Construction Of Dual-Drive Silk Protein Microneedles For H. Pylori-Infected Gastric Bleeding And Its Antibacterial Hemostasis Mechanism

H.pylori is highly pathogenic and contagious,which is considered as one of the most infectious bacteria in the world.It is the main pathogen causing inflammation of the stomach,gastric ulcer,gastric bleeding,gastric cancer and other diseases in patients,which not only threatens the life safety and health of human beings,but also brings a huge economic burden to the global health care system.H.pylori has become a public health problem of common concern to researchers,health care professionals and patients worldwide.Therefore,how to treat ulcerative bleeding caused by H.pylori and eradicate H.pylori is an urgent problem that needs to be solved to save lives.Although various drugs and carriers（including:antibiotics,liposomes,micro/nanoparticles,nanomotors）have been widely developed for the treatment of H.pylori,there are still many shortcomings,such as:1)difficulties in resisting the acidic environment of the stomach and therefore easy to inactivate medicines;2)short retention time in the stomach leading to low utilization of medicines;and 3)neglecting the bleeding of recurrent gastric ulcers caused by H.pylori infection.Therefore,the development of a substance with multifunction to stop bleeding and infection is of great significance for the effective treatment of H.pylori infection and its complications.This study coupled hemostatic and antibacterial substrates to prepare dual-driven filament protein microneedles（MNs-D）with self-propelled and magnetically guided properties.For the hemostatic substrate,hemostatic microspheres（SEC-Fe@Ca T）with self-propulsion and magnetic guidance properties were prepared by in situ growth method and template method.While,hollow magnetic fibroin microneedles（MNs）,as the antibacterial substrate,were prepared by mold casting method.Subsequently,SEC-Fe@Ca T were filled into the hollow structure of the MNs to construct the MNs-D.This thesis systematically investigated its preparation conditions,physicochemical properties,hemostatic and antimicrobial capacities and biocompatibility.Also,the hemostatic and antimicrobial mechinsm of MNs-D was investigated by a rat H.pylori infection model.On the basis of the content,this study is divided into four parts as follows:1)Preparation of MNs-D:Preparation of SEC-Fe@CaT（hemastatic substrate）:SEC-Fe was initially prepared by in situ growth of Fe₃O₄ on the surface of SEC substrate.Subsequently,Janus microspheres（SEC-Fe@Ca）were prepared by template method with flower-type of Ca CO₃ clusters growing on the unilateral side of SEC-Fe.SEC-Fe@Ca was able to react with H⁺in an acidic environment（gastric juice）to produce CO₂,which provided gas propulsion for the movement of SEC-Fe@Ca.To obtain SEC-Fe@Ca T,thrombin is finally vacuumloaded inside SEC-Fe@Ca..In this process,the preparation conditions for SEC,SEC-Fe,and SEC-Fe@Ca were systermatically explored to optimize the hemostatic capacity.Preparation of MNs（antimicrobial substrate）:fibroin-based microneedles with magnetic tips were prepared by using the mold casting method.By examining the impact of fibroin concentration on the durability and microphysical morphology of the antimicrobial microneedles,the ideal preparation conditions were discovered.When the concentration of fibroin protein is 10%,MNs have the most complete structure and fracture strength strong enough to puncture the skin layer,optimizing the preparation conditions for MNs.Coupling of SEC-Fe@CaT and MNs:the SEC-Fe@CaT is coupled into the hollow structure of the MNs,followed by encapsulation with soluble fibroin.Finally,the MNs-D was obtained.2)Physicochemical properties of the MNs-DGeneral physicochemical properties:the chemical structures of the MNs and SEC-Fe@Ca T in the MNs-D were characterized by Fourier infrared spectroscopy（FT-IR）,X-ray diffraction mapping（XRD）,and high-resolution X-ray photoelectron surface spectroscopy（XPS）,respectively,and the drug-carrying capacity of the SEC-Fe@Ca T was investigated.In addition,through in vitro drug release model,it was