| Background and objective: Currently,lymphoma is one of the most rapidly growing malignant tumors in the world with increasing at an annual rate of four percent,which is a serious threat to public health.Chemotherapy is the main treatment strategy for patients with lymphoma.The clinical application of traditional chemotherapy drugs has been,to some extent,limited,because of nontargeted and significant toxic side effects.Cisplatin(CDDP)is a common anticancer drug,which plays a pivotal role in the treatment of many malignant tumors.However,up to now,CDDP is still used as a second-line drug for lymphoma due to its own significant dose-limiting toxicity.Therefore,for patients with lymphoma,further reducing cytotoxicity and improving anti-cancer efficacy by optimizating drug delivery system of traditional CDDP through the nanotechnology,has remarkable clinical significance.In this study,cisplatin loaded poly(L-glutamic acid)-g-methoxy poly(ethylene glycol)complex nanoparticles(L-CDDP)was synthesized,and at the same time,animal model for lymphoma(BJAB cell)was constructed.On this basis,we systematically explored the efficacy and safety of GEM combined with L-CDDP anti lymphoma in vivo and vitro studies.This study can provide a theoretical evidence for reducing cisplatin-induced cytotoxicity and improving its anti-cancer efficacy.Methods: At first,L-CDDP was prepared,and relevant characterization of L-CDDP was evaluated,including drug loading content,drug loading efficiency,particles size and release data.At the cell level,CCK-8 assay was used to investigate the effects of gemcitabin(GEM),CDDP and L-CDDP on the BJAB and Raji cell viability at 24 h,48 h.What's more,the effects of GEM + CDDP group and GEM + L-CDDP group on BJAB cell viability at 48 h were also studied,and the molar ratio of drugs in these two groups was 13:1(GEM:CDDP).According to the results of CCK-8 assay,the combination index(CI)of GEM + CDDP group and GEM + L-CDDP group was obtained.Annexin-FITC/PI double staining was performed to detect the effects of each experimental group on BJAB and Raji cell apoptosis at 48 h,and meanwhile,the effects of GEM on the cell cycle of BJAB and Raji cells were also investigated by flow cytometry.At last,western blot and q PCR were used to compare the relative expression of some apoptotic proteins and the transcriptional level of target genes in each experimental group,respectively.At the animal level,at first,the maximal tolerable dose of Kunming mice in L-CDDP group,GEM + CDDP group and GEM + L-CDDP group was evaluated.And then,Kunming mice received toxicity test at the dose of MTD.Finally,anti-tumor experiments were conducted on the animal model for lymphoma(BJAB cell)in each experimental group.Results: 1.The drug loading content,drug loading efficiency and hydrodynamic diameter of L-CDDP was 18.3%,89.7% and 20.6 nm,respectively.The release of platinum(Pt)from L-CDDP in p H7.4 PBS and p H5.5 PBS at 180 h was 62.4% and 96.0%(P<0.001),respectively.2.In BJAB and Raji cells,half maximal inhibitory concentration(IC50)values of L-CDDP at 24 h and 48 h were significantly higher than that of CDDP.The IC50 of L-CDDP at 24 h was about 10 times higher than that of CDDP(BJAB,223.2±8.5 vs 15.3±0.9;Raji,993.8±3.8 vs 89.4±0.9);and this ratio at 48 h exceeded 20 times of CDDP(BJAB,116.8±5.9 vs 4.5±0.7;Raji,403.9±5.2 vs 10.9±0.9).3.When the molar ratio of GEM and CDDP was 13:1,the CI of GEM + CDDP group and GEM + L-CDDP group in BJAB cell at 48 h was 0.28 and 0.88,respectively.4.The results from flow cytometry indicated that the percent of BJAB cell apoptosis at 48 h in control group,GEM group,CDDP group,L-CDDP group,GEM + CDDP group and GEM + L-CDDP group was 1.8±0.1,13.8±0.4,18.4±0.3,14.4±0.4,26.7±0.2 and 42.1±1.5,respectively;however,the percent of Raji cell apoptosis at 48 h in control group,GEM group,CDDP group,L-CDDP group,GEM + CDDP group and GEM + L-CDDP group was 0.4±0.1,15.0±0.2,20.9±0.3,21.8±1.4,35.6±0.6 and 44.5±1.2,respectively.As can be seen from the above results,the proportion of cell apoptosis in GEM + L-CDDP group was significantly higher than that in other experimental groups(P<0.05).5.Results from western blot revealed that the expression of Bid,P53,Caspase 3(c)and PARP(c)protein all increased in each experimental group in comparison to control group for BJAB and Raji cells,except Bcl2 protein,and among these groups,GEM + CDDP group and GEM + L-CDDP group changed significantly.This phenomenon was consistent with q PCR results.6.Cell cycle test results showed that GEM mainly arrested BJAB and Raji cells in the G0 / G1 phase.The proportion of BJAB cells in the G0 / G1 phase and S phase in GEM group at 48 h was 64.9±0.3 and 14.9±0.3,respectively,however,the results in control group was 48.1±0.3 and 29.6±0.1,respectively,there was a statistical significance between these two groups(P=0.006);For Raji cells,the proportion of cells in the G0 / G1 phase and S phase in GEM group at 48 h was 63.1±0.3 and 24.0±0.2,respectively,and the results in control group was 33.4±0.2 and 35.0±0.1,respectively.There was also a statistical significance between these two groups(P=0.002).7.Results from MTD assay demonstrated that the MTD of Kunming mice in L-CDDP group,GEM + CDDP group and GEM + L-CDDP was 30 mg/kg,91 mg/kg + 7mg/kg and 208 mg/kg + 16 mg/kg,respectively.Toxicity test showed that renal toxicity in CDDP group and GEM + CDDP group was significantly higher than that in other experimental groups;and there was also an obvious hematological toxicity in CDDP group and hepatotoxicity in GEM + CDDP group.However,hematological toxicity,hepatotoxicity and renal toxicity in GEM + L-CDDP group were not significantly different from those in control group(P > 0.05).The antitumor experiment indicated that the antitumor effects in GEM + CDDP group and GEM + L-CDDP group were remarkably better than those in other groups.At the end of antitumor experiment,tumor volume in GEM + CDDP group and GEM + L-CDDP group was(119.1±54.3)mm3 and(150.3±35.9)mm3,respectively.Although tumor volume in GEM + CDDP group was slightly smaller than that in GEM + L-CDDP group by the end of the experiment;but,tumor had already progressed in GEM + CDDP group.However,tumor in GEM + L-CDDP group was still shrinking.Body weight of tumor-bearing SCID mice in GEM + CDDP group decreased more than twenty percent during antitumor experiment,and body weight in GEM + L-CDDP group merely decreased by fifteen percent.There had statistical significance between the two groups for body weight changes(P<0.05).Conclusions: 1.Compared to traditional CDDP,L-CDDP,namely cisplatin loaded poly(L-glutamic acid)-g-methoxy poly(ethylene glycol)complex nanoparticles,can significantly prolong time in blood circulation.2.BJAB and Raji cells were more tolerant to L-CDDP as compared to the traditional CDDP.3.Under the mole of ratio of GEM:CDDP=13:1,there had synergistic effects on inhibiting lymphoma cells proliferation in GEM + CDDP group and GEM + L-CDDP group.4.BJAB and Raji cell apoptosis in GEM + L-CDDP group was obviously higher than that in other groups,which may be related to P53 gene,BID gene and BCL2 gene.5.Compared with GEM + CDDP group,GEM + L-CDDP group not only reduced the toxicity of chemotherapy,but also prolonged tumor inhibition time. |
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