Study Of Drug Co-Delivery System Mediated By Positioning Remodeling Nanogels

Tumor is one of the culprits leading to the increasing human's mortality.The proliferation and metastasis of tumor mainly rely on tumor blood vessels to provide the necessary oxygen and nutrition.Vascular deficient tumor is characterized by self-limiting growth.Nowadays,many anti-vascular drugs have been developed for the treatment of tumor starvation by blocking the external nutritional supply.However,after this drug is effective,tumor cells will provide endogenous nutrients for their own proliferation through the autophagy enhancement effect.This self-protection will lead to a serious decline in antitumor effects.Then,destroying the tumor vasculature and inhibiting enhanced autophagy collectively block the tumor's exogenous and endogenous nutritional sources.It will be a promising antitumor method.Therefore,in this subject,we used sodium alginate(Alg)as the matrix material,Fe3+as the cross-linking agent and the gate switch,combretastatin A4(CA4)and hydroxychloroquine(HCQ)as the therapeutic drugs.A smart nanogel CA4-FeAlg/HCQ with positioning and remodeling function was constructed.It can be used for the co-delivery of vascular blocker CA4 and autophagy inhibitor HCQ to achieve the sequential release of CA4 in tumor blood vessels and HCQ in tumor cells,as well as synergistic treatment of A549 non-small cell lung cancer.In this study,the vascular blocker CA4 was grafted onto Alg to synthesize Alg-CA4 conjugate with a grafting yield of 14%,which improved the bioavailability and stability of CA4.Then,it was self-assembled with Fe3+to form CA4-FeAlg nanogels,and the autophagy inhibitor HCQ was loaded inside the nanogel to obtain CA4-FeAlg/HCQ for tumor synergistic treatment.Experimental results displayed that the hydrated particle size of CA4-FeAlg was about 150 nm.Under the condition of tumor microenvironment(TME)in vitro,Fe3+was reduced to Fe2+,the phase transition from gel to sol occured.The nanogel was dissociated from the outside to the inside,and its particle size decreased(? 27 nm),indicating that the nanogel has the characteristic of TME responsive particle size transformation(from large to small),which is beneficial to tumor deep penetration.The three-dimensional network structure of CA4-FeAlg could effectively load HCQ,and the entrapment efficiency and loading efficiency were 13.8%and 21.6%,respectively.The release results of CA4-FeAlg/HCQ investigated that the release behaviors of CA4 and HCQ were significantly different.CA4-FeAlg/HCQ was stable in the medium of pH 7.4+10%BSA(simulated blood circulation),while CA4 rapidly released in the medium of pH 7.4(simulated tumor vascular environment),and HCQ rapidly released in the medium of pH 5.5+5 mM glutathione(GSH,simulated tumor cell microenvironment).The above results proved that CA4-FeAlg/HCQ could sequentially release CA4 and HCQ at two active targets o.f tumor blood vessels and tumor cells,which provided a new idea for the combination of drugs on the nano-codelivery platform.The results of in vitro cell uptake and distribution revealed that FeAlg could be largely uptake by A549 lung cancer cells within 4 h.The uptake amount reached 84%.After entering the cells,FeAlg was mainly distributed in lysosomes.The average colocalization coefficient was 0.74 ± 0.09 at 3 h,indicating that FeAlg could quickly carry HCQ into tumor cells and locate at lysosomes.In tumor cells,FeAlg could be efficiently disproportionate H2O2 to produce-OH by Fenton's reaction.The generated·OH and released HCQ acted together on lysosomes,causing lysosomal damage,and then hindering the formation and degradation of autolysosomes.Then,this could exert autophagy inhibition to synergistic promote the antitumor effect of CA4.Immunohistochemistry and ELISA results showed that CA4-FeAlg/HCQ could notably inhibit the autophagy of tumor cells,which were significantly different from other groups.In vitro 3DMCS results exhibited that compared with mesoporous SiO2 with stable particle size,the penetration depth of FeAlg with particle size transformation characteristics increased by about 2-folds.In vitro cytotoxicity results showed that the cell inhibition rate of CA4-FeAlg/HCQ(20 ?g/mL,48 h)reached 93.86%.It could effectively kill A549 tumor cells.The Calcein-AM/PI staining also proved that the CA4-FeAlg/HCQ had the lowest cell activity.In vivo,the animal model was A549 tumor-bearing nude mice.Optical imaging results revealed that CA4-FeAlg/HCQ could accumulate in the tumor sites quickly and retain for a long time.Furthermore,CA4-FeAlg/HCQ had excellent deep penetration due to its particle size conversion characteristics in response to TME.Pharmacodynamic experiments showed that CA4-FeAlg/HCQ had the best treatment effect in vivo,the relative tumor volume eventually decreased to 0.40±0.10,which was significantly different from the CA4-FeAlg group.Immunofluorescence results showed that CA4-FeAlg could significantly reduce the density of tumor blood vessels,to block the nutrient pathway of tumor proliferation and induce tumor cell death.Active oxygen species results showed that FeAlg could produce·OH by disproportionating H2O2 in tumor to further kill tumor cells.Immunohistochemistry and TEM showed that CA4-FeAlg/HCQ could effectively inhibit autophagy induced by CA4-FeAlg antivascular therapy,thereby achieving synergetic treatment of tumor.