Research On PH-responsive Nano-delivery System Based On Enhancing Tumor Penetration And Improving Anti-tumor Efficacy

Despite numerous studies conducted on cancer over the past few decades,cancer has been one of the leading causes of high morbidity and mortality worldwide in recent years.Dense extracellular matrix(ECM)severely impedes the spread of the drug in solid tumors and leads to decrease in the oxygen density inside the tumor,reducing the efficiency of chemotherapy.Studies have shown that different proteolytic enzymes such as collagenase(Col)or bromelain can directly attached to the surface of the nanoparticles,and improve their diffusion,but the method of ligation may also impair the enzyme activity due to conformational changes or blockage of the active site.Therefore Col-nc was synthesized by a radical polymerization method forms an engineered polymer coat on the surface of Col.It could protect the activity of the enzyme before reaching the action site while being degraded under mild acidic conditions in tumor,and the released proteolytic enzyme could digest collagen.In addition,HFn as a carrier could effectively load DOX and had a self-targeting ability,enabling nanoparticles internalized into cancer cells more effectively.Then,collagenase nanocapsules(Col-nc)was combined with heavy chain ferritin(HFn)the chemotherapy drug doxorubicin(DOX)encapsulated into HFn to enhance tumor penetration of nanomedicine by hydrolyzing collagen from ECM.From in vivo and in vitro studies,we found that the collagen was effectively degraded by Col-nc/HFn(DOX)to increase the accumulation and penetration of nanoparticles in solid tumor site,and could alleviate hypoxia inside the tumor to enhance the antitumor effect of DOX.Therefore,the strategy of increasing nanoparticle penetration and alleviating tumor hypoxia in this system is expected to provide ideas for the clinical application of solid tumors.First,Col-nc were synthesized by free radical polymerization to form a polymer coating on the surface of free Col.Acrylamide and glycerol dimethacrylate were used as structural monomer and pH-degradable cross-linker,respectively.An amino gro up was formed on the surface of the capsule for the next reaction,which was provided by hydrochloride salt of 2-aminoethyl methacrylate.The average size of Col-nc was?60 nm and TEM showed that the particles were spherical morphology and uniformly dispersed without significant aggregation.The polymer coat was broken under acidic conditions and released Col to hydrolyze the collagen of ECM and enhance tumor penetration.To demonstrate acid responsiveness,Col-nc was treated in PBS at pH 6.5 for different time.Then,the Col-nc was characterized by dynamic light scattering(DLS),we found that the particle size gradually decreased from?62 nm to?7 nm,the final particle size was the same as that of Col.It indicated that the coating was broken and Col was released.The results of TEM were consistent with the particle size change.The degradation of the coating and the change of Col-nc surface could be observed from the TEM,indicaed that Col-nc could respond to pH 6.5.Then,The carboxyl group on DOX@HFn is then linked to the amino group on Con-nc to form the final carrier.The FT-IR spectra and UV-Vis absorption spectra image of Col-nc/HFn(DOX)showed the successful connection of the carrier and used to characterize the encapsulation of DOX,respectively.Then the encapsulation efficiency was measured to be 75.0%.The TEM and DLS showed the diameter of Col-nc/HFn(DOX)was?164 nm.The TEM image of Col-nc/HFn(DOX)showed the attachment of HFn particles around the Col-nc,confirming the connection of HFn to Col-nc.In addition,we used the ninhydrin method to measure the enzymatic activity of Col,Col-nc,and Col-nc/HFn(DOX)by calculating the released free amino acids.The 4T1 breast cancer cells were used as a cell model to investigate the distribution of the preparation Col-nc/HF n(DOX)in the cell by ingestion experiments.It showed from the ingestion results that the final preparation had a significantly higher intracellular uptake than free DOX,and most of it was distributed in the nucleus,mainly because HFn had a targeting effect allowing more preparations to be taken up.Then,the survival rate of Col-nc and Col-nc/HFn on cancer cells and normal cells were investigated.The results showed that the carrier had almost no inhibition on 4T1 and HUVEC cells,indicating that it had good biocompatibility.In addition,the MTT method was used to examine the inhibitory effect of the preparation on tumor cells.It could be seen from the experimental results that the cell inhibition rate reached 89.5%after the cells were treated with the final preparation for 48 h.It was indicated that the inhibitory effect of each preparation increases with time,and the inhibitory effect of each preparation increases as the concentration increases.Finally,we also evaluated the penetration of the formulation Col-nc/HFn(DOX)in 3D tumor spheres and the inhibition of 3D tumor spheres as well as the in vitro antitumor activity.From the results,it could be shown that the Col-nc in the final preparation were still presence of enzymatic activity and could increase the penetration of DOX@HFn in the 3D tumor sphere.After 12 h of action,the final formulation penetrated almost completely into the tumor sphere.The inhibition of 3D tumor spheres showed that Col-nc increased the penetration of DOX@HFn in the 3D tumor sphere,thereby enhancing the antitumor efficacy of the chemotherapy drug.Then,we selected female BABL/c mice as experimental animals and 4T1 cell lines to construct breast cancer animal models.The imaging and in vivo pharmacodynamics of Col-nc/HFn(DOX)in tumor-bearing mice were investigated.The distribution of Col-nc/HFn(DOX)in mice was studied by in vivo imaging experiments in mice.It could be seen from the in vivo imaging that the final formulation was mainly distributed in the tumor site and the accumulated fluorescence intensity was stronger than DOX@HFn.It was again proved that Col-nc/HFn(DOX)had a good targeting effect.The in vivo pharmacodynamics of the drug was studied by body weight,tumor size change before and after administration of the mouse,HE section and TUNEL experiment.It could be seen from the results that Col-nc/HFn(DOX)had a good antitumor effect.In addition,the Masson's staining analysis and immunofluorescence experiments were used to assess the ability of the preparation to degrade collagen,reduce tumor hypoxia and tumor penetration,respectively.The results were consistent with expectations,Col-nc/HFn(DOX)could effectively degrade collagen,increase the distribution of nanoparticles in tumors,and alleviate hypoxia inside the tumor,thereby increasing the antitumor effect of nanoparticles.