Regular Article
Liposomal Pemetrexed: Formulation, Characterization and in Vitro Cytotoxicity Studies for Effective Management of Malignant Pleural Mesothelioma
2015 年 38 巻 3 号 p. 461-469
詳細
2015 年 38 巻 3 号 p. 461-469
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.
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 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.
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), dioleoyl-phosphatidylethanolamine (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′-ditetradecanoyl-N-(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 LinesA 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% heat-inactivated 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 LiposomesAll 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” (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)
| Formulation | Composition | (Molar ratio) |
|---|---|---|
| F-1 | HSPC : CHOL : mPEG2000-DSPE | 5 : 3 : 0.25 |
| F-2 | HSPC : mPEG2000-DSPE | 5 : 0.25 |
| F-3 | HSPC : CHOL : DC-6-14 : mPEG2000-DSPE | 5 : 3 : 2 : 0.25 |
| F-4 | DOPE : POPC : CHOL : mPEG2000-DSPE | 3 : 2 : 3 : 0.25 |
| F-5 | DOPE : POPC : mPEG2000-DSPE | 3 : 2 : 0.25 |
| F-6 | DOPE : POPC : CHOL : DC-6-14 : mPEG2000-DSPE | 3 : 2 : 3 : 2 : 0.25 |
| F-7 | DOPE : DOPC : CHOL : mPEG2000-DSPE | 3 : 2 : 3 : 0.25 |
| F-8 | DOPE : DOPC : mPEG2000-DSPE | 3 : 2 : 0.25 |
| F-9 | DOPE : DOPC : CHOL : DC-6-14 : mPEG2000-DSPE | 3 : 2 : 3 : 2 : 0.25 |
The total phospholipid concentration was kept constant through all the formulation.
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 LiposomesThe 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:
 | (1) |
 | (2) |
Entrapment efficiency of PMX in liposomes was calculated from the following equation:
 |
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 StudyThe in vitro leakage of PMX from liposomes was measured using a dialysis method.28) Liposomes, at a concentration of 0.5 mM 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 AssayCytotoxicity 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 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 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.47 mM 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 Sunrise-R (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:
 |
The IC50, defined as the concentration of a drug that is required for 50% inhibition in vitro, was also calculated.
Statistical AnalysisAll values are expressed as mean±S.D. Statistical analysis was performed with a two-tailed unpaired t test and one way ANOVA using Graphpad InStat software (Graphpad Software, CA, U.S.A.). The level of significance was set at p<0.05.
The particle sizes of the prepared liposomes determined by the particle size analyzer are shown in Table 2, which clearly shows particle size increasing with the addition of cholesterol. Cholesterol was reported to affect the lipid packing structure and the orientation of the head groups along the membrane interface region.31) We assume that the increase in size of cholesterol-containing liposomes, compared to cholesterol-free counterparts, could be attributed to the presence of cholesterol between the lipid molecules, which straightens the acyl chains that were tilting sideway in cholesterol-free liposomes and fills the gaps between the acyl chains leading to the thickening of the bilayer membrane, and thereby, increasing the total liposome diameter.32) In addition to lipid packing changes, cholesterol is reported to enhance the extent of hydration around the interfacial membrane region, which also might contribute to the enlargement of the liposome membrane.33) These results are consistent with those of Tseng et al.34) who reported that liposomes incorporated with cholesterol showed higher particle sizes than cholesterol-free counterparts.
| F | Composition (molar ratio) | Size (nm) | Zeta potential (mV) |
|---|---|---|---|
| F-1 | HSPC : CHOL : mPEG2000-DSPE 5 : 3 : 0.25 | 134.5±17.6 | −6.8±0.7 |
| F-2 | HSPC : mPEG2000-DSPE 5 : 0.25 | 125.4±12.7 | −6.2±1.3 |
| F-3 | HSPC : CHOL : DC-6-14 : mPEG2000-DSPE 5 : 3 : 2 : 0.25 | 139.6±9.4 | 12.1±1.2 |
| F-4 | DOPE : POPC : CHOL : mPEG2000-DSPE 3 : 2 : 3 : 0.25 | 152.9±29.7 | −4.1±0.9 |
| F-5 | DOPE : POPC : mPEG2000-DSPE 3 : 2 : 0.25 | 129.5±11.6 | −4.8±1.0 |
| F-6 | DOPE : POPC : CHOL : DC-6-14 : mPEG2000-DSPE 3 : 2 : 3 : 2 : 0.25 | 134.5±17.6 | 21.7±2.9 |
| F-7 | DOPE : DOPC : CHOL : mPEG2000-DSPE 3 : 2 : 3 : 0.25 | 149.3±21.3 | −6.1±0.6 |
| F-8 | DOPE : DOPC : mPEG2000-DSPE 3 : 2 : 0.25 | 119.5±12.1 | −7.9±1.3 |
| F-9 | DOPE : DOPC : CHOL : DC-6-14 : mPEG2000-DSPE 3 : 2 : 3 : 2 : 0.25 | 142.6±18.5 | 19.2±2.1 |
Data were obtained with three liposome preparations which were prepared independently.
