°C under vacuum at 250 hPa for 1 h. Liposomes were sized by subsequent extrusion through polycarbonate membrane filters (Nuclepore, CA, U.S.A.) with pore sizes of 400 (×1), 200 (×2), 100 (×2) and 80 (×2) nm using an extruder device (Lipex Biomembranes Inc., Canada). The temperature of extrusion depended on the phosphatidylcholine component of the mixture. Fluid lipids whose phase transition temperatures are below room temperature (such as POPC, DOPC) were extruded at room temperature; other lipids were also extruded slightly above their phase transition temperatures (such as HSPC) at 65°C. The phospholipid concentration was evaluated by a phosphorus determination through an acidic digestion.26) Determination of Size and Zeta-Potential of PMXEntrapped Liposomes The average size and zeta potential of different formulations of PMX-entrapped liposomes were determined by a NICOMP 370 HPL submicron particle analyzer (Particle Sizing System, CA, U.S.A.) at 25±0.5°C. The mean particle size was measured based on photo correlation spectroscopy (dynamic light scattering, DLS) technique. The zeta potential was determined based on an electrophoretic light scattering (ELS) technique. The experiment was independently performed for 3 repeating samples per experimental group (n=3). Loading Percentage and Entrapment Efficiency of PMX in Liposomes The encapsulation efficiency of liposomes was examined after separating free PMX from liposomes by Sepharose CL-4B (Amersham Bioscience, Uppsala, Sweden) column chromatography.27) Entrapped PMX was then determined by lysis of liposomes with methanol. The PMX content was analyzed by a high performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with a C18 column (TSKgel ODS120T, TOSOH Bioscience) of a 4.6 mm×150 mm size. Phosphate buffer–acetronitrile (80 : 20) was used as a mobile phase at flow rate of 1 mL/min, an injection volume of 5 µL and at a wavelength of 254 nm. The PMX concentration was determined from the calibration curve of PMX at various concentrations. The experiment was independently performed for 3 repeating samples per experimental group (n=3). Experimental and theoretical percentages of PMX loading were calculated from Eqs. 1 and 2, respectively:
Composition of Different Liposomal PMX Formulations Formulation F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9
Composition HSPC : CHOL : mPEG2000-DSPE HSPC : mPEG2000-DSPE HSPC : CHOL : DC-6-14 : mPEG2000-DSPE DOPE : POPC : CHOL : mPEG2000-DSPE DOPE : POPC : mPEG2000-DSPE DOPE : POPC : CHOL : DC-6-14 : mPEG2000-DSPE DOPE : DOPC : CHOL : mPEG2000-DSPE DOPE : DOPC : mPEG2000-DSPE DOPE : DOPC : CHOL : DC-6-14 : mPEG2000-DSPE
The total phospholipid concentration was kept constant through all the formulation.
(Molar ratio) 5 : 3 : 0.25 5 : 0.25 5 : 3 : 2 : 0.25 3 : 2 : 3 : 0.25 3 : 2 : 0.25 3 : 2 : 3 : 2 : 0.25 3 : 2 : 3 : 0.25 3 : 2 : 0.25 3 : 2 : 3 : 2 : 0.25
Biol. Pharm. Bull. Vol. 38, No. 3 (2015)463
Experimental percentage of PMX loading PMX L (mg/mL) = ×100 PL L (mol) Theoretical percentage of PMX loading PMXinitial (mg/mL) = ×100 PLinitial (mol)
(1)
(2)
where PMX L and PLL represent the amounts of the entrapped PMX and phospholipids after the column separation of free PMX, respectively. PMXinitial and PLinitial represent the initial amounts of PMX and phospholipids used, respectively. Entrapment efficiency of PMX in liposomes was calculated from the following equation: Entrapment efficiency (%) Experimental percentage of PMX loading from Eq. 1 = ×100 Theoretical percentage of PMX loading from Eq. 2
