Study On The Antitumor Activity And Mechanism Of Responsive Engineered Bacterial Drug Delivery System

Due to the characteristics of hypoxia and immunosuppression in the microenvironment of solid tumor,the effectiveness and bioavailability of antitumor drugs are reduced in tumor treatment.Therefore,it is an urgent problem to seek more effective anti-tumor strategies.In recent years,eukaryotic cells,bacteria,viruses and other microorganisms have attracted extensive attention because of their tendency to target into the tumor microenvironment,and therefore can be used as excellent biological vectors.On this platform,nano-composite materials are further attached to form a multi-functional engineered bacterial drug delivery system,which can more effectively improve the anti-tumor effect and achieve targeted and precise treatment of tumors.Based on this,this thesis constructs two biodegradable multifunctional responsive bacterial drug delivery systems for tumor treatment.（1）Study on antitumor activity and mechanism of engineered bacterial drug delivery system with sound sensitive effect.Firstly,nano-gold（Au NPs）was coated on the surface of nano-RuO₂（RuO₂ NPs）with acoustic sensitive function to form modified acoustic sensitive agent Au-RuO₂ NPs.In addition,nano-selenium（Se NPs）,which can induce endogenous ROS production in cells,was self-assembled on the surface of attenuated Listeria innocua（LI）together with Au-RuO₂ NPs to form an engineered bacterial drug delivery system with complex sound sensitive effects（Bac@ARS）.The morphology of each component of Bac@ARS was observed by transmission electron microscopy.Au-RuO₂NPs was composed of the surface of the core gold nanosphere coated with RuO₂,showing the core-shell structure.Bac@ARS is a rod-like structure on which a large number of Au-RuO₂ NPs and spherical Se NPs are adsorbed.The formation of metal oxide by Ru as Ru⁴⁺was confirmed by X-ray diffraction analysis.Anoxic cell model of Fa Du cells was established in vitro.Bac@ARS can accumulate in the cell interior and catalyze H₂O₂ to produce O₂ to relieve cell hypoxia.After ultrasonic（US）treatment Bac@ARS can produce a large number of ROS to kill Fa Du cells.The exogenous ROS produced by acoustodynamic therapy and the endogenous ROS produced by Se NPs work together to activate anti-tumour immunity based on immunogenic cell death（ICD）,exerting a synergistic therapeutic effect.The in vivo therapeutic effects of Bac@ARS were explored using an ectopic tumour-bearing mouse model.After tail vein injection of Bac@ARS,Bac@ARS was traced by photoacoustic imaging system using the tumour tropism of LI to selectively accumulate at the tumour site and catalyse high concentrations of H₂O₂ at the tumor site to produce O₂,thereby alleviating hypoxia in TME.Bac@ARS was treated with US to produce large amounts of ROS and induce anti-tumour immune activation to synergistically treat the tumour.By analysing the distribution as well as the number of bacteria in vivo,it was demonstrated that the activity of LI was significantly reduced in all major organs after US irradiation,ensuring the biosafety of Bac@ARS.Thus,Bac@ARS is a promising microbial sonosensitiser and provides a new platform for optimising sonosensitisation for tumour therapy.（2）Photosynthetically engineered bacteria with photosensitising effects enhance photodynamic therapy by alleviating the tumour hypoxic microenvironment.The photosensitiser Ce6 was electrostatically adsorbed onto the surface of the photothermal material Au NPs to form Au-Ce6 NPs.Au-Ce6 NPs self-assembled on the surface of photosynthetic bacteria（Synechococcus 7942,Syne）to form Bac@Au-Ce6.The external morphology of Bac@Au-Ce6 was observed by transmission electron microscopy,and a large number of Au-Ce6 NPs could be observed adsorbed on the surface of Syne.In vitro oxygen production experiments demonstrated that Bac@Au-Ce6 was able to produce oxygen stably by photosynthesis of Syne under laser irradiation at 660 nm.By establishing a hypoxia model in 4T1 mouse cells,it was demonstrated that Bac@Au-Ce6was able to efficiently oxygenate cells under 660 nm laser irradiation and induce cell death through photodynamic generation of sufficient ¹O₂.An ectopic model was established by injection of 4T1 cells in a lotic mouse.This significant increase in ¹O₂ quantum yield was demonstrated to be attributed to the fact that active Syne can act as an oxygen generator,generating O₂ in situ in hypoxic solid tumors via photosynthesis and modulating TME,thereby increasing the effectiveness of ¹O₂.Bac@Au-Ce6 utilizes the photoacoustic imaging capabilities of Au NPs and photothermal imaging activated under 808 nm laser irradiation to further precisely monitor the mouse model of tumor treatment.In addition,ICP-MS analysis demonstrated that this engineered bacterium can be effectively metabolised from the body by excretion,ensuring the biosafety of Bac@Au-Ce6.Taken together,the results validate the rational design of photo-triggered photosynthetic engineered bacteria to enhance photodynamic therapy by alleviating the tumour hypoxic microenvironment.