Au@TiO₂-Hemoglobin Loaded Liposome Alleviating Tumor Hypoxia And Enhancing The Sonodynamic Effect With Low Ultrasonic Dose

In recent years,phototherapy,including photodynamic therapy（PDT）and photothermal therapy（PTT）,has been proved to an effective way for cancer treatment.However,due to the limited penetration depth of laser in tissue,its therapeutic effect on deep tumor is limited.Due to high penetration depth of ultrasound,SDT provide a new modality for the treatment of deep tumor.Sonodynamic therapy（SDT）a combination of low-intensity ultrasound with sonosensitizer,to produce reactive oxygen species（ROS）,which in turn kill tumor cells.However,the low efficiency of traditional sonosensitizer and the hypoxia environment in deep tumor site confines the therapeutic efficiency of sonodynamic therapy.In this paper,we will propose solutions to these two problems.Titanium dioxide（TiO₂）is a representative inorganic nanosensitizer commonly used in SDT.However,due to the quick recombination of electron-hole limits the ROS production of TiO₂.As previously reported,the presence of noble metal nanoparticles can improve the ROS production of TiO₂ by lowering the recombination rate of electrons and holes through electron trapping.Herein,Au@TiO₂-Hemoglobin loaded liposome nano system（Au@TiO₂-Hb-Lip）was developed to improve the efficiency of sonodynamic therapy against hypoxic deep tumor.First,to improve the ROS production of TiO₂,Au@TiO₂ core-shell nanoparticles were synthesized by coating TiO₂ onto the surface of Aunanospheres.Then,hemoglobin（Hb）which can serve as an oxygen supplier was conjugated onto the surface of Au@TiO₂ to get Au@TiO₂-Hb nanoparticles.At last,Au@TiO₂-Hb was encapsulted into liposomes to form Au@TiO₂-Hb-Lip nanoparticles.Here,the existence of Auimproves the ROS production of TiO₂ upon ultrasound activation by electron trapping.On the other hand,hemoglobin in the nano system serves as an oxygen supplier,improves the oxygen content in hypoxic tumor tissue,hence enhances the effectiveness of sonodynamic therapy to deep tumor.In addition,the liposome prevents Au@TiO₂ from aggregating in blood circulation hence prolong its circulation time and penetration depth in circulatory system.The constructed Au@TiO₂-Hb-Lip might be used as a highly effective sonosensitizer in cancer treatment.The detailed work are as follows:1.We first synthesized a sonodynamic agent based on Au@TiO₂ core-shell nanoparticles with controllable shell thickness at room temperature using hydrolysis method and detect the production of reactive ROS induced by Au@TiO₂ with different shell thickness.Firstly,AuNS were prepared based on the"seed mediated growth method".Before TiO₂ was coated on the surface of TiO₂,PSS was modified on the surface of Aunanospheres to remove CTAB which has biotoxicity.Then Au@TiO₂ core-shell nanoparticles were synthesized,and the shell thickness was controlled by the adding amount of NaHCO₃ which determines the hydrolytic rate of TiCl₃.DPBF was taken as an indicator probe to investigate the ROS production of Au@TiO₂.The results showed that Au@TiO₂with the shell thickness of 18.54 nm had the highest reactive oxygen species yield under ultrasonic excitation.Moreover,the ROS yield of Au@TiO₂ is much higher than that of TiO₂ at the same concentration,confirming that the presence of gold can significantly improve the sonodynamic effect of TiO₂.At last,the in vitro cell experiments showed that Au@TiO₂ nanoparticles had high sonodynamic inhibition effect.2.Au@TiO₂-Hb-Lip with good bio-compatibility and high tumor penetrability was further synthesized for effective SDT against hypoxic tumor.Hb which served as an oxygen sever was first linked to the surface of Au@TiO₂ to form Au@TiO₂-Hb nanoparticles.Then Au@TiO₂-Hb was encapsulated in liposome to prolong the circulation time and penetration depth of Au@TiO₂-Hb.Dynamic light scattering and transmission electron microscopy showed that the synthesized Au@TiO₂-Hb-Lip nanoparticles were uniform and well dispersed.Confocal laser scanning microscopy and the fourier transform infrared（FTIR）spectra confirmed the encapsulation of Au@TiO₂-Hb in liposome.The results of oxygen release test showed that Au@TiO₂-Hb encapsulated liposomes maintained high ROS production and oxygen carrying and releasing capacity.Finally,the sonodynamic inactivation effect of the sonodynamic effect mediated by Au@TiO₂-Hb-Lip was measured in vitro.The results showed that Au@TiO₂-Hb-Lip had high biocompatibility and the sonodynamic effect mediated by Au@TiO₂-Hb-Lip could effectively kill tumor cells.It is confirmed that the oxygen carried by Hb can effectively improve the hypoxia of tumor microenvironment.More than 80%of cells were killed after exposed to the irradiation of ultrasound with a power density of 1W/cm² for 3 minutes.This confirms that Au@TiO₂-Hb-Lip can be used as a highly effective acoustic sensitizer for the treatment of deep tumors.