Stability of PMX-Entrapped Liposomes The stability of PMX-entrapped liposomes was evaluated after storage at 4.5°C, under nitrogen gas. At days 0, 7, and 14, size of liposomes was determined by a particle size analyzer as described above. The experiment was independently performed for 3 repeating samples per experimental group (n=3). In Vitro Release Study The in vitro leakage of PMX from liposomes was measured using a dialysis method.28) Liposomes, at a concentration of 0.5 m M phospholipid, were diluted in 50% mouse serum, placed in a dialysis cassette with a molecular weight cutoff of 10 kDa, and dialyzed against 200 mL of isotonic phosphate buffer (pH 7.4) at 37°C. The concentration of lipid was selected to approximate the liposome concentration expected in the blood compartment of a 20 g mouse receiving liposomal PMX at a dose of 25 mg PMX/kg body weight. At various time points, aliquots (300–500 µL) were withdrawn from the cassette and stored at 4°C until analysis. The removed samples were replaced by equal volumes of isotonic phosphate buffer (pH 7.4) to maintain a constant volume for the receiving medium. PMX was quantified by RP-HPLC as described above. In Vitro Cytotoxicity Assay Cytotoxicity of various PMX liposomal formulations was determined by MTT assay, as described previously.29) Briefly, MSTO-211H cells (2×103) were seeded onto 96-well plates in 200 µL RPMI-1640 medium containing 10% fetal bovine serum (FBS) and incubated for 24 h prior to drug addition. The culture medium Table 2. F F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9
was replaced with fresh medium containing either free PMX or different PMX liposomal formulations at a concentration range of 0.01 to 10 µg/mL PMX. At 72 h post-incubation, the cells were washed twice with cold phosphate buffered saline (PBS, 37 m M NaCl, 2.7 m M KCl, 8.1 m M Na2HPO4 and 1.47 m M KH2PO4; pH 7.4) and cell viability was determined by MTT assay. Tumor cells were incubated with 50 µL MTT solution (5 mg/mL in PBS) for 4 h at 37°C. Then 150 µL of an acidic isopropanol solution (containing 0.04 N HCl) was added to each well to dissolve formazan crystals. The absorbance of each well was read at 570 nm on a microplate reader SunriseR (TEKAN Japan, Kanagawa, Japan). Data shown are representative of three independent experiments. The untreated cells served as 100% cell viability, and the viability percentage was calculated as follows: Viability (%) =(AbsT /AbsC ×100% where AbsT and AbsC are the absorbance of the treated well and control well, respectively.30) The IC50, defined as the concentration of a drug that is required for 50% inhibition in vitro, was also calculated. Statistical Analysis All values are expressed as mean±S.D. Statistical analysis was performed with a twotailed unpaired t test and one way ANOVA using Graphpad InStat software (Graphpad Software, CA, U.S.A.). The level of significance was set at pHSPC. These results were consistent with previously published data for doxorubicin and other drugs, which demonstrated that the fluidity of the liposomal membrane plays an important role in the release of liposomal contents.28,42) Noticeably, the release of PMX from HSPC liposomes was not observed even after 24 h. The saturated fatty acid residues in HSPC might result in increased organization of the phospholipid molecules in the bilayer structure, increase stability/phase transition temperature, and thus, hinder the release of PMX from the interior aqueous phase of liposomes.43) Cholesterol has been reported to increase the stability of liposomes via forming hydrogen bonding with liposomal membrane phospholipids.33) This condensing effect of cholesterol is assumed to increase the rigidity of liposomal membrane and thus reduce the premature release of liposomal contents. Consequently, we investigating the effect of the incorporation of cholesterol within liposomal membrane on the release profiles of PMX from fluid liposomes (i.e., liposomes containing either POPC or DOPC as the membrane forming phospholipid). As shown in Fig. 3, incorporation of cholesterol into bilayer membranes of fluid liposomes greatly reduces the release rates of
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Fig. 4. Effect of the Presence of a Cationic Lipid, DC-6-14, on Release Profiles of PMX from Different PMX Liposomal Formulation (A) PMX-containing cationic HSPC liposomes, (B) PMX-containing cationic POPC liposomes or (C) PMX-containing cationic DOPC liposomes. Data represent mean±S.D. (n=3).
