Cisplatin was purchased from Wako Pure Chemical Industries Co., Ltd. (Tokyo, Japan). EPC (Coatsome NC-50) was obtained from NOF Corporation (Tokyo, Japan). PEO-b-PMAA (Mw of PEO = 7,500, Mn of PMAA = 11,000) was purchased from Polymer Source, Inc. (Quebec, Canada). All other reagents used in this study were the finest grade.

For preparation of PM, PEO-b-PMAA was dissolved in acetone. For preparation of HNP, the PEO-b-PMAA solution was added into EPC solution dissolved in methanol at a molar ratio of EPC/PEO-b-PMAA of 50/2.8 or 50/50 (HNP-P5 and HNP-P50, respectively). The solution of PEO-b-PMAA or mixture of EPC and PEO-b- PMAA was added dropwise into water and stirred continuously for 30 min at room temperature. In order to remove the organic solvents, these mixtures were dialyzed against water using Spectra Por^®7 dialysis membrane with molecular weight cut-off (MWCO) 2,000 (Spectrum Laboratories, Inc., CA, USA).

Cisplatin was incorporated into PM, HNP-P5 and HNP-P50 by direct mixing of cisplatin solution at pH 9 adjusted with ammonia solution at a molar ratio of carboxylic acid of PEO-b-PMAA/cisplatin of 1/2, and then incubated with shaking at 37˚C for 48 h. The concentration of carboxylic acid was determined by an acid-base titration method. Finally, free cisplatin was removed from the cisplatin-loaded PM, HNP-P5 and HNP-P50 by centrifugation at 2500 g for 20 min using an Amicon^® Ultra filter (MWCO 30,000, Merck Millipore Ltd, Carrigtwohill, Ireland).

The particle sizes of PM, HNP-P5 and HNP-P50 were measured by the cumulant method using a light-scattering photometer (ELS-Z2, Otsuka Electronics Co, Ltd., Osaka, Japan) at 25˚C after diluting the dispersion to an appropriate volume with water. The ζ-potentials were measured by electrophoresis light-scattering methods using ELS-Z2 at 25˚C after diluting the dispersion to an appropriate volume with water. For calculating the entrapment efficiency of cisplatin, cisplatin was extracted from PM, HNP-P5, and HNP-P50 by solubilizing lipid layer using the Bligh and Dyer method [18] . Briefly, 20 μL of PM, HNP-P5, or HNP-P50 solution was mixed with 180 μL 0.1 N HCl solution. A 750 μL aliquot of a solution of methanol: chloroform (1:2), 250 μL chloroform and 250 μL 0.1 N HCl was added and then centrifuged to separate the aqueous (upper) and organic solvent (lower) phases. The cisplatin content of the upper phase was measured with a high-performance liquid chromatography (HPLC) system (Shimadzu Co. Kyoto, Japan) composed of an LC-10 ATVP pump, an SIL-10 AF autoinjector, and an SPD-10 AVP UV detector at an absorbance at 306 nm. The upper phase was applied to an anion exchange column Inertsil AX^® (250 mm × 4.6 mm, GL Sciences, Tokyo, Japan) equilibrated in a mixture of 8:40:10:20 (v/v) of ethyl acetate, methanol, water and N, N-dimethylformamide at a flow rate of 1 mL/min. The entrapment efficiency (EE%) of cisplatin in PM, HNP-P5 and HNP-P50 was calculated using the following equation.

EE% = (Amount of drug entrapped)/(Amount of drug added) × 100.

The release studies of cisplatin were performed by placing 200 µL of PM, HNP-P5, or HNP-P50 solution into Spectra Por^®7 dialysis tubing with MWCO 3500 (Spectrum Laboratories, Inc.). The PM and HNPs were then immersed in 50 mL of phosphate-buffered saline (PBS; pH 5.5 or 7.4) with continuous stirring in a water bath at 37˚C. At various time points, 200 μL aliquots were withdrawn from the outer aqueous solution and replaced with 200 μL of PBS. The Pt concentration was measured by a graphite furnace atomic absorption spectrophotometry (GF-AAS) method after diluting with 0.1 N HCl to an appropriate volume. The analysis program of the GF-AAS involved three steps: 1) a drying stage at 80˚C - 100˚C for 40 s, 2) an ashing stage at 800˚C for 30 s, 3) an atomization stage by heating at 3000˚C for 7 s, and then cooling down. The concentration of Pt was measured at 265.9 nm with a slit bandwidth of 0.4 nm, and then converted into cisplatin concentration. Wurster correction was used for calculating the cumulative amount of cisplatin released.

