Preliminary Studies On Anti-Helicobacter Pylori Bioadhesive Drug Delivery System Based On Surface Molecular Imprinting Technology

Helicobacter pylori (H. pylori) infection is currently one of the greatest threats to people’s health owing to its high infection rate and low eradication rate. One of the challenges in H. pylori treatment is that most of the pharmaceuticals, which lack specific affinity toward their target biomembrane, are incapable of anchoring on the surface of the bacteria and maintaining the local drug concentration, thus failing to eradicate the bacteria. Studies on H. pylori have demonstrated that Lpp20 is a conserved 20-kDa outer membrane lipoprotein antigen specifically produced by all strains of H. pylori. And there exists an exposed antigenic domain (83-115 from N-terminus sequence, abbreviated as NQA) on it, which acts as a potential target for vaccine and therapy development.For the past few decades, molecularly imprinted polymers, especially those imprinted with proteins or peptides, have attracted much attention and been widely recognized as a promising alternative to natural antibodies due to their specificity, robustness and reusability.In the present study, we aimed to fabricate a nano-structured anti-H. pylori bioadhesive drug delivery system by means of surface imprinting technique. NQA, which was expressed as an antigenic domain of surface lipoprotein on H. pylori, was employed as a template molecule to create specific binding sites right on the surface of the nanoparticles.It was hypothesized that when the imprinted nanoparticles get close to H. pylori, the antigenic domain of surface lipoprotein on H. pylori may insert into the imprinted cavities on the nanoparticles. Thus, the anti-H. pylori drug loaded nanoparticles tend to anchor on the surface of H. pylori and remain a prolonged adhesion and maintain effective local drug concentration.In a word, the surface imprinted nanoparticles, which are characterized by an "antigen-anyibody"-like specificity and free from immunogenicity or instability, indicate potential for a novel bioadhesive drug delivery system for bacterium targeting.Our work includes the following two parts.Part I:Study on surface imprinted microspheres of model proteinIn this part, lysozyme was employed as a substitute for in vivo drug targets to prepare molecularly imprinted microspheres. Lysozyme is an easily obtainable amphiphilic protein with suitable mass and good stability. Thanks to its properties, lysozyme imprinted microspheres may possess "surface imprinting" characteristics to some extent. And this part of study intends to lay a good foundation for further developing specific antigen or receptor (or their epitopes) imprinted particulate carrier for drug targeting.An inverse-phase suspension polymerization method was applied to prepare lysozyme (template protein) imprinted microspheres (Lyz-MIP), using acrylamide as functional monomer and N, N’-methylene bisacrylamide as cross-linker. Morphological observation of the template protein imprinted polymeric microspheres was performed with optical microscopy as well as scanning electron microscopy (SEM). The average particle size and zeta potential were also determined. The adsorption kinetics of Lyz-MIP was investigated, and its adsorption capacity, selectivity and even specific recognition of template protein in competitive rebinding test were evaluated using water and physiological saline as experimental media respectively.Results demonstrated that Lyz-MIP had a spherical morphology with an average particle size of about 35μm and zeta potential about-30mV. Adsorption kinetics study indicated fast adsorption equilibrium of Lyz-MIP within 40 minutes. Whether in single or binary protein competitive rebinding test, Lyz-MIP showed a preferential rebinding toward Lyz, which became more significant when measured in physiological saline, probably due to the reduction of non-specific adsorption.PartⅡ:Study on anti-Helicobacter pylori bioadhesive nanoparticles based on surface molecular imprinting technologyIn this part, an inverse microemulsion polymerization method was applied to prepare NQA-surface imprinted nanoparticles, using acrylamide as functional monomer and N, N’-methylene bisacrylamide as cross-linker. To reach the aim of "surface imprinting", the hydrophilic peptide NQA was modified with a long hydrocarbon tail to form an amphiphilic molecule, and used as the template to imprint the surface capture sites for the recognition of the entire bacterium.Besides the conventional batch rebinding test, several approaches, frequently used as assessments of protein-protein interaction or the bioadhesive properties of polymers, such as SPR, FP and "zeta potential discrepancy", were first applied to evaluate the rebinding of template on surface imprinted nanoparticles.All these approaches led to the same conclusion that surface imprinted nanoparticles possess specific recognition capacity toward the template compared with the other two controls. The preliminary in vitro experiment also indicated that the surface imprinted nanoparticles succeeded in targeting H. pylori by means of their specific recognition toward the template.In a word, enlightened by the research on molecularly imprinted microspheres of model protein, we embarked on a series of preliminary studies by introducing the technique of molecular imprinting into anti-H. pylori bioadhesive drug delivery system. More importantly, "suface imprinting" is the key consideration of the study. Beneficial attempts have been explored both in protein/peptide imprinted micro-/nanoparticle preparation and in the evaluation of imprinting efficacy of template molecules.