The Experimental Study Of The Human A₅₄₉ Cell Xenografts In Nude Mice After Intratumoral Injection Of Microspheres Containing Carboplatin

Objective: 1. To fabricate Poly(lactide-co-glycolide) microspheres containing carboplatin and to investigate their general properties, such as the morphology, the diameter distributions, the loading capacity, the encapsulation efficiency, the kinetics of drug release process in vitro. 2. To evaluate the effects of the microspheres containing carboplatin on human lung adenocarcinoma cell line A₅₄₉ in vitro. 3. To evaluate the ability of the microspheres releasing carboplatin to suppress the growth of the A₅₄₉ cell xenografts implanted subcutaneously in nude mice and investigate the effects on tumor cell apoptosis, proliferation and properties of drug-resistance in vivo.Methods: 1. Double emulsion method was adopted to fabricate the microspheres releasing carboplatin. The morphology and the diameter distributions of the microspheres was investigated by scanning electron microscope and particle size analyse apparatus. The loading capacity, the encapsulation efficiency, and the kinetics of drug release process in the PBS solution in vitro was investigated with atomic absorption spectrophotometry (AAS). 2. The3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to estimate the number of viable cells in drug screening trials. A human lung adenocarcinoma cell line A₅₄₉ was used in this study. Microspheres releasing carboplatin were administered to the culture solution for culturing A₅₄₉ cell. The values of optic density(OD) of viable cell, cell inhibition rate(IR), and the values of 50% inhibition concentration (IC₅₀) were assayed. 3. BALBnu/nunude mice were implanted with human lung adenocarcinoma cell line A₅₄₉ subcutaneously to establish human lung adenocarcinoma model. When the volume of tumor reaching about 450～650mm³, the mice loaded tumor were randomly divided into 5 groups of 12 each. (1) A group received intratumoral injection of saline; (2) B group received intratumoral injection of microspheres with no carbplatin; (3) C group received intraperitoneal injection of carboplatin; (4) D group received intratumoral bolus injection of carboplatin; (5) E group received intratumoral injection of microsphers releasing carboplatin. The mice in every group were subdivided randomly into 3 subgroups of 4 each. The volume of tumor was measured twice every week. For each group, a subgroup were euthanized at the end of the 1st week, and the others were did so at the end of the 3rd and 5th week after treatment respectively. Half of tumor, plasma, liver, kidney and one femur in every mice were collected for measuring the concentration of carboplatin in these tissues. Apoptosis of tumor cell was assayed by TUNEL in situ. PCNA immunohistochemistry was adopted to assess the ability of tumor cell proliferation.P-gp and GST-Ï€immunohistochemistry was used to evaluate the drugresistance of tumor cell, and the positive expression was analyzed quantitatively by using a computer image process system.Results: 1. The morphology of the microspheres containing carboplatin was good without accretion at their appearance and the size distribution was within a narrow range; The average loading capacity of drug was 11.72±0.56%, the average encapsulation efficiency of drug was 58.44±1.77%; The microspheres had good redispersion after freeze-drying; The cumulative drug release ratio was about 91.45% during 18 days with a stable release process, and the kinetics of the release process could be described by the Higuchi equation: Q(t)=24.7871×t^1/2-9.9260, R=0.9958. 2. Microspheres releasing carboplatin showed effective anti-tumor activity to A₅₄₉ cell in vitro. The value of IC₅₀ was about 136.03ug/ml and the regression equation was Y=0.3304X±5.0553 (n=6, R=0.9639). Microspheres with no carboplatin did not show the activity of anti-tumor. The volumes of tumors in A and B groups were larger than those of other groups, and the values were beyond 7000mm³ at the end of the 5th week. The average of tumor volume in E group was the smallest among these groups, followed by D group. No platinum was detected in the tumors of A, B, and C group. The concentration of carboplatin was 0.83±0.24ug/g in the tumors of D group at the end of the 1st week and no platinum was detected in the following two subgroups of D group. For the three subgroups of E group, the carboplatin concentration of the tumors was 403.95±99.71ug/g, 68.51±12.41ug/g, 1.40±0.79ug/g respectively, and the average concentration of each subgroup was higher compared to those of the other subgroups euthanized at the same time. No platinum was detected in the samples of plasma, livers, kidneys and femurs of these groups. No apoptosis was observed in the tumors of A and B group at the end of the 1st and the 3rd week. Apoptosis was observed occasionally in the tumors of the same groups at the end of the 5th week. The apoptosis indexes(AI) of C, D, and E groups were larger than those of the A group, and the AIs of three subgroups in E group were the largest among these subgroups. With time passing, the AIs of the subgroups were decreasing gradually in C, D, and E group. The expression of PCNA was very high in the tumors of A and B group at the end of the 1st week, and the proliferation indexes (PIs) were 71% and 73.8 % respectively, and then the PIs were decreasing gradually. The PIs of C and D groups were 52.8% and 45.4% at the end of the 1st week respectively, showing significant difference compared to that of A group. However, no significant difference about the PIs was discovered between C and D group. The expression of PCNA in the tumors of E group was the lowest, and the PIs were 24.8 %, 27%, 38% at the end of the 1st, 3rd, and 5th week respectively. No significant change was observed about the expression of P-gp in the tumors of A, B, C, and D groups. The values of integrated optical density (IOD) of P-gp in the tumors of E group were somewhat increasing with time, but no significant difference was detected among either the subgroups in E group or other groups. No significant change was discovered about the expression of GST-Ï€in the tumors of A, B, and C groups. The value of IOD of the GST-Ï€in the tumors of D group was increasing and significant difference was detected compared to that of A group at the end of the 5th week. The value of the IOD of E group was significantly higher than those of other groups and increased significantly with time. Furthermore, significant difference was observed compared to A, B, and C groups at the end of the 5th week, but no significant difference was detected compared to D group at the same time.Conclusions: 1. The microspheres containing carboplatin fabricated by double emulsion technique had a rather good loading capacity with stable drug release properties, showing better pharmaceutic characteristic, which could maintain two weeks therapeutic effects in vitro. 2. Microspheres releasing carboplatin showed effective anti-tumor activity to A₅₄₉ cell in vitro, and the efficacy of anti-tumor was enhanced by increasing the amount of the microspheres. 3. The growth of the A₅₄₉ cell xenografts in the nude mice was suppressed by the intratumoral injection of microspheres loading carboplatin. The efficacy of suppression was superior to that of carboplatin administered by injection intraperitoneally and intratumorally. 4.There is some influence on the drug-resistance properties of the surviving A₅₄₉ cells and the ability of detoxification may augment after intratumoral chemotherapy with micro-spheres releasing carboplatin...