Murine Lewis lung carcinoma (LLC) and colon carcinoma Colon 26 cells were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Miyagi, Japan). The cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum and kanamycin (100 μg/mL) in a humidified atmosphere containing 5% CO₂ at 37˚C. For the in vitro cytotoxic assay, LLC and Colon 26 cells were seeded separately at a density of 1 × 10⁴ cells per well in 96-well plates, respectively, and maintained in the medium for 24 h before treatment.

To examine the cytotoxicity of cisplatin, the cells were treated with medium containing various concentrations of cisplatin in PM, HNP-P5, and HNP-P50, and they were then incubated for 48 h. After treatment, the cell number was determined using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cell viability (%) was expressed relative to the absorbance of untreated cells at 450 nm.

All animal experiments were performed with approval from the Institutional Animal Care and Use Committee of Hoshi University. To generate Colon 26 tumors, 1 × 10⁶ cells were inoculated subcutaneously into the flank of female BALB/c mice (female, 6 weeks old, Sankyo Lab. Service Corp., Tokyo, Japan). After the tumor size had reached 100 mm³, PM, HNP-P5, and HNP-P50 were administered intravenously via the tail vein at a dose equivalent to 4 mg cisplatin per kg. Twenty-four hours after injection, blood was collected in heparinized tubes by decapitation to obtain plasma, and the tumor and kidneys were excised.

For analysis of Pt levels, plasma, tumor, and kidneys were digested with concentrated HNO₃, and then heated at 70˚C for 1 h followed by heating at 120˚C overnight. The concentration of Pt was measured by GF-AAS method after diluting with 0.1 N HCl in appropriate volume as described above section, and calculated as μg∙Pt/mL plasma or µg∙Pt/g tissue.

All data were produced in replicates and presented as the mean ± SD. To evaluate the significance of differences, data were analyzed by one-way ANOVA, followed by Tukey’s HSD post-hoc test, with P values less than 0.05 considered as statistically significant.

PM, HNP-P5, and HNP-P50 were prepared by the nanoprecipitation method. PM consisted of 100% PEO-b- PMAA, and HNP-P5 and HNP-P50 contained at 5.3 and 50 mol% PEO-b-PMAA with EPC, respectively. EPC comprises a polar head of choline, with fatty acid forming the double tail chains [19] . These lipophilic chains are expected to associate with the hydrophobic segment of PEO-b-PMAA to form an additional protective layer of the micelle and avoid micelle breakage during the excessive dilution in body fluids.

The sizes of PM, HNP-P5, and HNP-P50 before inclusion of cisplatin were approximately 220 nm, 110 nm, and 65 nm, respectively, and their ζ-potentials were approximately −21 mV, −13 mV, and −17 mV, respectively (Table 1). Generally, the size of PM was smaller than the vesicles of liposomes [20] . However, the size of PM was large compared with HNP-P5 and HNP-P50. It has been reported previously that divalent cation metal is needed to form self-assembly ionomer complexes with PEO-b-PMAA [21] . The large size of PM may relate to the hydration of PEO-b-PMAA by water without divalent cation metals. In contrast, in HNPs, the hydrophobicity of EPC might change the amphiphatic balance of PEO-b-PMAA.

After cisplatin loading, the particle sizes of PM, HNP-P5, and HNP-P50 were not significantly changed, but their ζ-potentials were increased (Table 1), indicating that the negatively charged carboxylic acid of PMAA interacted electrostatically with cisplatin. In contrast, increasing the EPC content in HNP slightly reduced entrapment efficiency (Table 1), suggesting that the high content of PEO-b-PMAA in PM and HNP-P50 could interact well with cisplatin.

