Construction Of Functionalized Liposomes And Application Research In Inhibiting Streptococcus Mutans Biofilm & Clinical Case Report

Streptococcus mutans biofilm is one of the important factors leading to dental caries,and plaque control is an effective treatment.Antimicrobial agents are the traditional treatment for plaque control.However,there are some problems such as increased microleakage,discoloration of teeth,increased bacterial resistance,and influence on oral flora,which force researchers to constantly search for new antimicrobial agents.Previous studies from our research group have shown that many plant extracts have the ability to inhibit plaque growth.However,some potential antimicrobial agents,including plant extracts,are often not retained in the oral cavity for a long time due to saliva washout or have low concentrations due to poor water solubility.Thus,researchers need to develop new drug delivery methods according to the characteristics of the oral microenvironment.Curcumin is a plant extract with photosensitive properties,which can produce a large number of reactive oxygen species under light and play an antibacterial role in photodynamic therapy.At present,curcumin has been widely studied in the medical field because of its antibacterial,anti-inflammatory,antiviral and anti-cancer effects.However,its lipid solubility and easy decomposition,as well as the particularity of the oral environment,limit its application in the field of dentistry.Therefore,there is an urgent need to develop special drug delivery systems based on the oral microenvironment to improve the drug utilization efficiency of potential antibiotics including curcumin in the oral cavity.In recent years,liposomes,as an amphiphilic drug carrier,have been widely used in drug delivery due to their advantages of high biocompatibility,improved drug stability,and reduced toxic and side effects.With the development of materials science,more and more liposomes with special functions for different disease microenvironments have been gradually designed and developed to improve the targeting and drug delivery ability of liposomes in different disease microenvironments.In this thesis,different functionalized liposomes were constructed based on the characteristics of dental caries microenvironment.In view of the side effects of commonly used oral antibacterial agents in clinic,we continued the previous study of our research team on plant extracts.In this study,curcumin was loaded into a functionalized liposome system to investigate its antimicrobial delivery ability and its effect on inhibiting Streptococcus mutans biofilm and preventing dental caries.In the review,we introduce the composition of liposomes and their advantages as drug delivery systems,and summarize the current application of liposome drug delivery systems in various oral diseases.In the first part,according to the characteristics of the Streptococcus mutans biofilm in the dental microenvironment of 37 ℃,low pH and rich in-NH₂ groups,two kinds of functionalized liposomes were constructed: temperature-pH-sensitive liposomes and N-hydroxy succinimide（NHS）functionalized liposomes.Among them,the temperature-pH-sensitive liposomes have the characteristics of releasing drugs at appropriate temperature and pH conditions,while NHS-functionalized liposomes have the characteristics of specific adhesion to-NH₂-rich surfaces.In the second part,to address the problem that some potential antibacterial drugs cannot be retained in the oral cavity for a long time due to their properties,curcumin,a phytoextract with photodynamic properties,was loaded into NHS-functionalized liposomes.As the surface of Streptococcus mutans biofilm is rich in-NH₂ and other functional groups,NHS-functionalized liposomes can effectively bind to-NH₂ to form chemical bonds,so that the loaded drug can stay on the surface of the biofilm for a longer time and the NHS-functionalized liposomes can play a role in sustained drug release.At the same time,curcumin itself is photosensitive and can produce reactive oxygen species under blue light,which plays a role in inhibiting bacteria through photodynamic effect.This drug delivery system significantly inhibited the biofilm growth of Streptococcus mutans.This part of experiments continued our group’s longterm research on plant extracts,explored the potential application of NHSfunctionalized liposomes loaded with curcumin in plaque control,and also provided experience for the future use of temperature-pH-sensitive liposomes to deliver drugs.Part