PMX, which diffuse across the lipid membrane down a PMX concentration gradient, presumably due to increase the rigidity of a liposomal membrane. After 24 h, only 20% of PMX was
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Fig. 5.
In Vitro Cytotoxicity of Various PMX Formulations
MSTO-211H cells (2×103) were incubated with media containing serial dilutions of various PMX formulations (free PMX or liposomal PMX formulations). Following a 72-h incubation at 37°C, cell survival was determined by the MTT assay. Data represent mean±S.D. (n=3).
released from cholesterol-containing POPC liposomes compared to 80% of PMX released from cholesterol-free POPC liposomes. Similarly, incorporation of cholesterol into bilayer membranes of DOPC liposomes significantly decreased the release rate of PMX from liposomes, compared to cholesterolfree DOPC liposomes (13% vs. 65%, respectively). Finally, we investigated the effect of the incorporation of a cationic lipid, DC-6-14, into the liposomal membrane on PMX release. As shown in Fig. 4, incorporation of DC-6-14 was found not to affect the release profile of PMX from all the liposomal formulations. These results are consistent with those of Abu Lila et al.25) who reported that no remarkable differences were observed on the oxaliplatin release profiles HSPC liposomes. In Vitro Cytotoxicity of PMX Formulations Recent studies have shown that cationic liposomes, by virtue of their surface positive charge, have a propensity to selectively bind and/or internalize into tumor cells, compared to neutral counterparts. Therefore, we investigated the in vitro cytotoxicity of free PMX and different cationic liposomal formulations of PMX using MTT assay. As shown in Fig. 5, PMX encapsulated within fluid-phase cationic liposomes (cationic POPC- and DOPC-liposomes) showed a remarkable in vitro cytotoxic effect against MSTO-211 H cells. The IC50 values were 61.46±2.1 ng/mL for PMX-containing cationic POPC liposomes and 129.55±3.2 ng/mL for PMX-containing cationic DOPC liposomes. Surprisingly, PMX encapsulated within solid-phase cationic liposomes (HSPC liposomes) failed to exert any cytotoxic effect against MSTO-211H cells at the tested concentration range of PMX. These results might be attributed to the slowest release rate of PMX from HSPC liposomes, compared to that from fluid-phase liposomes, as manifested in Fig. 4. All liposomal PMX formulations were significantly less cytotoxic against MSTO-211H cells than free PMX (p
Regular Article
Liposomal Pemetrexed: Formulation, Characterization and in Vitro Cytotoxicity Studies for Effective Management of Malignant Pleural Mesothelioma Noha Essam Eldin,a,b Hanan Mohamed Elnahas,a Mahmoud Abd-Elghany Mahdy,a and Tatsuhiro Ishida*,b a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University; Zagazig 44 519, Egypt: and b Department of Pharmacokinetics and Biopharmaceutics, Institute of Health Biosciences, The University of Tokushima; Tokushima 770–8505, Japan. Received November 9, 2014; accepted December 8, 2014 Pemetrexed (PMX) is a newly developed multi-targeted anti-folate with promising clinical activity in many solid tumors including malignant pleural mesothelioma (MPM). However, PMX does not show sufficient anti-tumor activity in vivo when used alone either due to inefficient delivery of adequate concentrations to tumor tissue or dose-limiting side effects. In order to overcome these problems and to achieve potent anti-tumor activity, PMX was encapsulated into a liposomal delivery system. In the present study, various formulations of liposomal PMX were prepared. The effect of formulation parameters on the encapsulation efficiency of PMX within liposomes was evaluated. In addition, the influence of drug release rate on the in vitro cytotoxicity was investigated. Encapsulation of PMX within liposomes was remarkably increased by the incorporation of cholesterol within liposomal membranes and by increasing the total lipid concentration. Encapsulation efficiency was found to be unaffected by the type of phospholipid used or the inclusion of a cation lipid, DC-6-14. Interestingly, encapsulation of PMX within “fluid” liposomes was found to allow efficient release of PMX from liposomes resulting in a potent in vitro cytotoxicity against MPM MSTO-211H cell line. On the other hand, entrapment of PMX within “solid” liposomes substantially hindered PMX release from liposomes, and thus PMX failed to exert any in vitro cytotoxicity. These results suggest that encapsulation of PMX within “fluid” liposomes might represent a novel strategy to enhance the therapeutic efficacy of PMX while minimizing the side effect encountered by the non selective delivery of free PMX to various body tissues. Key words