The liposome’s zeta potential, which is directly related to its net charge, was also measured by the NICOMP 370 HPL zeta-sizer. Inclusion of a positively charged lipid (DC-6-14) was found to substantially increase the zeta potentials of the prepared liposomes (Table 2).
Effect of Phospholipid Type and the Presence of Cholesterol and/or Cationic Lipids on Encapsulation Efficiency of PMXThe ability of liposomes to encapsulate drugs within a closed lipid bilayer membrane was found to be affected by each of the type of the phospholipid used and the presence or absence of cholesterol within the bilayer membrane.35,36) Therefore, we investigated the effect of different types of phospholipids, solid-phase phospholipid (HSPC) or fluid-phase phospholipids (POPC or DOPC), on the encapsulation efficiency of PMX. Results in Table 3 show that the encapsulation efficiency of PMX within liposomes prepared from either HSPC, POPC or DOPC was comparable. However, the encapsulation efficiency of PMX was increased significantly by the incorporation of cholesterol in the liposomal membrane, rather than the type of phospholipids (Table 3). The effect of cholesterol on increasing the encapsulation efficiency of PMX within liposomes was found to be a function of cholesterol induced increase in the bilayer surface area and the overall liposomal size.31)
| Formulation | Composition (molar ratio) | Encapsulation efficiency (EE (%)) |
|---|---|---|
| F-1 | HSPC : CHOL : mPEG2000-DSPE(5 : 3 : 0.25) | 14.8±2.8 |
| F-2 | HSPC : mPEG2000-DSPE(5 : 0.25) | 10.2±1.3 |
| F-3 | HSPC : CHOL : DC-6-14 : mPEG2000-DSPE(5 : 3 : 2 : 0.25) | 13.9±1.9 |
| F-4 | DOPE : POPC : CHOL : mPEG2000-DSPE(3 : 2 : 3 : 0.25) | 14.1±2.1 |
| F-5 | DOPE : POPC : mPEG2000-DSPE(3 : 2 : 0.25) | 9.9±1.7 |
| F-6 | DOPE : POPC : CHOL : DC-6-14 : mPEG2000-DSPE(3 : 2 : 3 : 2 : 0.25) | 13.6±1.3 |
| F-7 | DOPE : DOPC : CHOL : mPEG2000-DSPE(3 : 2 : 3 : 0.25) | 13.8±2.3 |
| F-8 | DOPE : DOPC : mPEG2000-DSPE(3 : 2 : 0.25) | 8.6±1.2 |
| F-9 | DOPE : DOPC : CHOL : DC-6-14 : mPEG2000-DSPE(3 : 2 : 3 : 2 : 0.25) | 12.9±2.9 |
The inclusion of a cationic lipid, DC-6-14, in the liposomal membrane was found not to affect the entrapment of PMX within the liposomes (Table 3). Similar results were observed by Abu Lila et al.25) who reported that the encapsulation efficiency of oxaliplatin within HSPC/Chol liposomes was not affected by the inclusion of DC-6-14, as a positively charge imparting lipid.
Effect of Total Lipid Concentration on Encapsulation Efficiency of PMXThe effect of total lipid concentration on the percent PMX encapsulated into either HSPC, POPC or DOPC liposomes is illustrated in Table 4. The entrapment efficiency of PMX was increased from 4.2±0.7 to 14.8±2.8%, from 3.9±0.6 to 14.1±2.18%, and from 3.7±1.1 to 13.8±2.3%, for HSPC-, POPC- and DOPC-liposomes, respectively, as the total lipid concentration was increased from 10 to 50 µmol/mL. These results are consistent with previous reports revealing that increasing the total lipid concentration favors the formation of large unilamellar vesicles/multilamellar vesicles (MLV), and thereby, increases the trapped volume for the encapsulated drug within liposomes.37,38)
| Formulation | Lipid concentration (µmol/mL) | Encapsulation efficiency (EE (%)) |
|---|---|---|
| HSPC liposome | 10 | 4.2±0.7 |
| 25 | 6.7±1.5 | |
| 50 | 14.8±2.8 | |
| POPC liposomes | 10 | 3.9±0.6 |
| 25 | 8.1±1.8 | |
| 50 | 14.1±2.1 | |
| DOPC liposomes | 10 | 3.7±1.1 |
| 25 | 7.3±0.8 | |
| 50 | 13.8±2.3 |
Increasing PMX concentration from 5 to 25 mg/mL was found to increase the percentage of PMX loading, however, the entrapment efficiency of PMX tended to decrease for all formulations (Table 5). The highest entrapment efficiencies of PMX were obtained for PMX at a dose of 5 mg/mL. These results are in agreement with those of Srisuk et al.39) who found that, while the percentage loading of methotrexate increased with increasing methotrexate dose, the entrapment efficiency decreased with increasing methotrexate dose.
