The Study On The Anti-lymphoma Effect Of Polymer Nanoparticle Co-loaded With Doxorubicin And Curcumin

Lymphoma has occupied almost 50% of newly diagnosed hematopoietic malignancies every year in worldwide.The incidence of Non-Hodgkin Lymphoma has increased to be the senventh,and the mortality rate is at the ninth place.Lymphoma has been a serious threat to human health.The traditional chemotherapy is still the foundation treatment,including Adriamycin(Doxorubicin,DOX),which is one of the most widespread drug,and taking an important part of the first-line chemotherapy of invasive B cell lymphoma.DOX is a kind of nonselective cytotoxic drug,and may lead to adverse reactions,that affected clinical application.Change the dosage form,optimize the transmission system and joint sensitization or protectant together are feasible effective means and may have significant clinical significance.Purpose:The polymer nano materials [mPEG-b-P(Glu-co-Phe)] was used as the carrier to co-loaded DOX and Curcumin(CUR),that was L-DOX+CUR.We evaluated the efficacy and toxicity of this nanomaterial system in the treatment of invasive B cell lymphoma both in vivo and vitro,then attempted to explain the mechanism.Methods:First of all,sample preparation of L-DOX,we took invasive B cell lymphoma cell as model,including Burkitt lymphoma cell(Raji),large B cell lymphoma cell(BJAB and Pfeiffer),preliminarily evaluated L-DOX treatment of lymphoma in vitro experiments.Then,on this basis,we co-loaded DOX+CUR with the mechanism of electrostatic and physical embedding,completed the preparation of polymer nanoparticle system(L-DOX+CUR)innovatively.We took Raji,BJAB and Pfeiffer cell as the model.DOX signal were tested to determine the ability of the drugs entering the cells by flow cytometry,the different enrichment areas in the cells directly observed by confocal microscope.The toxicity of co-loaded system was tested by CCK-8 assay in different cells,the synergistic coefficients were calculated.Then we analyzed the apoptosis ability of the co-loaded system and the possible mechanisms of apoptosis pathways regulation by flow cytometry and Western Blot.At the same time,the MTD(maximum tolerable dose)test was perforemed on Kunming rats in order to evaluate the safety in vivo.The next,based on the pre-experiments,we established the best way to bear SCID mice by BJAB cell,then took it as animal model for the next vivo experiment.During the inhibition experiment,the tumor volume and the body weight were tested,and the pathological analysis of tumor tissues was completed.Finally,we evaluated the application value of mPEG-b-P(Glu-co-Phe)co-loaded DOX+CUR in the treatment of invasive B cell lymphoma extensively based on in vivo and vitro results.Results:1.By simply mixed mPEG-b-P(Glu-co-Phe)and DOX powder in the water phase,we successfully prepared L-DOX,the drug-loading rate was 10.6%.In vitro experiments,L-DOX showed stronger ability to enter cells and higher toxicity than DOX with Raji,BJAB and Pfeiffer cells.2.The co-loaded system,L-DOX+CUR,was prepared successfully,and the mole ratio of DOX and CUR fixed in 1:1.23.(DOX loading rate 9.7%,CUR loading rate 8.1%).3.The toxicity of L-DOX+CUR system,in vitro(Raji,BJAB and Pfeiffer cell as model):(1)L-DOX+CUR had stronger entry cell capability than small molecules combined drug(DOX+CUR)or the single drug(DOX).(2)The main enrichment area of DOX was nucleus,which is different of CUR.