Ⅰ Construction and Characterization of Different Functionalized LiposomesObjective: According to the temperature and pH range of Streptococcus mutans biofilm in the dental caries microenvironment,the temperature-pH sensitive liposomes were constructed.According to the-NH₂ group on the surface of Streptococcus mutans biofilm,the N-hydroxy succinimide（NHS）functionalized liposomes were constructed by utilizing the characteristic that the ester group formed after modification of DSPE-PEG terminal by NHS can amidate with-NH₂.Methods: Synthesis of temperature-pH sensitive liposomes: Temperature-pHsensitive liposomes were obtained by thin-film hydration method after dissolving soybean lecithin and P（NIPAM-co-MAA-co-ODA）in a mixture of chloroform and methanol（volume ratio 2:1）.The morphology was detected by TEM,the particle size and potential were detected by DLS,and the phase transition temperature and phase transition pH were detected by DSC.Synthesis of NHS-functionalized liposomes: NHSfunctionalized liposomes were obtained by thin-film hydration method after dissolving L-α-phosphatidylcholine,cholesterol and DSPE-PEGNHS in dichloromethane.The morphology was detected by TEM,and the particle size,potential and stability were detected by DLS.Results: The morphology of the temperature-pH sensitive liposomes and NHSfunctionalized liposomes appeared as spherical vesicles with good dispersion.The particle size of the temperature-pH-sensitive liposomes was 95 ± 14 nm,and the ζ-potential was-13.2 ± 3.15 mV.DSC results showed that the phase transition temperature and pH of the liposomes were 38 ℃ and 6.5,respectively.The particle size of NHS-functionalized liposomes was 178.51 ± 54.28 nm,and the ζ-potential was-19.33 ± 0.34 mV.The PDI of NHS-functionalized liposomes was stable within a week.Conclusion: The temperature-pH-sensitive liposomes and NHS-functionalized liposomes were successfully constructed.The characterization results showed that these two liposomes were spherical vesicles with good dispersion,strong negative charge and good stability,and both of them had the potential to deliver drugs in the oral environment.Part Ⅱ The effect of adhesion and inhibition of curcumin-loaded NHS-functionalized liposomes（Cur@LP）on Streptococcus mutans biofilmObjective: This part of the experiment continued our previous research on plant extracts.Curcumin,a photosensitive plant extract,was selected.Aiming at the problems such as the short half-life and the limited movement radius of ROS produced by curcumin after photoactivation,a curcumin loaded NHS functionalized liposome system（Cur@LP）was constructed.The ability of the system to adhere to Streptococcus mutans biofilm,deliver and sustained-release curcumin,and the inhibitory effect of the system on Streptococcus mutans biofilm were investigated.methods: Curcumin loaded NHS-functionalized liposomes（Cur@LP）were synthesized by thin-film hydration method.TEM was used to observe its morphology,and DLS was used to determine its particle size distribution and zeta potential.The cytotoxicity of Cur@LP was assessed by CCK-8 assay and LDH assay.Confocal laser scanning microscopy（CLSM）was used to observe the adhesion ability of Cur@LP to Streptococcus mutans biofilm.Crystal violet staining and CLSM staining were used to evaluate the anti-Streptococcus mutans biofilm effect of Cur@LP.SEM was used to observe the removal effect of Cur@LP on the Streptococcus mutans biofilm on the tooth in vitro surface.Results: The particle sizes of LP and Cur@LP were 206.67 ± 8.38 nm and 312 ± 18.78 nm,respectively,which proved that curcumin was successfully loaded.The ζ-potential of Cur@LP was-20.83 ± 0.59 mV.The encapsulation efficiency of Cur@LP was（42.61 ± 2.19）%,and the release rate of curcumin in Cur@LP was rapidly released to 21% within 2 hours.The Cur@LP system exhibited negligible cytotoxicity and was effective in adhering to Streptococcus mutans biofilms,facilitating the retention of curcumin within the biofilms.When compared with curcumin alone,Cur@LP showed a stronger anti-biofilm effect.Conclusion: In this study,we successfully constructed Cur@LP,which demonstrated the reduced cytotoxicity of curcumin loaded into NHS-functionalized liposomes.Compared with curcumin alone,Cur@LP adhered to Streptococcus mutans biofilms effectively,delivered curcumin and inhibited biofilm growth.This system has the potential application to the field of plaque control in oral environment,and also provides experience for the future use of temperature-pH-sensitive liposome-loaded drugs.