Study On The Nano-drug-targeted Delivery System For Anti-biofilm

Salmonella enterica serovar Typhimurium(S.Typhimurium)is a typical food-borne pathogen,that has always seriously threatened the health and life of humans and animals.During acute infections,they rapidly proliferate and largely spread as unicellular organisms,whereas in chronic infections,and they predominantly colonize body surfaces and tissues as multicellular aggregates termed biofilms.Biofilm formation allows the bacterial population to be resistant to multiple environmental and therapeutic damages,thus increasing the difficulty and cost of treatment.Therefore,to prevent and treat biofilm-related diseases,it is imperative to develop antibacterial strategies against biofilm.Because the biofilm are generally resistant to antibiotics,new methods are required for their removal.In recent years,nanomaterials involving the combination of multiple treatment modalities have been recognized as an effective strategy against biofilm,however,their targeted and long time controlled release in bacterial infection remain a major challenge.As a class of chemical antibodies,aptamers have the characteristics of high specificity and affinity in pathogen detection and biomarker screening,and are often used as targeting molecules in tumor therapy to assist drug delivery to tumor cells.The microenvironment of the biofilm is highly similar to that of a tumor,and some similar methods of treating tumors are suitable for the biofilm as well.Based on the above mentioned research background,we have studied the role of targeted nano-drug delivery system against biofilm formation.The main works are as follows:(1)As flagellar motility is a potential target of biofilm treatment,it is necessary to explore the influence of flagellar motility on the development of biofilm,and provide a theoretical basis for combating biofilm strategies.S.Typhimurium was used as a model bacteria,and their flagella mutants(?flg E and?fli C)with impaired flagellar motility were constructed through Red recombination technology,and the development process of their biofilms was well explored in detail.By comparing them with wild-type strains,we found that these mutants lacking flagellar motility form fewer biofilms in the early stage,and the formed mature biofilms contain more cells and extracellular polymeric substances(EPS).In addition,fewer mutant cells adhered to glass plates compared with wild-type cells even after 6 h incubation,suggesting that flagellar motility play a significant role in preliminary cell-surface interactions.Overall,it was also indicated that flagellar motility play an important role in Salmonella biofilm initiation and maturation,which can help us to clarify the mechanisms involved in biofilm formation and to develop more rational control strategies.(2)The aptamer has the advantage of specific recognition,which can help to accurately deliver drug molecules to the site infected by pathogenic bacteria.In order to obtain a perfect aptamer,we purified the exogenous flagellin from the bacteria.Continually,a series of screens were made by SELEX for their aptamers.Finally,an aptamer ST-1 with high specificity and affinity for the bacteria was obtained successfuly.The recombinant plasmid p ET28a-flg E was transformed into E.coli BL21(DE3),and the recombinant protein Flg E was induced by IPTG.After purification and concentration on a nickel column,Flg E protein with a purity more than 90%was obtained.The flagellin was screened by SELEX using His-tagged magnetic beads and an aptamer library containing 10⁸ different sequences,and finally PCR products were obtained for high-throughput sequencing.The top ten sequences with the best binding ability were analyzed for homology and family,and three representative aptamers(ST-1,ST-3,ST-4)were screened for affinity and specificity assays.Among them,ST-1(Kd value of 25.25±2 n M)has the best affinity and specificity,which provides a good recognition probe for subsequent research on aptamer-based biofilm therapy.(3)Biofilms are usually resistant to antibiotics,so it is necessary to develop strategies to replace antibiotics.Here,we established a nano-drug-targeted delivery system consisting of an aptamer(Apt)?indocyanine green(ICG)and carboxyl-functionalized graphene oxide(GO-COOH),namely,ICG@GO-Apt,for targeted treatment of the biofilm formed by S.Typhimurium.Apt-conjugated nanosheets of high special recognizing properties can largely accumulate near abscess caused by the pathogens,thus increasing greatly the local drug molecule concentration and promoting precisely their delivery.They can simultaneously generate heat and reactive oxygen species under near-infrared irradiation for photothermal/photodynamic therapy,thereby significantly enhancing biofilm elimination.Both cell viability and hemolysis experiments show that the nanosheets have good biocompatibility.More importantly,the multi-function phototherapeutic platform shows an efficient biofilm elimination with an efficiency of about 99.99%in a abscess formation model.Overall,the proposed phototherapy platform provides a large possibility for eliminating biofilms.(4)Although two-dimensional nanomaterials have good photo-thermal stability and biocompatibility,they have fluorescence quenching characteristics,and do not use conjugated fluorescent dyes for real-time imaging.In order to develop a nano-drug-targeted delivery system capable of imaging to treat biofilms,we used Zn O as a carrier to load the photosensitizer ICG and Apt targeting S.Typhimurium,and constructed the ICG@Zn O-Apt nanotherapeutic system.To perform real-time trace imaging of ICG@Zn O-Apt,tetramethylrhodamine(TMRA)modified Apt was selected as a fluorescent molecule and incubated with S.Typhimurium biofilm.The confocal fluorescence scanning microscope tracks,in real time,that the nanoparticles have the ability to penetrate biofilm.Modified Aptamers can specifically recognize and target S.Typhimurium,and thus help in greatly increasing the local drug molecule concentration and ensuring that these drugs delivered precisely.In acidic conditions,ICG@Zn O-Apt can produce Zn²⁺for chemotherapy.Under the near-infrared light irradiation,they simultaneously produce ¹O₂ and heat for photodynamic therapy and photothermal therapy.The synergistic effect of three sterilization modes can effectively kill planktonic bacteria before the formation of the biofilm and destroy the established biofilm.Reportedly,ICG@Zn O-Apt nanoparticles have an excellent biocompatibility and low toxicity to host organs and tissues.The ICG@Zn O-Apt nanoparticle was found to be effective and safe in a skin wound abscess model in mice.This research shows that the integrated imaging and treatment platform provides a new technical method for the clinical applications of combating bacterial infections.