Application And Mechanism Of Chloroquine And Doxorubicin Loaded Reduction-responsive Nanogels For The Synergistic Treatment Of Osteosarcoma

BackgroundOsteosarcoma is a primary malignant bone tumor with high malignancy,rapid growth,early metastasis and poor prognosis.It is the main cause of cancer death in adolescents.At present,there are still many problems to be solved in the pathogenesis,biological behavior,adjuvant treatment and evaluation methods of osteosarcoma.Among them,The side effects and multi-drug resistance of chemotherapy drugs are the main deficiencies of chemotherapy,so an improved strategy is urgently needed to improve the treatment effect and safety.Compared with traditional chemotherapy drugs,nanodrugs have the characteristics of long blood circulation time,high tumor selectivity,sensitivity to tumor microenvironment and controlled release.The stimuli-responsive polyamino acid nanogels can undergo morphological or functional changes in stimulating conditions(pH,glutathione,enzyme,etc.),such as swelling or disassembly,which can help nanodrugs overcome multiple barriers in vivo and significantly improve the targeted delivery and release of nanodrugs,thereby improving the treatment of nanodrugs.It can also reduce the side effects on normal tissues.Chloroquine(CQ)can not only increase the pH value of lysosome,but also prevent the fusion of lysosome and autophagosome.In theory,CQ can help nanodrugs escape from lysosome and accelerate the release of nanodrugs in cells.In recent years,studies have found that the abnormal autophagy in cells is closely related to the occurrence and development of tumor.As a classical autophagy inhibitor,CQ has been widely studied on its anti-tumor effect.In the development of osteosarcoma,whether autophagy promotes cell death as a tumor inhibition mechanism or mediates drug resistance as a tumor survival mechanism remains controversial.To sum up,in view of the bottleneck of the treatment of osteosarcoma,a polyamino acid nanogel with reduction-responsiveness was designed and prepared as a nano carrier to load the traditional small molecule chemotherapeutic drug doxorubicin(Dox)to avoid the early clearance of doxorubicin and prolong its time in blood circulation.Through the enhanced permeability and retention(EPR)effect,nanodrugs selectively accumulate in tumor tissues.Under the condition of high glutathione(GSH),the two sulfur bonds within the nanogels break down,and release doxorubicin responsiveness to achieve the precise delivery of doxorubicin in tumor tissue specific accumulation and responsive release in tumor cells,thereby enhancing the therapeutic effect and safety of doxorubicin.To elucidate the mechanism of anti-osteosarcoma effect of chloroquine,a broader idea is provided for optimizing nanocarrier and improving anti-osteosarcoma efficiency.On the one hand,combined application of chloroquine can help nanodrugs achieve lysosomal escape,which makes doxorubicin release more quickly in cells and play a therapeutic role.On the other hand,it can play a synergistic anti-tumor effect through the autophagy inhibition of chloroquine,which provides new evidence for autophagy involved in the occurrence and development of osteosarcoma.In this regard,further research may provide new therapeutic strategies and targets for osteosarcoma.ObjectiveIn this study,a nanocarrier was designed reasonably to overcome the obstacles of doxorubicin circulation in vivo,tumor accumulation,tumor infiltration,endocytosis and intracellular release,so as to achieve the purpose of enhancing efficiency and reducing toxicity,and clarify the mechanism of chloroquine's anti-osteosarcoma effect.Methods1.The copolymer mPEG113-P(Phe-co-Cys)was prepared by ring-opening polymerization of L-Phe NCA and L-Cys NCA with mPEG113-NH2 as macromolecular initiator.The structure,particle size and morphology of the copolymer were further analyzed by NMR,DLS,TEM,FT-IR,elemental analysis and GPC,and its stability and reduction-responsiveness were verified.MTT method was used to detect its biocompatibility in vitro.2.The drug loading content and drug loading efficiency of NGs/Dox were measured.The particle size and morphology were analyzed by DLS and TEM.The in vitro release behavior of Dox was evaluated by Dox cumulative release curve,CLSM and FCM,and its cytotoxicity and reduction-responsiveness was detected by MTT method.3.The anti-osteosarcoma effect of CQ combined with NGs/Dox in vitro was detected by MTT method,and the properties of the combination of two drugs were evaluated.4.The effect of CQ on the uptake of NGs/Dox by osteosarcoma cells was observed by FCM,and the effect of CQ on lysosome escape of NGs/Dox was observed by CLSM.TEM was used to observe the changes of autophagosomes in osteosarcoma cells before and after CQ treatment.Western Blot was used to detect the changes of LC3 ?,LC3 ? and P62 proteins in osteosarcoma cells treated with CQ.5.The concentration of Dox in serum of SD rats was detected by HPLC.The data were processed by PK solver program to obtain the half-life,area under the time concentration curve and clearance of NGs/Dox and doxorubicin.6.Maestro fluorescence imaging system was used to observe the distribution of Dox in various organs and tumors,and semi-quantitative analysis was carried out.7.The general situation and body weight and tumor volume of osteosarcoma-bearing mice were observed,and the serum,tumor tissue and main organs were obtained after treatment for biochemical,histopathological and immunohistochemical analysis,respectively.Results1.The copolymer mPEG113-P(Phe10-co-Cys5)was successfully prepared.The results of TEM showed that the copolymer mPEG113-P(Phe10-co-Cys5)could be self-assembled into spherical nanogel with a radius of 30.56±3.21nm in aqueous solution.The radius of nanogel in PBS by DLS was 40.2±22.7nm,and the particle size