Metal(Pd,Ir) Based Nanocatalytic Platform For The Prevention And Therapy Of Helicobacter Pylori Infection

Helicobacter pylori（H.pylori）is a common infectious Gram-negative bacterium that colonizes the gastric mucosa.Due to the strong acid resistance,adhesion,motility and pathogenicity,H.pylori can remain latent in the stomach for a long time and cause infection of at least half of the world’s population.It is reported that H.pylori infection has become an important cause of stomach diseases such as gastric mucosal inflammation,peptic ulcer and gastric cancer.At present,triple therapy（proton pump inhibitor/bismuth+two antibiotics）or quadruple therapy（proton pump inhibitor+bismuth+two antibiotics）is mainly used in the clinical treatment of relevant diseases caused by H.pylori infection.Although the above-mentioned therapies can eliminate more than 90%of H.pylori,there are still some disadvantages:1.Drugs cannot effectively penetrate the gastric mucus layer（50-300μm）.Meanwhile,the regular gastric emptying and constant changes of the mucus layer prevent the drug from staying in the stomach for a long time,resulting in a low accumulation concentration of the drug that cannot kill H.pylori effectively,thereby leading to a low cure rate and a high recurrence rate.2.The problem of drug resistance caused by strain variation and biofilm cannot be effectively solved.3.Excessive inflammatory response and oxidative stress-mediated gastric mucosa damage caused by H.pylori infection cannot be effectively alleviated.4.Antibiotics lack targeting,which can easily cause side effects such as intestinal flora disorder.5.Antibiotic therapy cannot achieve effective prevention of H.pylori.Therefore,it is of great significance to develop non-antibiotic therapies that integrate multiple functional modes into a single therapeutic platform for H.pylori infection.Nanocatalytic therapy,as an emerging method of using nanocatalysts to initiate catalytic reactions in vivo to treat various diseases,has attracted extensive attention.Among them,metal nanocatalysts are widely applied to anti-tumor,antibacterial,anti-inflammatory,antioxidant and other medical fields due to their remarkable biosafety,stability and biocatalytic activity of imitating natural enzymes.However,on account of the complex physiological and pathological microenvironment of H.pylori infection sites,pure metal nanocatalysts are difficult to perform efficient biocatalytic functions,which are specific in three aspects:Ⅰ.Lack of gastric retention and inflammatory targeting.Ⅱ.Weak mucus penetration.Ⅲ.Easy to be cleared by immune system.As a result,it is vital to construct multifunctional composite materials based on metal nanocatalysts for the precise,effective and safe treatment and of H.pylori infection.In this thesis,from the perspective of the complex microenvironment of H.pylori infection site,we used metal nanocatalysts as substrates,and combined them with metal-organic frameworks,hydrogels,therapeutic gases and probiotics,aiming to construct a series of versatile platforms with inflammatory microenvironment-responsiveness for H.pylori infection.Then,multiple properties of the platforms and the effects on animals were systematically studied.This study broadens the application of nanocatalytic therapy in the prevention and treatment and prevention of H.pylori infection.The main contents of this study are summarized as follows:Chapter 1:Firstly,we summarized the physiological characteristics,pathogenic mechanism and detection methods of H.pylori,and focused on the current research and treatment progress of H.pylori infection.Secondly,we introduced the research status of nanocatalytic therapy and its application in the field of biomedicine.Thirdly,the basis for selecting the topic and the main study content of this thesis were emphatically clarified.Chapter 2:We first synthesized a palladium（Pd）nanocatalyst and encapsulated it in zeolite imidazole metal-organic framework（ZIF-8）in situ to form Pd@ZIF-8nanoparticle.Then,the therapeutic gas（hydrogen）was introduced into the lattice gap of Pd nanocatalyst through the pore structure of the ZIF-8 to form Pd（H）@ZIF-8nanoparticle.Finally,the Pd（H）@ZIF-8@AP platform was synthesized by self-assembly of Pd（H）@ZIF-8 with negatively charged ascorbyl palmitate（AP）hydrogels.Both in vitro and in vivo experiments demonstrated that Pd（H）@ZIF-8@AP could target and adhere to the inflammatory site through electrostatic interactions.Then,Pd（H）@ZIF-8@AP could be hydrolyzed by matrix metalloproteinase（MMP）enriching in inflammatory sites,realizing the targeted delivery and in situ release of Pd（H）@ZIF-8 nanoparticle.The released Pd（H）@ZIF-8 nanoparticle was further decomposed by gastric acid to generate zinc ions(Zn²⁺),and the Pd nanocatalyst catalyzed hydrogen to generate highly reducing active hydrogen atoms,thereby effectively killing H.pylori and avoiding the drug resistance.Meanwhile,the active hydrogen could regulate the expression of inflammatory factors to inhibit excessive inflammatory responses,remove excessive oxygen free radicals to reduce the apoptosis of gastric mucosal epithelial cells caused by oxidative stress,and up-regulate the expression of gastric mucosal repair proteins to repair impaired gastric mucosa.Significantly,this versatile nanocatalytic platform avoided the imbalance of intestinal flora,providing a promising strategy for non-antibiotic treatment of H.pylori infection.Chapter 3:The previous work in Chapter 2 mainly aimed at clearing free H.pylori in the superficial layer of gastric mucus,which could not effectively break through the gastric mucus barrier to kill H.pylori efficiently,and it was difficult to prevent H.pylori colonization and regulate intestinal flora.Therefore,we synthesized a cysteine-modified iridium nanozyme（Ir C NPs）with multiple enzymatic activities and the adhesive polymer amino-polyethylene glycol-maleimide（NH₂-PEG-NHS）,which were then grafted in the surface of Lactobacillus rhamnosus GG（LGG）via disulfide bond and amide bond respectively,to prepare the multifunctional engineered probiotic（LGG@Ir C/NH₂-PEG-NHS,LIP）.LIP had three advantages:1.Mucus layer penetration and mucosal adhesion:LIP could successfully penetrate the mucus layer smoothly and adhere to the surface of the gastric mucosa through non-covalent（hydrophobic and self-coagulation）and covalent（sulfide bond and hydrogen bond）forces.2.Synergistic antibacterial effects:LIP could hinder the colonization of H.pylori by competing for adhesion and up-regulating the expression of mucin and heat shock protein 70（HSP 70）.In addition,H.pylori infection results in the damage of mucus layer and low pH of the gastric mucosal surface.LIP exhibited impressive peroxidase and oxidase activity in response to the low pH,and then produced oxygen free radicals and antiseptics,thereby effectively killing H.pylori and removing bacterial biofilm without drug resistance.3.Anti-inflammatory and antioxidant stress effects:In a wide pH range,LIP showed catalase（CAT）activity,which could reduce the inflammatory response mediated by the nuclear factor-kappa-B（NF-κB）p65signaling pathway and inhibit the recruitment of neutrophils and macrophages,thereby down-regulating the expression of nicotinamide adenine dinucleotide phosphate oxidase 2（NOX2）and the production of reactive oxygen species（ROS）.Hence,the apoptosis of normal gastric epithelial cells could be prevented,and the gastric mucosal damage caused by oxidative stress was reduced.4.Intestinal flora regulation:LIP could maintain intestinal homeostasis and reduce the occurrence of intestinal diseases by increasing the richness and diversity of intestinal flora.