Mechanism Investigation Of Tumor Microenvironment Response Multistage Nanoparticles For Deep Tumor Penetration

Hyaluronic acid(HA)is a major component of extracellular matrix(ECM)which over expressed in tumor tissues.The over expression of HA not only contributes to the increase of interstitial fluid pressure(IFP)but also hinders the penetration of nanoparticles in tumors.In this study,we developed a hyaluronidase-embedded drug delivery system which could enhance tumor penetration by modulating tumor microenvironment.Specifically,amphiphilic triblock polymer mPEG-b-poly(2-diisopropylaminoethyl methacrylate)-b-poly(2-guanidinoethyl methacrylate)(mPEG-PDPA-PG,PEDG)was synthesized by RAFT polymerization.Then the epirubicin-loaded cationic micelles were formed by self-assembling mPEG-PDPA-PG.Afterwards,hyaluronidase-embedded nanoparticles were formed via electrostatic interaction of epirubicin-loaded cationic micelles(NPs-EPI)with hyaluronidase(HAase).HAase could degrade HA to further improve tumor penetration efficiency and the PDPA block of mPEG-PDPA-PG has desirable p H-responsive capability,therefore,the prepared micelles can be prone to swell or dissolution at acidic endosomal/lysosomal due to the pH-sensitive PDPA segment,which could be utilized as an effective means to trigger rapid drug release.~1H NMR and Gel Permeation Chromatography(GPC)were used to investigate the synthesis of mPEG-PDPA-PtBAM.Dynamic light scattering(DLS)was used to measure the particle size,zeta potential of EPI-loaded micelles(NPs-EPI)and nanoparticles(NPs-EPI/HAase).Besides,the encapsulation efficiency(EE)and loading capacity(LC)of EPI in NPs-EPI was determined by Flex Station 3 multifunction reader.Moreover,the mechanism of NPs-EPI uptake by HepG2 cells was investigated by TEM and flow cytometry.HepG2 cells were used to establish tumor spheroids in vitro,the effects of NPs-EPI combined with different concentrations of HAase on the permeability and structure of tumor spheroids was investigated by confocal microscopy.HepG2 tumor spheroids and HepG2 tumor xenograft models were constructed to investigate the penetration mechanism of NPs-EPI/HAase.The results of ~1H NMR and GPC indicated that mPEG-PDPA-PtBAM was successfully synthesized.Results of DLS showed the size of NPs-EPI and NPs-EPI/HAase were 96.6±0.4 nm and 117.1±0.5 nm,respectively,while zeta potential of NPs-EPI and NPs-EPI/HAase were 34.6±0.4 mV and 19.6±0.4 m V,respectively.TEM results demonstrated that NPs-EPI and NPs-EPI/HAase were regular spheres.The EE and LC of EPI in the NPs-EPI were 40.57±0.62%and 8.28±0.13%,respectively,and flow cytometry results revealed that NPs-EPI entered the cells through the endocytosis pathway mediated by trellis protein and megapinocytosis,and then the micelles were transported to lysosomes for drug release.CLSM results indicated that with the increase of the HAase combined concentration,the fluorescence of the EPI distributed more extensively.Besides,NPs-EPI combined with HAase at low concentration did not significantly affect the structure of tumor spheroids,while the structure of tumor spheroids with HAase at high concentration became loose.In addition,by treating tumor spheroids with NPs and NPs/HAase and labeling HA in ECM,it can be confirmed that HAase could effectively degraded HA in ECM to enhance the penetration efficiency of NPs-EPI.Penetration mechanism investigation of NPS-EPI/HAase in solid tumors showed that after the degradation of HA,the drug-loaded micelles could enter the solid tumor effectively and inhibit tumor growth,and the degradation of HA could also increase tumor vascular density and induce tumor vascular normalization.In conclusion,we developed a hyaluronidase-embedded drug delivery system which could enhance tumor penetration by modulating the tumor microenvironment.NPs-EPI/HAase could degraded the HA in ECM,by which adjusting the tumor microenvironment to enhance the penetration efficiency of NPs-EPI.Subsequently,NPs-EPI mainly employed clathrin-mediated and macropinocytosis-mediated endocytic pathways for cellular uptake and were further directed to the lysosomes for further drug release triggered by proton sponge effect.Compared with NPs-EPI,NPs-EPI/HAase showed deeper tumor penetration and better tumor growth inhibition in solid tumors,while showing higher degree of tumor tissue damage and lower Ki-67 positive cells.In summary,coating nanoparticles with HAase can provide a simple but efficient strategy for nano-drug carriers to enhance solid tumor penetration and chemotherapeutic efficacy.