| Formulation | PMX concentration (mg/mL) | PMX loading (%) | Encapsulation efficiency (%) |
|---|---|---|---|
| HSPC liposome | 5 | 9.4±1.5 | 20.8±1.1 |
| 10 | 12.8±1.3 | 18.1±1.4 | |
| 25 | 16.5±2.2 | 14.8±2.8 | |
| POPC liposomes | 5 | 8.2±1.1 | 19.6±0.8 |
| 10 | 11.5±1.6 | 17.5±0.4 | |
| 25 | 18.2±1.8 | 14.1±2.1 | |
| DOPC liposomes | 5 | 8.1±1.9 | 18.9±1.3 |
| 10 | 9.1±2.7 | 16.4±0.9 | |
| 25 | 10.7±1.8 | 13.8±2.3 |
One of the most important properties of liposomal lipid bilayer is the relative fluidity/mobility of each individual lipid molecule in the bilayer, which can significantly affect the physical stability of the prepared liposomes. Phase transition temperature (Tm), the temperature at which phospholipids changed from solid crystalline state into liquid crystalline state, has been reported to determine the fluidity/mobility of liposomal lipid bilayer.40) Therefore, the effect of phospholipids type, with different Tm values, on the liposomal physical stability was investigated.
Following storage at 4°C for 7 and 14 d, the particle sizes of different liposomal PMX formulations were similar to their initial sizes, although some significant differences were observed in some formulations especially those composing of DOPC as a membrane-forming lipid (Fig. 1). The increase in the size of DOPC liposomes may be attributed to the higher fluidity of DOPC (Tm −20°C), compared to either POPC (Tm −2°C) and HSPC (Tm 55°C), which may enhance liposomal aggregation and/or fusion.
* p<0.05.
Many reports have demonstrated that the release of encapsulated drug from liposomes is substantially affected by the composition of liposomes.28,41) Therefore, at first, we investigated the effect of phospholipid type on PMX release from liposomes. As shown in Fig. 2, incorporating fluid-phase phospholipids (i.e., phosphatidylcholines with lower phase transition temperature into the liposomal membrane) such as POPC with Tm −2°C or DOPC with Tm −20°C, increased drug leakage rates compared to liposomes containing solid-phase phospholipids (i.e., phosphatidylcholines with higher phase transition temperatures) such as HSPC whose Tm is 55°C. The release rates from the different liposomal formulations can be arranged in the following rank order POPC>DOPC>HSPC. 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)
Data represent mean±S.D. (n=3).
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 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 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 cholesterol-free DOPC liposomes (13% vs. 65%, respectively).
Data represent mean±S.D. (n=3).
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.
(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).
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<0.001). In addition, empty PEG-coated cationic liposomes did not show any cytotoxicity at lipid concentrations corresponding to the PMX containing PEG-coated cationic liposomes we tested (not shown).
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).
Liposomes have been acknowledged as successful drug carriers for a wide variety of anti-cancer agents. The market availability of liposomes, including Doxil®, DaunoXome® and DepoCyt®, shows that liposome technology can further mature into even more highly sophisticated pharmaceutical products.44,45) In the current study, we developed the first liposomal formulation of the anti-folate agent PMX. In addition, we emphasized the potency of such a liposomal formulation to inhibit the growth of MPM cells, (MSTO-211H) in vitro. In consideration of such promising results, liposomal PMX surely holds great promise in conquering aggressive MPM, in vivo, by potentiating the efficacy of the entrapped PMX at the target organ/tissue while minimizing its adverse side effects. Further studies are currently being conducted in our laboratory to emphasize the in vivo potential of our liposomal PMX formulation against MPM.
In the present study, we have developed the PMX loaded liposomes. PMX encapsulated within fluid liposomes (POPC- or DOPC-liposomes) showed a superior in vitro cytotoxic effect against mesotheliomal MSTO-211H tumor cells, compared to solid liposomes (HSPC-liposomes) encapsulating PMX. These results suggest that PMX encapsulated within fluid liposomes might exert a potent therapeutic efficacy, superior to free PMX, when applied in vivo via ensuring targeted delivery of PMX to tumor tissue while minimizing the side effects encountered with free form of PMX.
The authors thank Mr. James L. McDonald for his helpful advice in developing the English manuscript. This work was partially supported by a research program for development of intelligent Tokushima artificial exosome (iTEX) from Tokushima University.
The authors declare no conflict of interest.