(3)CCK-8 results shown the L-DOX+CUR group with the minimum IC50 after 48 hrs in all the three cells,and,by calculating,both the pure combined and co-loaded DOX+CUR had synergistic effect,especially in BJAB cells(min CI 0.019).4.L-DOX+CUR system can induce apoptosis,in vitro(Raji and BJAB cell as model):(1)The four treatment groups(DOX,L-DOX,DOX+CUR and L-DOX+CUR)were dealt with Raji and BJAB cell for 24 or 48 hours,all groups showed rising rates of apoptosis(P= 0.000).(2)In Raji cell,combined drug groups showed higher apoptosis rate than DOX group(P< 0.05);in BJAB cell,the rates were L-DOX+CUR > DOX+CUR > DOX(P<0.05),L-DOX+CUR > L-DOX(P<0.001),the differences were significant.(3)Compared with control group,Raji cell and BJAB cell both showed PARP up-regulation in loaded groups(P<0.001),and Bcl-2 down-regulation with Bax increased significantly(P<0.05).(4)BJAB cell showed differences among groups on PARP expression,that were L-DOX>DOX and L-DOX+CUR>L-DOX(P<0.05).Except DOX group,all the groups of Raji cell showed Bcl-2 down-regulation with Bax and caspase3 increased significantly(P<0.01).To be interesting,Bid significantly express higher in loaded groups of BJAB,wihle in pured groups of Raji cell(P<0.01).5.L-DOX+CUR system,the pre-experiment of animals in vivo:(1)MTD on Kunming mice,DOX group was 10mg/kg,L-DOX 20mg/kg and L-DOX+CUR group in 25mg/kg.(2)The possible best way to contribute SCID mice model was implantate 5 ×106 BJAB cells subcutaneously,which model was confirmed by pathlogic test,which was IHC: CD20+,PAX5+,KI67+ 70-90%,CD3-.6.L-DOX+CUR system anti-tumor activity on BJAB bearing SCID mice :(1)All the mice accepted DOX,L-DOX,CUR,DOX+CUR or L-DOX+CUR(accumulated DOX 9mg/kg),the inhibition effects started on the eighth day,P=0.031.(2)The growth rate of tumors` volume were significantly different among all the six groups(P= 0.000).All groups showed significant in suppressing tumor growth,P<0.05,except CUR group,(P value: CUR 0.235,DOX 0.021,L-DOX 0.003,DOX+CUR 0.003,L-DOX+CUR 0.001).Until the twelfth day,the two polymer nanoparticle groups showed negative growth trend and it happened earlier in L-DOX+CUR group than L-DOX.(3)After the different treatments above,mice in each group showed weight loss,P<0.05.Weight loss appeared earliest in DOX group,last in L-DOX+CUR group,and the most severe in DOX group,the lest degree in L-DOX+CUR group.(4)Observed the pathological H-E sections,Compared with DOX,the necrosis area of the L-DOX+CUR group and the DOX+CUR group were significantly larger than the DOX group,and the former was obvious most.Conclusion:1.A new polymer nanoparticles-mPEG-b-P(Glu-co-Phe),can simply static load DOX(L-DOX).L-DOX had higher ability to entering Raji,BJAB and Pfeiffer cells by swallowing function,quickly release after entering and more capable of killing cells.We innovatively created co-loaded system,L-DOX+CUR,inhanced the abiblity further more.2.Pured or nanoparticled formation of DOX and CUR both had synergistc effect by 1:1.23 mole ratio,and the effect was the most prominent in the BJAB cell CI=0.019.3.The cell toxicity on Raji,BJAB and Pfeiffer cells,the nanoparticled drug were superior to the pured ones,48 hours was stronger than 24 hours.The higher toxicity may because of the higher apoptosis inducing ability from L-DOX+CUR than other groups.The main mechanism of apoptosis was intrinstic pathway,and maybe extrinstic pathway as well.4.Implantate 5×106 BJAB cells subcutaneously on SCID mice is a possible way to contribute lymphoma mice model.5.L-DOX+CUR system increased the MTD more than 4 times compared with pure DOX in Kunming mice.The toxicity of nanoparticl-drug decreased and tolerance improved.Apply L-DOX+CUR to treat BJAB bearing SCID mice could guarantee higher efficacy and less toxicity.