Cholesterol, and soy phosphatidylcholine were purchased from Shanghai Chemical Agent company. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) , cyanuric chloride, O-(2-aminoethyl) polyethylene glycol 3000, N,N-diisopropylethylamine, dimethyl sulfoxide (DMSO), 1.2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) were purchased from Sigma. Docetaxel was kindly supplied by Jiangsu Hengrui Medicine Co. Monoclonal antibody specific for CEA was purchased from Beijing Zhongshan Biotechnology Co. Immunocyt-ochemical detection kit was from Wuhan Boster Biological Technology Co.

Immunoliposomal docetaxel was prepared according to the method described by Bendas[14-17] with minor modifications. Briefly, a 2-fold molar of N, N-diisopropylethylamine and a 3-fold molar cyanuric chloride were added to chloroform solution of DPPE, mixed for 12 h, then DPPE-cyanuric chloride was isolated. DPPE-PEG-OH was prepared by adding a 1-fold molar aminopolyethylene glycol 3000 and a 2-fold molar of N,N-diisopropylethylamine to DPPE-cyanuric chloride. PEG terminus was activated by reaction with 1-fold molar of cyanuric chloride in addition to a 2-fold molar of N,N-diisopropylethylamine. DPPE-cyanuric chloride (cyanur-PEG-PE) was synthesized.

Liposomal docetaxel was prepared with soy phosphati-dylcholine cholesterol 2:1 (molar ratio), cyanur-PEG-PE (0.5 mmoL), docetaxel (1 µmoL). Multilamellar vesicles were extruded through a 200 nm polycarbonate membrane. Antibody conjugated with docetaxel liposomes: anti-CEA-antibody molar ratio of 1 000:1 was prepared in 0.15 mol/L NaCl (pH 8.8). Unbound antibodies were separated by gel permeation chromatography using Sepharose 4B.

LoVo cells, the CEA-expressing human colon adenocarcinoma cell line, were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum, 100 μg/mL streptomycin, 100 units/mL penicillin. The population doubling time was approximately 30 h. LoVo cells were grown at 37°C in a humidified atmosphere of 50 mL/L CO₂.

LoVo cells (5 × 10³) were plated onto 96-well plates or 6-well plates. Before the administration of appropriate doses of immunoliposomal docetaxel or radiation, cells were plated for 24 h to allow attachment. LoVo cells were incubated with different concentrations of immunoliposomal docetaxel or liposomal docetaxel as equivalent docetaxel doses of 1 nmol/L, 2 nmol/L, 10 nmol/L, 100 nmol/L and 1 000 nmol/L for 2 h at 37°C. After washing thrice with culture medium, the cells with or without radiation were incubated for 24 h or 48 h at 37°C. Cells were X-irradiated at 0, 2, 4, 6 and 8 Gy respectively . Irradiation was performed with 6-MV photons generated by a Philips SL-75 linear accelerator at a dose rate of 1.3 Gy/min. In the combined treatment modality studies, the treated cells were incubated for 24 h before radiation treatment. All doses were delivered without interruption and with the same focus-target distance.

LoVo cells were treated with various concentrations of immunoliposomal docetaxel, and some were exposed to radiation. To measure cell proliferation, the colorimetric method based on MTT assay was used[18,19]. Cells were stained with MTT (50 mg/mL) and incubated for 4 h at 37°C in a humidified atmosphere of 50 mL/L CO_2. Then the cells were lysed in DMSO, and the solutions were read in a Bio-Tek spectrophotometer at 570 nm. The production of formazan crystals, and the intensity of color after their dissolution, were proportional to the number of viable cells.

At the end of each treatment, LoVo cells were harvested with 0.5 g/L trypsin and 0.2 g/L EDTA. Cells were fixed in 2 mL of 70% cold ethanol at 4°C for 2 h, centrifuged, washed twice with phosphate buffered saline (PBS), and resuspended in 0.5 mL propidium iodide (PI)/RNase A solution. The mixed cells were incubated in the dark at room temperature for 10 min. The fluorescence emission of stained cells was measured with a Becton Dickinson flow cytometer. The number of cells and DNA content were detected by flow cytometry, the fraction of cells in each phase of cell cycle was analyzed. Apoptotic cells were determined by the hypodiploid peak.