The in vitro profiles of cisplatin release from PM, HNP-P5, and HNP-P50 were evaluated by immersing nanoparticles in PBS (pH 5.5 and 7.4). Decreasing the pH from 7.4 to 5.5 increased the release of cisplatin from PM, HNP-P5, and HNP-P50 (Figure 1(a), Figure 1(b)). It has been reported that dissociation of cisplatin from PM- AA depended on the pH and ionic strength of the environment [15] . HNP-P5 and HNP-P50 showed low release of cisplatin at approximately 40% and 67%, respectively, of the cumulative dose over 120 h in PBS pH 5.5, whereas PM exhibited the highest release of up to 90% (Figure 1(b)). HNP-P5 exhibited reduced cisplatin release compared with HNP-P50, indicated that increasing EPC content in HNPs reduced cisplatin release. The presence of the lipid layer could limit water diffusion into the inner part of particles [22] ; therefore, the barrier of EPC in HNPs might reduce cisplatin release.

Next, we evaluated the cytotoxicity of PM, HNP-P5, and HNP-P50 on Colon 26 and LLC cells by 48 h exposure. PM and HNP-P50 had similar cytotoxic effects on Colon 26 (IC₅₀ = 1.79 and 1.71 μg/mL, respectively) (Figure 2(a)) and LLC cells (IC50 = 0.42 and 0.87 μg/mL, respectively) (Figure 2(b)) compared with cisplatin solution

^a)Each value represents the mean ± SD (n = 3). HNP-P5 and HNP-P50, hybrid nanoparticles (HNPs) composed of egg phosphatidylcholine/ poly(ethylene oxide)-block-poly(methacrylic acid) at molar ratios of 50/2.8 and 50/50, respectively.

(IC₅₀ = 1.10 μg/mL for Colon 26 and <0.1 μg/mL for LLC cells). In contrast, HNP-P5 decreased cytotoxic effects (IC₅₀ ≥ 10 μg/mL for Colon 26 and 2.56 μg/mL for LLC) compared with PM and HNP-P50. These results correlated well with the slow release of cisplatin from HNPs. Furthermore, to confirm the effect of carriers on the cell viability, LLC cells were exposed to PM, HNP-P5, and HNP-P50 without cisplatin, and no effects were observed on the cell viability (Figure 2(c)).

Finally, we evaluated the biodistribution 24 h after injection of cisplatin-loaded PM, HNP-P5, and HNP-P50 on Colon 26 tumor-bearing mice. Compared with cisplatin solution (0.1 μg∙Pt/mL plasma), which was almost clear- ed from the systemic circulation, the concentration of Pt in plasma after injection of PM remained at 5.3 μg∙Pt/mL at 24 h (Figure 3(a)). Higher tumor accumulation of cisplatin was also observed with PM (1.7 μg∙Pt/g tumor) than HNP-P5 or HNP-P50 (0.5 and 0.9 μg∙Pt/g tumor, respectively) (Figure 3(b)). This suggested that PM could significantly improve tumor accumulation of cisplatin by prolonged circulation in the bloodstream (EPR effect), compared with cisplatin solution. Although HNP-P5 and HNP-P5 did not significantly increase cisplatin levels in plasma and tumors compared with cisplatin solution, they were still better than cisplatin solution. We speculated that EPC in HNPs might be destabilized in the circulation by interaction with blood components, although it could reduce the release of cisplatin in the in vitro experiments (Figure 1 and Figure 2). In contrast, the accumulation of cisplatin in the kidneys after injection of PM, HNP-P5, and HNP-P50 was low and did not show significant differences (Figure 3(c)). We also prepared other types of HNPs composed of EPC/ PEO-b-PMAA/sodium deoxycholate or EPC/PEO-b-PMAA/Tween 80 at a molar ratio of 50/2.8/5, and evaluated their biodistribution at 24 h after intravenous injection into mice; however, their cisplatin levels in plasma (1.1 and 0.8 μg∙Pt/mL, respectively) and tumor (0.3 and 0.3 μg∙Pt/g tumor, respectively) were low compared with PM (data not shown). In HNPs, further improvement might be required to obtain better stability in vivo. Using lipids with higher phase transition temperature such as HSPC for the formulation of HNPs may be able to improve in vivo tumor delivery.