Investigation On Hemoglobin-based Bio-mimetic Drug Delivery System For Combined Sonodynamic-chemotherapy

Hollow mesoporous titanium dioxide?TiO₂?as the sonosensitizer could produce reactive oxygen species?ROS?under ultrasound irradiation for sonodynamic therapy?SDT?.However,the nanoparticles accumulate less in the tumor site,and the SDT efficiency is limited by the hypoxic microenvironment of deep tumors.To tackle these issues,the hemoglobin?Hb?-based bio-mimetic drug delivery system,CCM-Hb-TiO₂/RRx-001,was constructed for combined sonodynamic-chemotherapy.This system had the following characteristics:1)The nanoparticles camouflaged with MCF-7 source cancer cell membranes?CCM?would accumulate to the tumor site due to the immune escape and homologous targeting abilities.2)Hb could release oxygen in the hypoxic tumor site to sensitize the SDT efficiency of TiO₂.3)The nitrite reductase activity of the resulting Deoxy-Hb was enhanced by hemoglobin-RRx-001 adduct,which could greatly accelerate the reduction of nitrite?NO₂^??to nitric oxide?NO?in tumor site for vasodilation and anti-tumor effects.Meanwhile,the level of the cellular ROS was elevated due to glutathione?GSH?depletion by glutathione-RRx-001 adduct.Therefore,CCM-Hb-TiO₂/RRx-001 has the potential to synergize SDT with chemotherapy to increase the contents of reactive oxygen and nitrogen species?RONS?for enhanced anti-tumor efficacy.The main researches were as follows:1.Firstly,hollow mesoporous titanium dioxide?TiO₂?nanoparticles were synthesized by using synthetic silica?SiO₂?as templates,following by sol-gel method,calcination and etching processes.Then Hb was covalently modified onto aminated-TiO₂ nanoparticles via amide reaction.Subsequently,RRx-001,an anti-cancer drug,was loaded into the TiO₂ nanoparticles via ultrasonic dispersion method.CCM was obtained by hypotonic lysis,homogenization and differential centrifugation of MCF-7 human breast cancer cells.Finally,Hb-TiO₂/RRx-001 and CCM were co-extruded to acquire CCM-Hb-TiO₂/RRx-001.The successful preparation of CCM-Hb-TiO₂/RRx-001 was characterized by transmission electron microscopy,Fourier transform infrared spectra and UV-visible spectra.The zeta potential and hydrodynamic size of CCM-Hb-TiO₂/RRx-001 were?21.7±1.0 mV and 145.6±3.6 nm respectively.The oxygen release ability was evaluated using Ru?dpp?₃Cl₂ as oxygen-quenching probe,which demonstrated that the oxygen loading amount in CCM-Hb-TiO₂/RRx-001 was about 2.5 times more than that of free Hb.Then,we used singlet oxygen sensor green?SOSG?to detect the ROS generation,the results showed that there was higher ROS production in CCM-Hb-TiO₂ under US irradiation than that of CCM-TiO₂,although both of them exhibited US time-dependent behaviors.Furthermore,GSH level was decreased along with the increased RRx-001 concentration.2.The cellular uptake of Hb-TiO₂ loading Nile red among different cell lines?MCF-7,MDA-MB-231,Hs538Bst,HepG2,NIH 3T3,RAW 264.7?was investigated in vitro.The result confirmed that CCM-Hb-TiO₂ possessed the specifically homologous targeting and immune escape capability compared with bare Hb-TiO₂.Then,MCF-7 cells were employed to examine the activities of different formulations.DHE and DAF-FM DA were used to detect cellular ROS and NO respectively by fluorescent microscope and flow cytometry.Both of ROS and NO levels had increased in CCM-Hb-TiO₂/RRx-001+US group.The level of GSH dropped by 60%in CCM-Hb-TiO₂/RRx-001+US group,which was propitious to elevate intracellular ROS.The cell viabilities were assessed using SRB method.CCM-Hb-TiO₂hadnoobvioustoxicitytoMCF-7cells,while CCM-Hb-TiO₂/RRx-001 showed a significant decrease in cell survival rate?19.0±2.2%?under ultrasound irradiation.3.The distribution of Hb-TiO₂ loading IR783 after intravenously administration in single tumor-bearing and double tumor-bearing nude mice was further investigated in vivo.The fluorescent imaging results showed that CCM-Hb-TiO₂could enhance the accumulation in MCF-7 tumor sites than bare Hb-TiO₂,and preferentially targeted to the MCF-7 tumor in the competition of HepG2 tumor.The results of tumor slices confirmed that the treatment of CCM-Hb-TiO₂/RRx-001could dilate the tumor blood vessels,increase the drug accumulation in tumor site and decrease the expression of HIF-1?.The enhanced fluorescence intensity of DHE and DAF-FM in the frozen tumor region were simultaneously occurred in CCM-Hb-TiO₂/RRx-001+US group.Furthermore,the anti-tumor efficacy assay demonstrated that the relative tumor volume in CCM-Hb-TiO₂/RRx-001+US group decreased to 1.17±0.10,significantly inhibiting the growth of tumor.Meanwhile,the treatment of CCM-Hb-TiO₂/RRx-001+US caused considerable disrupted cells in H&E staining,substantial TUNEL-positive and?-H2AX-positive tumor cells.In addition,no obviously abnormal change was found in tumor weight and in H&E staining of major organs for each group,implying the good biosafety of CCM-Hb-TiO₂/RRx-001.To sum up,the designed CCM-Hb-TiO₂/RRx-001 could enhance its accumulation in MCF-7 tumor sites due to the specifically recognition to homologous tumor and immune escape.The Hb-based combination of SDT and chemotherapy would synergistically elevate RONS for enhanced anti-tumor efficacy.Consequently,the system of CCM-Hb-TiO₂/RRx-001 made it more promising to targetedly enhance the cancer therapy in future.