Streptoavidin-biotin peroxidase complex technique was used for staining cells. The treated LoVo cells were harvested and plated on slides. The cells were fixed in 95% aqueous alcohol. The endogenous peroxidase activity was blocked by a 3% solution of hydrogen peroxide in methanol for 30 min. Cells were immunostained using monoclonal antibody against human survivin for 1 h at 37°C. After three further washes with PBS, a second biotinylated rabbit anti-mouse antibody was applied for 1 h at room temperature. Following extensive washes with PBS, color development was demonstrated with diaminobenzidine tetrahydrochloride chromagen. The slides were lightly counterstained with hematoxylin. Stained cells were examined using light microscopy and photographed. JD801 morphologic analysis system for color-image measurement was used to semi-quantify immunohistochemical staining of survivin.

To determine the cytotoxicity of immunoliposomal docetaxel to human colon cancer cells, LoVo cells were treated with various concentrations of immunoliposomal docetaxel for 24 or 48 h. Relative survival data for LoVo cells are shown in Figure 1. Under the treatment with 2 nmol/(L·d) immunoliposomal docetaxel for 24 h, survival was decreased 12% as compared with 1 nmol/L a immunoliposomal docetaxel. The results for Figure 1 also indicate that LoVo cells were more sensitive to immunoliposomal docetaxel treatment in 24 h than for 48 h. The cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. The immunoliposomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L.

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Figure 1 Cytotoxicity of immunoliposomal docetaxel to LoVo cells.

LoVo cells received a series of test doses of radiation, ranging from 2 to 8 Gy. The radiotoxicity generated is shown in Figure 2. At the doses of 4 to 8 Gy, the reaction to irradiation appeared less pronounced.

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Figure 2 Radiotoxicity to LoVo cells irradiated with single doses.

To examine whether immunoliposomal docetaxel could target LoVo cells and reveal radiopotentiating effect, these cells were treated with immunoliposomal docetaxel and liposomal docetaxel, respectively, then irradiated at the dose of 2 Gy radiation doses. The control cells were irradiated alone. Using MTT assay, the relative surviving fractions were obtained. The results in Figure 3 showed that immunoliposomal docetaxel was strongly radiopotentiated in LoVo cells, compared with the effects of liposomal docetaxel or single irradiation (P < 0.05). No significant radiopotentiating effects were found after treatment with liposomal docetaxel.

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Figure 3 Target radiopotentiating effects of docetaxel immunoliposomes on LoVo cells.

To determine whether immunoliposomal docetaxel in combination with radiation could increase cellular sensitivity to radiation through cell cycle redistribution, we analyzed the LoVo cells by flow cytometry. After treatment with immunoliposomal docetaxel or liposomal docetaxel, all cells were irradiated at 2 Gy. The response of LoVo cell cycle to radiation is shown in Figure 4. Compared to treatment with liposomal docetaxel, the percentage of G₂/M cells treated with immunoliposomal docetaxel was significantly increased (P < 0.01), but the percentages of cells both in G₂/G₁ phase and in S phase were decreased significantly (P < 0.05). Apoptosis was also monitored by flow cytometry (Figure 4). Apoptosis was significantly increased in LoVo cells due to the combined effects of immunoliposomal docetaxel and radiation.

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Figure 4 Combined of effect immunoliposomal docetaxel and radiation on cell cycle distribution and apoptosis.

Survivin expression in LoVo cells after irradiation and treatment with immunoliposomal docetaxel was verified by immunocytochemistry. Survivin was positively stained with anti-survivin monoclonal antibody. Representative results are shown in Figure 5.

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Figure 5 Expression of survivin in LoVo cells.

Positive staining of survivin was mainly present as diffuse cytoplasmic staining with variable intensity. Integral optical density of survivin was detected semiquantitatively by immunohistochemical staining combined with image analysis. For density measurement, color-images were directly analyzed using D801 morphologic analysis system. The semiquantitative data reported here were directly comparable to image analysis data. Survivin expression in LoVo cells after irradiation and treatment with immunoliposomal docetaxel was significantly decreased in comparision to treatment with liposomal docetaxel (P < 0.001, Figure 6).

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Figure 6 Survivin expression in LoVo cells were determined by quantitative image analysis.