Construction Of A Hierarchical Responsive Delivery System Targeting Tumor-associated Macrophages And Its Application In Tumor Treatment In Vitro And In Vivo

Objective:A stepwise stimulus-responsive nanoparticle carrying anti-tumor drug,doxorubicin(DOX),was designed and synthesized to inhibit the growth,invasion and metastasis of-breast cancer and improve the poor prognosis of breast cancer by clearing M2 type-TAMs.Methods:based on M2-type TAMs in tumor has the characteristics of-high expression of mannose receptor,Mannan was used to target M2 type-TAMs.methoxy polyethylene glycol amine(Methoxypolyethylene glycol amine,MW=2000,PEG2000)was used to modify mannan to escape of macrophage cells in the body's normal tissues,such as Kupffer cells of liver cells.Then Mannan combined with hydrophobic benzene boric acid pinacol ester(phenylboronic acid pinacol ester hydrochloride,B).A hypoxia and ROS responsive nanoparticle PEG200-Azo-mannan-B(PAMB)obtained and loaded DOX.The structure of PAMB was analyzed by nuclear magnetic resonance(NMR)and infrared spectroscopy(IR).Its physical and chemical properties(particle size,potential,etc.)were characterized.MTT method was used to investigate the cytotoxicity of different formulations on M2-type TAMs under hypoxic and normoxic conditions.Confocal microscope was used to investigate the uptake capacity of cells to diffient formulations under hypoxic and normoxic conditions.4T1 tumor-bearing mice model was established to investigate the targeting effect of formulations in vivo.The effect of removing M2-type TAMs,inhibiting tumor invasion and metastasis were studied.The relevant factors in tumor were investigated.Results:The prepared nanoparticles were uniform in shape and spherical.The particle size was around 200nm.PAMB could fracture successfully under hypoxic conditions.PAMB/DOX had a strong ability to release DOX under ROS.Cellular uptake experiment showed that cells exposed to PAMB/DOX had more uptake under hypoxic conditidion than those exposed to normoxic condition.Cytotoxicity experiments showed that PAMB had stronger cytotoxicity in hypoxic condition than normoxic condition.The results of in vivo distribution showed that PAMB/ICG had better tumor targeting,and there was less liver distribution compared with non-PEG mannan nanoparticles,indicating that PAMB effectively reduced liver capture.In vivo effect results showed that PAMB/DOX could remove M2-type TAMs better than free DOX.PAMB/DOX showed good anti-tumor effect and significant inhibition of lung metastasis.Further studies on the changes of tumor microenvironmental enzymes showed that PAMB/DOX reduced the expression of matrix metalloproteinase MMP-9,inhibited tumor angiogenesis and so on.Conclusion:PAMB/DOX effectively inhibited lung metastasis of 4T1 tumor by Clearing M2-type TAMs.Potential mechanism studies found that PAMB/DOX significantly inhibited the expression of MMP-9 and angiogenesis.The results showed that removing M2-type TAMs could improve tumor microenvironment and reduce the harm caused by tumor metastasis.This is of great clinical significance for the prognosis and treatment of breast cancer.