liposome; malignant pleural mesothelioma; polyethylene glycol; pemetrexed
Pemetrexed (PMX) is a new-generation anti-folate, approved for the treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer, currently being evaluated for the treatment of a variety of other solid tumors.1,2) Unlike other classical anti-metabolites, such as methotrexate, which selectively target a single enzyme critical in purine and pyrimidine biosynthetic pathways, PMX has multi-targeted activity on the enzymes such as thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT), which participate in de novo purine/pyrimidine synthesis.3,4) This multiple enzyme–inhibitory properties of PMX create a combinatorial effect wherein inhibition of three enzymes at multiple sites gives an advantage in overcoming acquired or intrinsic resistance associated with over-expression or mutation of any one of the enzyme.5) However, like other chemotherapeutic agents, its cytotoxic efficacy is limited, in part, by the inadequate delivery to the target tissue and/or dose-limiting side effects.6,7) Currently, significant progress has been achieved by targeted delivery of cytotoxic drugs to tumor tissue, which could effectively minimize the adverse effects of chemotherapy,8,9) while maximizing its therapeutic efficacy via attaining adequate concentrations of the chemotherapeutic agent within the tumor tissue.10,11) Liposomes are one of the promising drug delivery systems used in cancer therapy, and a number of reviews have been published on the advancement of liposomal delivery as the
enabling technology for anti-cancer drug delivery.12,13) One of the major advantages of liposomal delivery is the ability of liposomes to alter the pharmacokinetics and biodistribution of the encapsulated agent.14,15) Liposomes of sizes ranging from 50 to 150 nm are able to capitalize on the discontinuities in the tumor vascular endothelium and extravagate more readily as compared to normal healthy endothelium.16,17) Combining with an impaired lymphatic system in tumor tissues, liposomal systems allow increased preferential accumulation of the encapsulated agent in tumor site with a concomitant decrease in the extent and types of non-specific toxicities. These advantages of liposomal delivery have culminated in two approved liposomal formulations of doxorubicin in the treatment of metastatic breast cancer, with the dose-limiting cardiotoxicity much reduced and tolerability greatly improved.18,19) Malignant pleural MPM is a locally invasive and rapidly fatal malignancy with a poor prognosis. The incidence of MPM is expected to peak at the coming decades especially in developing countries where the use of asbestos-containing materials is still very common.20) PMX, as a single agent or in combination with other chemotherapeutic agents such as platinum analogs or PEGylated liposomal doxorubicin,21,22) has been applied clinically as a first-line treatment of a wide variety of solid tumors, including MPM. However, the overall prognosis of patients with MPM remains very poor with response rates of approximately 40%.23) The objective of this study, therefore, is to evaluate the
To whom correspondence should be addressed. e-mail: [email protected] * © 2015 The Pharmaceutical Society of Japan
Biol. Pharm. Bull.
462
potential of liposome as a delivery system for PMX. The physicochemical properties, stability and in vitro release studies were investigated in detail. The in vitro cytotoxicity of the formulated liposomal PMX was also evaluated using a human MPM cell line.