did not change significantly as time goes on(0,2,6,12 and 24 hours).In the PBS containing 10.0mM GSH,the particle size increased with the passage of time.With the increase of nanogel concentration and the passage of time,there was no significant change in the survival rate of the two kinds of osteosarcoma cells detected by MTT method,and remained at a high level(more than 95%).2.Through the observation of TEM,it was found that NGs/Dox still showed a regular spherical appearance,with a radius of 41.67±7.17nm.The radius of nanogel in PBS determined by DLS was 62.4±32.2nm.3.Compared with Dox,NGs/Dox showed sustained release without obvious sudden release.The Dox release rate of GSH positive group was higher than that of GSH negative group(72 hours P<0.001).The results of co-culture of K7M2 cells and 143B cells with NGs/Dox by CLSM showed that with the passage of time,,the uptake of NGs/Dox by the two kinds of osteosarcoma cells gradually increased and gradually concentrated into the nucleus.Compared with the GSH negative groups,the Dox fluorescence intensity was stronger in the GSH positive groups,especially in the nucleus.The results of FCM detection of two kinds of osteosarcoma cells showed that under the condition of the same co-culture time,the proportion of Dox fluorescence positive cells in GSH positive groups were significantly higher than that in GSH negative groups.4.At different concentrations,NGs/Dox could inhibit the proliferation of two kinds of osteosarcoma cells to a certain extent.Compared with NGs/Dox(GSH-)group and Dox group,the results of NGs/Dox(GSH+)group showed that NGs/Dox could inhibit the proliferation of two kinds of osteosarcoma cells more efficiently in vitro at different concentrations.Compared with CQ negative groups,all the concentrations of NGs/Dox and Dox in CQ positive groups showed stronger inhibitory effect on the proliferation of two kinds of osteosarcoma cells,and the combination of Dox or NGs/Dox and CQ showed a synergistic effect.5.CLSM results showed that almost all of the two kinds of osteosarcoma cells co-cultured with NGs/Dox entered lysosome in 2 hours without CQ pretreatment,lysosome escape occurred in 4 hours,and lysosome escape occurred in most osteosarcoma cells after co-culture for 6 hours.Under the condition of CQ pretreatment,lysosome escape occurred after the uptake of nanodrug by cells for 2 hours,and then Dox was released into the nucleus.The Dox red fluorescence of osteosarcoma cells co-cultured for 4 hours or 6 hours was further enhanced and gradually concentrated in the nucleus.Compared with the Control group,a large number of autophagosomes could be observed under TEM.The results of Western blot showed that the expression of p62 and LC3 ? in CQ positive groups were higher than that in CQ negative groups.6.The results of pharmacokinetics showed that the t1/2 of NGs/Dox and Dox were 16.15±2.86 hours(P<0.01),5.64±0.7 hours,respectively,and the blood circulation time of NGs/Dox was significantly prolonged.The AUC0-t of NGs/Dox was 11.54 times that of Dox(P<0.001),while the clearance of Dox was 7.12 times that of NGs/Dox(P<0.01).7.The results of ex vivo fluorescence imaging showed that compared with Dox,NGs/Dox had higher fluorescence intensity in tumor at 1,6 and 12 hours after caudal vein administration.Semi-quantitative results at 12 hours showed that the fluorescence signal intensity of NGs/Dox group was significantly higher than that of Dox group(the P values of subcutaneous and orthotopic osteosarcoma models were P<0.01,P<0.001,respectively).8.The tumor volume of Control group treated with PBS increased most rapidly.Compared with the Control group,the tumor growth of other groups were inhibited in varying degrees,and the trend was NGs/Dox(CQ+)group<NGs/Dox group<Dox(CQ+)group<Dox group<CQ group<Control group.The results of the trend of relative tumor necrosis area between the two tumor-bearing models were as follows:NGs/Dox(CQ+)group>NGs/Dox group>Dox(CQ+)group>Dox group>CQ group>Control group.The trend of cleaved caspase-3 fluorescence intensity in each group was NGs/Dox group>Dox group>Control group,while the Ki-67 fluorescence intensity trend was opposite to that of cleaved caspase-3,NGs/Dox(CQ+)group<NGs/Dox group<Dox(CQ+)group<Dox group<CQ group<Control group.9.The osteosarcoma bearing mice in Dox group had the most weight loss and loss of appetite and activity,and the mice in NGs/Dox(CQ+)group had the least weight loss.Histopathological results showed that the heart,liver,lung and kidney of Dox group and Dox(CQ+)group showed more serious damage,while NGs/Dox group and NGs/Dox(CQ+)group were similar,and lighter than Control group and CQ group.Conclusions1.A kind of copolymer mPEG113-P(Phe10-co-Cys5)was designed and synthesized,which can be self-assembled into spherical nanogel in aqueous solution.The nanogel has suitable particle size and morphologic characteristics.It can be stable in physiological environment,and has good reduction-responsiveness and biocompatibility.It can be used as a nanocarrier for further research on osteosarcoma.2.The NGs/Dox with uniform and suitable size,which could prolong the time of Dox in the blood circulation,avoid its premature clearance,increase its accumulation in osteosarcoma tissue,and responsibly release Dox under the condition of high GSH in osteosarcoma cells.NGs/Dox,compared with Dox,can play a synergistic and attenuated role in treatment of osteosarcoma.3.In the subcutaneous model of osteosarcoma constructed by K7M2 cells and the orthotopic model of osteosarcoma constructed by 143B cells,the combination of NGs/Dox and CQ has more obvious advantages in anti-osteosarcoma effect,and no more serious toxic and side effects were observed compare with NGs/Dox alone.4.CQ plays a synergistic anti-osteosarcoma effect by helping NGs/Dox escape from lysosomes and inhibit autophagy.