Size-variable Nano-delivery System Based On Extracellular Matrix Reduction Strategy For Improving Chemotherapy

After activation by transforming growth factorβ(TGF-β),cancer-associated fibroblasts generate dense extracellular matrix that wraps around tumor cells.The extracellular matrix prevents drug penetration at the tumor site resulting in a significant reduction in the effectiveness of chemotherapy.Therefore,improving drug penetration at the tumor site is an urgent problem to be solved for improving the effectiveness of chemotherapy.In this study,a size-variable nano-delivery system based on remodeling the tumor microenvironment was constructed to enhance the penetration of doxorubicin(DOX)at the tumor site and improve the chemotherapeutic efficacy.The main research of this article included the following areas.1.Preparation and characterization of HA-DOX@GNPs-Met@HFnFirstly,hyaluronic acid(HA)and DOX were used to synthesize HA-DOX prodrug via amide reaction.Secondly,gelatin nanoparticles(GNPs)and heavy chain ferritin(HFn)were used as vectors to load HA-DOX and metformin(Met),respectively.Then,HA-DOX@GNPs and Met@HFn were connected to prepare HA-DOX@GNPs-Met@HFn nano-delivery system by amide reaction.The nano-system was characterized via ~1H NMR,FT-IR spectra,UV-vis absorption spectra,polyacrylamide gel electrophoresis and laser nanoparticle size measurement.The results showed that the average particle size and zeta potential of the nano-system were about 121 nm and-24 m V.In addition,the results of gel electrophoresis experiments and transmission electron microscopy showed that the nano-system degraded after incubation with matrix metalloproteinase-2(MMP-2),and the particle size was measured to decrease to about 40 nm after incubation 8 h.In vitro drug release experiments showed that the cumulative release rates of DOX and Met were approximately 80%and 72%,respectively.Finally,the results of stability experiments showed that the particle size and PDI of the nano-system remained basically unchanged in PBS and serum solution for a certain period of time,indicating that it had good stability.2.Study on antitumor effect and penetration ability in vitroThe mouse breast cancer 4T1 cells were used as a cell model to investigate antitumor effect and penetration ability of the nano-delivery system in vitro.Firstly,the results of cellular uptake experiments showed that HA-DOX and FITC@HFn were taken up by 4T1 cells significantly higher than free DOX and FITC.Secondly,the survival rates of normal cells after incubation with GNPs-HFn were higher than85%,indicating that the vector had excellent biocompatibility.In contrast,the survival rates of 4T1 cells after incubation with HA-DOX@GNPs-Met@HFn nano-system were the lowest,indicating that it was highly cytotoxic to 4T1 cells.In addition,MMP-2 pretreated nano-system showed excellent penetration effect in tumor spheroids.Meanwhile,the nano-system displayed significant inhibition effect on the growth of tumor spheroids,indicating it had good antitumor activity.Finally,the results of western blotting experiments showed that nano-system decreased expression of TGF-β,thus reducing the generation ofα-smooth muscle actin and Collagen I.3.Study on distribution in vivo and penetration in tumor tissueThe mouse model of 4T1 breast cancer was constructed to study distribution in vivo and penetration in tumor tissue of the nano-delivery system.The results of in vivo imaging showed that nano-system had excellent tumor targeting ability.Then,frozen sections of tumor tissues and immunofluorescence staining sections of tumor vessels were observed,and the results showed that nano-system had good penetration and retention effects at the tumor sites.In addition,Masson staining sections of tumor tissues were observed,and the results revealed that nano-system significantly reduced the generation of collagen fibers.Finally,immunofluorescence staining sections of tumor tissues were analyzed,and the results verified that nano-system down-regulated TGF-βto reduce the expression ofα-smooth muscle actin and Collagen I.4.Study on antitumor effect and safety in vivoThe mouse model of 4T1 breast cancer was established to investigate antitumor effect and safety in vivo of nano-delivery system.The results of relative tumor volume change and tumor inhibition rate showed that nano-system had strong inhibition effect on tumor growth,and tumor inhibition rate was about 87%.H&E staining,Ki67 immunohistochemical staining and TUNEL staining sections of tumor tissues were observed,and the results revealed that nano-system had excellent antitumor effect.In addition,the results of changes in body weight,visceral index,blood routine and blood biochemical data,the pathological analysis of H&E staining sections of major organs showed that nano-system had good biosafety.In summary,this study successfully constructed a size-variable nano-delivery system based on remodeling the tumor microenvironment to reduce extracellular matrix generation for facilitating deep penetration of HA-DOX,thus significantly improving chemotherapy efficacy.