MATERIALS AND METHODS Materials Pemetrexed disodium (PMX; Alimta®), a freely water soluble crystalline powder with a molecular weight of 471.37 g/mol, was purchased from Eli Lilly (Indianapolis, IN, U.S.A.). Dioleoyl phosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoyl phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-n[methoxy(polyethylene glycol)-2000] (mPEG2000 -DSPE) were generously donated by NOF (Tokyo, Japan). Cholesterol (CHOL) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). A cationic lipid, O,O′-ditetradecanoylN-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14) was purchased from Sogo Pharmaceutical (Tokyo, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesque (Kyoto, Japan). All other reagents were of analytical grade. Tumor Cell Lines A human malignant pleural MPM cell line, MSTO-211H, was purchased from the American Type Culture Collection (Manassas, VA, U.S.A.) and was maintained in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum (Japan Bioserum, Hiroshima, Japan) and gentamicin (10 µg/mL). Cells were incubated under standard culture conditions (20% O2, 5% CO2, 37°C). Preparation of Liposomes All liposomal formulations were composed of the bilayer-forming phospholipid phosphatidylcholine, with fatty acyl chains of various lengths and degrees of saturation (HSPC, POPC/DOPE or DOPC/DOPE), in combination with or without cholesterol and/or a cationic lipid, DC-6-14. DOPE was added to act as a membrane fusion promoter.24) All formulations contained 5 mol% (relative to phospholipid) of mPEG2000 -DSPE. The detailed composition of different liposomal formulations was summarized in Table 1. Liposomes were prepared using the reverse phase-evaporation method as described previously.25) Briefly, lipids (50 mmol) were dissolved in 6 mL of chloroform–diethyl ether (1 : 2 v/v) and then 2 mL of PMX solution (25 mg/mL) in phosphate buffer saline (pH 7.4) was dropped into the lipid mixture to form a water/oil (W/O) emulsion. For preparation of “empty” Table 1.
Vol. 38, No. 3 (2015)
(no drug-containing) PEG-coated liposomes, phosphate buffer saline (pH 7.4) was added instead of PMX solution. The volume ratio of the aqueous to the organic phase was maintained at 1 : 3. The emulsion was sonicated for 15 min and then the organic phase was removed to form liposomes by evaporation in a rotary evaporator at 40°C under vacuum at 250 hPa for 1 h. Liposomes were sized by subsequent extrusion through polycarbonate membrane filters (Nuclepore, CA, U.S.A.) with pore sizes of 400 (×1), 200 (×2), 100 (×2) and 80 (×2) nm using an extruder device (Lipex Biomembranes Inc., Canada). The temperature of extrusion depended on the phosphatidylcholine component of the mixture. Fluid lipids whose phase transition temperatures are below room temperature (such as POPC, DOPC) were extruded at room temperature; other lipids were also extruded slightly above their phase transition temperatures (such as HSPC) at 65°C. The phospholipid concentration was evaluated by a phosphorus determination through an acidic digestion.26) Determination of Size and Zeta-Potential of PMXEntrapped Liposomes The average size and zeta potential of different formulations of PMX-entrapped liposomes were determined by a NICOMP 370 HPL submicron particle analyzer (Particle Sizing System, CA, U.S.A.) at 25±0.5°C. The mean particle size was measured based on photo correlation spectroscopy (dynamic light scattering, DLS) technique. The zeta potential was determined based on an electrophoretic light scattering (ELS) technique. The experiment was independently performed for 3 repeating samples per experimental group (n=3). Loading Percentage and Entrapment Efficiency of PMX in Liposomes The encapsulation efficiency of liposomes was examined after separating free PMX from liposomes by Sepharose CL-4B (Amersham Bioscience, Uppsala, Sweden) column chromatography.27) Entrapped PMX was then determined by lysis of liposomes with methanol. The PMX content was analyzed by a high performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with a C18 column (TSKgel ODS120T, TOSOH Bioscience) of a 4.6 mm×150 mm size. Phosphate buffer–acetronitrile (80 : 20) was used as a mobile phase at flow rate of 1 mL/min, an injection volume of 5 µL and at a wavelength of 254 nm. The PMX concentration was determined from the calibration curve of PMX at various concentrations. The experiment was independently performed for 3 repeating samples per experimental group (n=3). Experimental and theoretical percentages of PMX loading were calculated from Eqs. 1 and 2, respectively:
Composition of Different Liposomal PMX Formulations Formulation F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9
Composition HSPC : CHOL : mPEG2000-DSPE HSPC : mPEG2000-DSPE HSPC : CHOL : DC-6-14 : mPEG2000-DSPE DOPE : POPC : CHOL : mPEG2000-DSPE DOPE : POPC : mPEG2000-DSPE DOPE : POPC : CHOL : DC-6-14 : mPEG2000-DSPE DOPE : DOPC : CHOL : mPEG2000-DSPE DOPE : DOPC : mPEG2000-DSPE DOPE : DOPC : CHOL : DC-6-14 : mPEG2000-DSPE
The total phospholipid concentration was kept constant through all the formulation.
