A Study On Doxorubicin-loaded Hyaluronidase Functionalized Silica Nanocarrier Enhancing Immunogenic Cell Death Of Tumor Cells

Backgroud and ObjectiveThe chemo-immunotherapy has become a highly prospective method for cancer treatment and it has been widely acknowledged that chemotherapeutic drugs such as doxorubicin(DOX)could trigger antitumor immune responses.However,chemotherapeutic drugs alone have many limitations such as insufficient tumor penetrability and appear to induce weak immunogenic cell death(ICD).Herein,we report the design of hyaluronidase functionalized silica nanoparticles encapsulating DOX(DOX@HMSPHs),which could enhance the permeability of drug and induce enhanced ICD effect,finally activating specific tumor immunity.Methods1.Hollow mesoporous silica(HMSN),hyaluronidase functionalized HMSHs(HMSPHs)and DOX-loaded nanoparticles(DOX@HMSPHs)were synthesized by chemical methods and their physical and chemical properties were tested.Then DOX loading and release in vitro was determined.The uptake of nanoparticles HMSPs and HMSPHs by melanoma cell line B16/F10 in mimic tumor extracellular matrix(ECM)was observed under confocal microscopy.2.The distribution of DOX in B16/F10 cells was observed after uptaking drug-loaded nanoparticles under the confocal microscope.Then the activity of B16/F10 cells was detected by CCK-8 kit and the apoptosis of B16/F10 cells induced by drug-loaded nanoparticles was detected by flow cytometry.The drug-loaded nanoparticles were incubated with B16/F10 cells for 24 h,then the release of DAMPs(including CRT,HMGB1 and ATP)in B16/F10 cells was tested by flow cytometry,Western-Blot and so on.B16/F10 cells,treated with drug-loaded nanoparticles for 24 h,were incubated with bone marrow-derived dendritic cells(BMDCs)for another 24 h and the expression of CD86 on BMDCs was determined by flow cytometry and the secretion levels of IL-12p40,IL-6,IL-1βand TNF-αin the supernatant of BMDCs was tested by ELISA kits.In addition,3days after intratumoral injection of drug-loaded nanoparticles into B16/F10melanoma-bearing mice,the ratio of CD11c~+CD86~+cells in tumor-draining lymph nodes(TDLNs)was measured by flow cytometry.3.A tumor model of C57BL/6 mice bearing B16/F10 melanoma was constructed and the mice were treated every other day with intratumor injection of drug-loading nanoparticles for four times,and the treatment dose was 1.5 mg/kg DOX.Then tumor growth during drug administration was monitored.On the 20th day,the experiment was terminated.The tumor volume and tumor weight of the mice in each treatment group were measured.The expression of HA in the tumor was detected by immunofluorescence,and the proportion of CD3~+CD8~+T cells in the tumor was detected by flow cytometry.In addition,vaccination assay was performed.B16/F10 cells,treated with different drug groups for 24 h,were subcutaneously injected into the left dorsal flanks of 5-week-old female C57BL/6 mice as a tumor vaccine.After 7 days,untreated B16/F10 cells were re-inoculated into the right dorsal flanks of mice,and the tumor growth of the right dorsal flanks of mice was monitored.At the end of the experiment,the proportion of CD3~+CD8~+T cells in the right dorsal flanks of mice was detected by flow cytometry,and the level of IFN-γin the serum of the mice was detected by ELISA kit.Results1.Successfully synthesized HMSNs had uniform diameters of~150-200 nm and a shell thickness of~30-40 nm.The specific surface area and pore volume of the HMSNs was 884.5 m~2/g and 0.65 cm~3/g,respectively.Then hyaluronidase functionalized nanoparticles(HMSPHs)were synthesized and HAase was shown to be successfully coated on HMSNs under transmission electron microscopy.The DOX@HMSPHs were obtained by loading DOX in the nanoparticles.After modified with HAase,the encapsulation efficiency(EE)and drug loading capacity(DL)of DOX@HMSPHs nanoparticles reached76.85±1.9%and 8.44±0.19%,respectively.In PBS(pH 5.5),the DOX release rate of DOX@HMSPHs in 8 h could reach~70.9%,which was significantly higher than the DOX release rate(~31.2%)in pH 7.4.In the mimic tumor extracellular matrix,the amount of HAase-modified HMSPHs taken up by B16/F10 cells was significantly increased compared with the HAase-free HMSPs at each time point.2.Confocal microscopy images showed that the fluorescence intensity of DOX significantly increased in B16/F10 cells treated with DOX@HMSPHs compared with the DOX@HMSPs group.Compared with the PBS group,HMSPs and HMSPHs had no significant effect on the cell viability of B16/F10 cells,while the survival rates of B16/F10cells treated with DOX,DOX@HMSPs and DOX@HMSPHs for 24 h were 63.2±9.5%,62.2±5.5%and 49.0±2.1%,respectively.Cell apoptosis experiments showed that the proportion of apoptosis in the DOX@HMSPHs group was 67.6±4.4%,which was significantly higher than that of DOX@HMSPs group(55.0±2.8%)and DOX group(35.2±8.8%).The nanocarriers(HMSNs,HMSPs and HMSPHs)had no obvious cytotoxic effects on the normal mouse fibroblast cell line NIH/3T3,the human melanoma cell line A375 and the mouse melanoma cell line B16/F10.Compared with the DOX and DOX@HMSPs groups,the DOX@HMSPHs group induced a significant increase in the positive rate of CRT on the surface of B16/F10 cells,a significant increase in the mean fluorescence intensity of CRT,and a significant increase in the secretion of HMGB1 and ATP in the supernatant.Compared with the other groups,the percentage of CD86~+BMDCs,induced by apoptotic B16/F10 after DOX@HMSPHs incubation was significantly increased,and the levels of IL-12p40,IL-6,IL-1βand TNF-αin the supernatant of BMDCs also significantly increased.Compared with the other groups,the percentage of CD11c~+CD86~+cells in TDLNs significantly increased 3 days after intratumoral injection of DOX@HMSPHs.3.Compared with other groups,DOX@HMSPHs intratumoral injection could significantly inhibit the growth of B16/F10 melanoma,degrade intratumoral HA and promote intratumoral CD3~+CD8~+T cell infiltration.The results of the vaccination assay showed that the tumor vaccine induced by DOX@HMSPHs could delay the growth of the re-inoculated tumor,promote the activation of CD3~+CD8~+T cells in re-inoculated tumor,and increase the level of IFN-γin the serum of mice.ConclusionsWe successfully synthesized DOX@HMSPHs nanoparticles,and the hyaluronidase on the surface could degradate HA in the ECM to enhance the permeability of the nanoparticles.DOX released from the nano drug-loading system induced ICD of tumor cells and forced the tumor cells to release tumor-associated antigens and DAMPs,thereby promoting the maturation of DCs and further activating tumor-infiltrating cytotoxic T lymphocytes(CTLs),finally activating specific anti-tumor immunity.In addition,the nanocarrier HMSPs could be used as immunological adjuvants to further enhance DC recognition of tumor antigens and enhance their antigen presentation ability,thereby enhancing the activation of tumor-infiltrating CTLs.In conclusion,our results indicated that the DOX@HMSPHs controlled release system might be an effective strategy to enhance ICD-induced specific tumor immunity.