Probiotics As Potential Carriers For Tagerted Delivery And Controlled Release Of Drugs

In the antitumor and anti-intracellular bacteria research,challenges such as poor durg targeting,low bioavailability and rapid blood clearance of traditionary drug carrier drugs have not been solved.Many bacteria have been found to have the ability for tumor targeting,and the use of these bacteria for drug loadin and target delivery has broad prospects.In this thesis,probiotics Escherichia coli Nissle 1917（EcN）were used to construct several drug carriers with tumor targeting and controlled release for treatment of tumor or intracellular bacteria.Antitumor drugs,drug-loaded hybrid micelles or microtubules were loaded on the surface of the bacteria.In addition,antibacterial drugs are loaded into the internal cavities of bacteria ghosts（BGs）and the tumor-related drugs are loaded into living bacteria to construct a series of bacterial delivery system of drugs.The prepared drug carriers have the ability for tumor targeting and stimuli-responsive drug release after exposure to the tumor microenvironment.The bacterial drug loading system EcN-ca-Dox was constructed by binding the antitumor drug doxorubicin（DOX）to the surface of EcNs by an acid-sensitive bond of cis-aconitic anhydride,and the drug loading was about 4.3%.The bacteria maintained the cell viability of about 71%after drug loading,and the construction process did not impair the ability of bacterial motility.In vitro drug release indicated that the EcN-ca-Dox complex could keep stable in the blood circulation,and release DOX rapidly in tumors.In vivo imaging proved the preferential accumulation of EcN-ca-Dox in tumors,and the result of in vivo distribution demonstrated EcN-ca-Dox as a drug carrier could achieve better drug retention and tumor targeting,and had a more significant antitumor effect than free drugs.DOX or?-tocopheryl succinate（TOS）were conjugated with poly（ethylene glycol）as micellar precursors(PM_Doxox and PM_TOS).The hybrid micelle precursors were bound to the EcN surface by the tetrazine/norbornene bioorthogonal reaction and acid-sensitive bonds to form EcN@PM_D/T.Under the stimulation of tumor pH,the hybrid micelle precursors were released from the complex and self-assembled into hybrid micelles.Hybrid micelles released active drugs in an intracellular redox-sensitive environment,and brought anti-tumor effects.The morphology of EcN in EcN@PM_D/T/T complex was integral with cell viability of 89.2%,motion speed of 8.5?m/s,and drug loadings of about 5.8%.When the content ratio of PM_Doxox and PM_TOSOS was 1:2,a synergistic effect was determined with a synergy index of 0.29.EcN@PM_D/T/T was used in tumor therapy to promote tumor apoptosis,inhibit tumor growth and proliferation,and effectively prolong the lifespan of tumor-bearing mice without obvious blood,liver and kidney toxicity.The DOX-loaded microtubules were prepared through layer by layer-by-layer assembly of the sodium alginate and chitosan in the pores of polycarbonate membranes,and EcNs were combined with the drug loaded microtubules to form MT_DOX@EcN microrockets with drug loading content of about 8.1%.The inherent motility and tumor targeting ability of the microrockets led to penetrate in the viscous matrix and reach the inside of tumors,subsequently release the drug after response to the slightly acidic tumor microenvironment.Due to the promotion of EcN,MT_DOX@EcN showed higher tumor cell phagocytosis,cytotoxicity and apoptosis compared to MT_Dox.With the self-propelled motion of bacteria,MT_DOX@EcN could target tumors,promote drug penetration and release in tumor areas to achieve antitumor effects.The bacterial envelope BGs was prepared from EcN,and antibacterial drugs ciprofloxacin（CIP）were loaded into BGs to successfully construct BG@Cip,and the drug loading content was 12.5%.Macrophages had a high phagocytic ability to BGs,which was 2.5 times than that of live bacteria.BG@Cip could effectively promote the macrophage internalization of drugs.In vitro release experiments showed that BG@Cip could rapidly release about 60%of drugs within 2 hours in the presence of intracellular acidic environment and lysozyme.As a natural immune activator,BG@Cip could increase the secreations of antibacterial cytokines TNF-?and IL-6 to help eliminating intracellular bacteria.Antitumor drugs 5-fluorocytosine（5FU）and the macrophage-polarized drugs zoledronic acid（ZOL）were loaded into the living bacteria EcNs by electroporation,and gold nanoparticles were modified on the EcN surface to prepare EcN_Z/F@Au with drug loadings content of 17.3%（8.2%for 5FU and 9.1%for ZOL）.EcN_Z/F@Au was irradiated by near infrared light for 30 minutes,and the heat production from gold nanoparticles substantially led to the conversion of bacteria to BGs.Drugs in inner of BGs were released through the pores to treat tumor cells and macrophages,causing high cytotoxicity and apoptosis and promoting the polarization of tumor-associated macrophages to M1 phenotype.The combinative antitumor effect of photothermal therapy,immunotherapy and chemotherapy after EcN_Z/F@Au treatment were demonstrated in vivo.In summary,this paper used the tumor targeting ability of EcNs as the drug carrier to prepare several drug carriers for tumor treatment,and used phagocytosis ability of BGs by macrophages to treat intracellular bacteria.EcNs or BGs could target the lesion and had the ability to control drug release in response to the pathological environment.This paper provides a novel and feasible strategy for the construction of antitumor or antibacterial drug carriers.