(Molar ratio) 5 : 3 : 0.25 5 : 0.25 5 : 3 : 2 : 0.25 3 : 2 : 3 : 0.25 3 : 2 : 0.25 3 : 2 : 3 : 2 : 0.25 3 : 2 : 3 : 0.25 3 : 2 : 0.25 3 : 2 : 3 : 2 : 0.25
Biol. Pharm. Bull. Vol. 38, No. 3 (2015)463
Experimental percentage of PMX loading PMX L (mg/mL) = ×100 PL L (mol) Theoretical percentage of PMX loading PMXinitial (mg/mL) = ×100 PLinitial (mol)
(1)
(2)
where PMX L and PLL represent the amounts of the entrapped PMX and phospholipids after the column separation of free PMX, respectively. PMXinitial and PLinitial represent the initial amounts of PMX and phospholipids used, respectively. Entrapment efficiency of PMX in liposomes was calculated from the following equation: Entrapment efficiency (%) Experimental percentage of PMX loading from Eq. 1 = ×100 Theoretical percentage of PMX loading from Eq. 2
Stability of PMX-Entrapped Liposomes The stability of PMX-entrapped liposomes was evaluated after storage at 4.5°C, under nitrogen gas. At days 0, 7, and 14, size of liposomes was determined by a particle size analyzer as described above. The experiment was independently performed for 3 repeating samples per experimental group (n=3). In Vitro Release Study The in vitro leakage of PMX from liposomes was measured using a dialysis method.28) Liposomes, at a concentration of 0.5 m M phospholipid, were diluted in 50% mouse serum, placed in a dialysis cassette with a molecular weight cutoff of 10 kDa, and dialyzed against 200 mL of isotonic phosphate buffer (pH 7.4) at 37°C. The concentration of lipid was selected to approximate the liposome concentration expected in the blood compartment of a 20 g mouse receiving liposomal PMX at a dose of 25 mg PMX/kg body weight. At various time points, aliquots (300–500 µL) were withdrawn from the cassette and stored at 4°C until analysis. The removed samples were replaced by equal volumes of isotonic phosphate buffer (pH 7.4) to maintain a constant volume for the receiving medium. PMX was quantified by RP-HPLC as described above. In Vitro Cytotoxicity Assay Cytotoxicity of various PMX liposomal formulations was determined by MTT assay, as described previously.29) Briefly, MSTO-211H cells (2×103) were seeded onto 96-well plates in 200 µL RPMI-1640 medium containing 10% fetal bovine serum (FBS) and incubated for 24 h prior to drug addition. The culture medium Table 2. F F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9
was replaced with fresh medium containing either free PMX or different PMX liposomal formulations at a concentration range of 0.01 to 10 µg/mL PMX. At 72 h post-incubation, the cells were washed twice with cold phosphate buffered saline (PBS, 37 m M NaCl, 2.7 m M KCl, 8.1 m M Na2HPO4 and 1.47 m M KH2PO4; pH 7.4) and cell viability was determined by MTT assay. Tumor cells were incubated with 50 µL MTT solution (5 mg/mL in PBS) for 4 h at 37°C. Then 150 µL of an acidic isopropanol solution (containing 0.04 N HCl) was added to each well to dissolve formazan crystals. The absorbance of each well was read at 570 nm on a microplate reader SunriseR (TEKAN Japan, Kanagawa, Japan). Data shown are representative of three independent experiments. The untreated cells served as 100% cell viability, and the viability percentage was calculated as follows: Viability (%) =(AbsT /AbsC ×100% where AbsT and AbsC are the absorbance of the treated well and control well, respectively.30) The IC50, defined as the concentration of a drug that is required for 50% inhibition in vitro, was also calculated. Statistical Analysis All values are expressed as mean±S.D. Statistical analysis was performed with a twotailed unpaired t test and one way ANOVA using Graphpad InStat software (Graphpad Software, CA, U.S.A.). The level of significance was set at pHSPC. These results were consistent with previously published data for doxorubicin and other drugs, which demonstrated that the fluidity of the liposomal