demonstrated that MNs-D could achieve a sustained 24 h drug releasing.The drug release efficiency of MNs in the simulated gastric fluid（p H=1）could reach 79.24%,and the release rate of SEC-Fe@Ca T could reach 92.15%.In addition,animal models were used to verify the gastric acid neutralization performance and retention performance of MNs-D.It was concluded that MNs-D was able to neutralize gastric acid rapidly and maintain this neutral environment for 1 h.Because of this,it could provid a gentler environment for the thrombin to maintain activity and halt bleeding.Also,the MNs-D signal could still be detected in the stomach after 24 hours,indicating a longer gastric retention performance.This facilitate MNs as a substrate to release drug slowly in the treatments of H.pylori in stomach.Dual-drive performance:MNs-D is able to move continuously for 12 s while using gas propulsion,covering a maximum distance of 3 cm.The SEC-Fe@Ca T is rapidly ejected from the tail of the MNs-D during this process,and quickly dispersed in the gastric juice.This provided a pathway to cure diffuse bleeding caused by H.pylori.MNs-D can move with a targeted motion of 2 cm/s under magnetic guidance.MNs-D has more good motion performance with dual-drive capacities,offering a reliable drug delivery platform for achieving quick hemostasis and antimicrobial.3)Hemostatic and antibacterial properties of MNs-DThe hemostatic performance of SEC-Fe@CaT was evaluated by an in vitro model and four in vivo animal models.The hemorrhage models showed that SEC-Fe@Ca T wase able to achieve hemostasis in 30 s,30 s,45 s and 85 s for the rat broken tail hemorrhage model,the rat femoral artery hemorrhage model,the rabbit liver hemorrhage model and the rabbit femoral artery hemorrhage model,respectively.The ability to achieve rapid hemostasis can be attributed to 1)the gas propulsion property of SEC-Fe@Ca T,which helps to improve the dispersion of the hemostatic agent and the contact with the occult bleeding sites;2)SEC-Fe@Ca T can puncture the red blood cells during the movements to promote the release of coagulation substances,which promotes platelet activation and accelerates the release of ADP and TXB₂ to achieve rapid hemostasis.The in vitro antimicrobial performance of MNs was evaluated by the inhibition circle test,bacterial survival rate test,and bacterial live/dead staining test.In addition,the in vivo antimicrobial performance of MNs-D was explored by constructing a rat in vivo H.pylori infection model.The results showed that MNs-D was able to eliminate H.pylori infection in rats,and H.pylori decreased to 8.18×10⁵ CFU/g in the stomach after the application of MNs-D.4)Biological and tissue compatibility of MNs-DThe hemocompatibility and cytocompatibility of the MNs-D and SEC-Fe@Ca T were tested by hemolysis rate test,cell viability test,respectively.What is more,subcutaneous implantation was used to investigate the degradation capability of SEC-Fe@Ca T in vivo.Furthermore,rat body weight change,major organ pathology,gastrointestinal histopathology,and blood biochemical analyses were used to show that MNs-D had good biochemical and histocompatible qualities.In summary,MNs-D with both hemostatic and antibacterial properties was prepared by coupling SEC-Fe@Ca T with MNs.It was systermatically investigated in terms of the preparation conditions,physicochemical properties,hemostatic properties and itsmechanisms,antibacterial properties and its mechanisms,and biocompatible properties.The MNs-D can 1)neutralize gastric acid in a short period of time and providing a mild environment for the hemostatic agent to work;2)release the hemostatic substrate rapidly and make extensive contact with the gastric wall to promote hemostasis under the dual-drive;3)targete the gastric mucus layer under the dual-drive,thus increasing the retention time of MNs-D in the stomach;4)increase the local antibiotic concentration,thus achieving efficient antibacterial.Therefore,the MNs-D system as a novel drug delivery platform and therapeutic strategy is expected to facilitate the treatment of H.pylori infection and bleeding complications in the stomach.