membrane plays an important role in the release of liposomal contents.28,42) Noticeably, the release of PMX from HSPC liposomes was not observed even after 24 h. The saturated fatty acid residues in HSPC might result in increased organization of the phospholipid molecules in the bilayer structure, increase stability/phase transition temperature, and thus, hinder the release of PMX from the interior aqueous phase of liposomes.43) Cholesterol has been reported to increase the stability of liposomes via forming hydrogen bonding with liposomal membrane phospholipids.33) This condensing effect of cholesterol is assumed to increase the rigidity of liposomal membrane and thus reduce the premature release of liposomal contents. Consequently, we investigating the effect of the incorporation of cholesterol within liposomal membrane on the release profiles of PMX from fluid liposomes (i.e., liposomes containing either POPC or DOPC as the membrane forming phospholipid). As shown in Fig. 3, incorporation of cholesterol into bilayer membranes of fluid liposomes greatly reduces the release rates of
Vol. 38, No. 3 (2015)
Fig. 4. Effect of the Presence of a Cationic Lipid, DC-6-14, on Release Profiles of PMX from Different PMX Liposomal Formulation (A) PMX-containing cationic HSPC liposomes, (B) PMX-containing cationic POPC liposomes or (C) PMX-containing cationic DOPC liposomes. Data represent mean±S.D. (n=3).
PMX, which diffuse across the lipid membrane down a PMX concentration gradient, presumably due to increase the rigidity of a liposomal membrane. After 24 h, only 20% of PMX was
Biol. Pharm. Bull. Vol. 38, No. 3 (2015)467
Fig. 5.
In Vitro Cytotoxicity of Various PMX Formulations
MSTO-211H cells (2×103) were incubated with media containing serial dilutions of various PMX formulations (free PMX or liposomal PMX formulations). Following a 72-h incubation at 37°C, cell survival was determined by the MTT assay. Data represent mean±S.D. (n=3).
released from cholesterol-containing POPC liposomes compared to 80% of PMX released from cholesterol-free POPC liposomes. Similarly, incorporation of cholesterol into bilayer membranes of DOPC liposomes significantly decreased the release rate of PMX from liposomes, compared to cholesterolfree DOPC liposomes (13% vs. 65%, respectively). Finally, we investigated the effect of the incorporation of a cationic lipid, DC-6-14, into the liposomal membrane on PMX release. As shown in Fig. 4, incorporation of DC-6-14 was found not to affect the release profile of PMX from all the liposomal formulations. These results are consistent with those of Abu Lila et al.25) who reported that no remarkable differences were observed on the oxaliplatin release profiles HSPC liposomes. In Vitro Cytotoxicity of PMX Formulations Recent studies have shown that cationic liposomes, by virtue of their surface positive charge, have a propensity to selectively bind and/or internalize into tumor cells, compared to neutral counterparts. Therefore, we investigated the in vitro cytotoxicity of free PMX and different cationic liposomal formulations of PMX using MTT assay. As shown in Fig. 5, PMX encapsulated within fluid-phase cationic liposomes (cationic POPC- and DOPC-liposomes) showed a remarkable in vitro cytotoxic effect against MSTO-211 H cells. The IC50 values were 61.46±2.1 ng/mL for PMX-containing cationic POPC liposomes and 129.55±3.2 ng/mL for PMX-containing cationic DOPC liposomes. Surprisingly, PMX encapsulated within solid-phase cationic liposomes (HSPC liposomes) failed to exert any cytotoxic effect against MSTO-211H cells at the tested concentration range of PMX. These results might be attributed to the slowest release rate of PMX from HSPC liposomes, compared to that from fluid-phase liposomes, as manifested in Fig. 4. All liposomal PMX formulations were significantly less cytotoxic against MSTO-211H cells than free PMX (p
