The Preparation Of PH/Temperature Dual-Responsive Injectable Hydrogel And Its Effect On Bacterial Biofilm

Bacterial biofilms are one of the major etiologies of chronic microbial infections,and persistent infections increase the chances of hospitalization and risk of death for patients.Increased biofilm virulence and resistance to external factors due to quorum sensing(QS)among bacteria within the biofilm,as well as their metabolic conditions that promote the development of drug-resistant bacteria,make biofilm infection difficult to be eradicated by conventional antibiotic therapy,leading to persistent chronic infectious diseases.Almost all biofilms exhibit severe drug resistance compared to planktonic bacteria,but conventional antibiotic therapy for biofilm infections has certain disadvantages: it fails to kill deep-seated bacteria and leads to a constant switch between resting and attack phases,eventually leading to chronic infections.Quorum sensing inhibitors(QSIs)can inhibit intra-biofilm community sensing to further inhibit biofilm formation and promote dispersion of mature biofilms.At the same time,quorum induction inhibitors can greatly reduce the virulence of bacteria in biofilm and kill bacteria without causing drug resistance.However,the direct application of quorum sensing inhibitors may reduce their biofilm disruption effect and even cause some side effects.Objective:In order to treat biofilm-induced chronic inflammation more efficiently,a drug delivery system based on quorum sensing inhibitors needs to be constructed using the structural characteristics of biofilms.In this study,we designed a populationsensing-based dual-response(p H/temperature)injectable hydrogel drug delivery system,tested its physicochemical properties,and verified its antibacterial properties through bacterial assays,and then verified the therapeutic effects of the drug delivery system on chronic inflammatory diseases caused by biofilm infections through animal experiments.Methods:(1)Preparation and characterization of materials: the co-blended phase change particles of cinnamaldehyde with lauric acid and stearic acid were prepared and mixed with the prepared chitosan particles,and the two were cross-linked to form hydrogels.The volume ratios of the two hydrogel precursors were controlled to prepare hydrogels with different ratios(precursor volume ratios of 4:6,5:5,and 6:4;named C4CP6,C5CP5,and C6CP4,respectively).The injectability and p H response characteristics and temperature response characteristics of the hydrogels were examined.The precursor particles and hydrogels with different ratios were characterized using zeta potential and particle size analysis,scanning electron microscopy(SEM),Fourier transform infrared spectroscopy(FT-IR),and X-ray diffractometer(XRD).The rheological properties,stress-strain,hydrophilic and hydrophobic properties,swelling properties and equilibrium water content,porosity,water retention properties,etc.of the hydrogels were tested to study the physical properties and slowly release properties of the hydrogels.(2)Evaluation of antibacterial properties and biocompatibility: The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of the precursor substances and hydrogels were measured by micro-broth dilution method and plate counting method.The short-term and long-term antibacterial effects of the hydrogels were evaluated using the inhibition ring diffusion method and long-term coculture method.Biofilm disruption assay was used to verify the biofilm disruption effect of the hydrogel.The contents of reactive oxygen species(ROS),NADPH oxidase(NOX)and malondialdehyde(MDA)in bacterial cells after the action of hydrogel were examined to investigate the antibacterial mechanism.(3)Biocompatibility evaluation: hemolysis test,the CCK-8 method and scratch test were used to detect the effect of hydrogel on fibroblast growth and migration.(4)Evaluation of the efficacy of chronic inflammation: A mouse model of chronic otitis media was established,and a blank control group,a negative control group and a hydrogel treatment group were set up,while the inflammation in the external ear canal of mice was observed on the 4th,7th and 14 th days of treatment and the effect of hydrogel on bacteria and biofilm was analyzed by bacterial count and scanning electron microscopy.Results:(1)The particle size and potential positive and negative of the two precursor particles were measured by zeta potential and particle size analysis.Both precursor particles were about 0.5 μm in size with opposite electrical properties,and the instantaneous increase in particle size of the two particles after mixing confirmed the electrostatic adsorption of the precursor particles.The relevant characterization and physicochemical property results confirmed the successful preparation of hydrogels and the formation of hydrogels by the reaction of Schiff base with the precursor particles.The rheological properties indicate the structural support properties and temperature responsiveness of the hydrogels,and the stress-strain indicates that the C5CP5 hydrogels are tightly cross-linked internally and the hydrogels are hydrophilic.The swelling rate of hydrogel can reach 800%,the porosity can reach 77%,and the water retention can reach more than 70% in 7 days.(2)The results of minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)confirmed that the hydrogel precursor particles CAPCM has broad-spectrum antibacterial effect and good bactericidal effect,and confirmed the enhanced antibacterial performance of CA-PCM particles at melting temperature.The inhibition ring diffusion method further confirmed the broadspectrum antibacterial property of hydrogel and the enhanced antibacterial performance at melting temperature,and the long-term co-culture confirmed the long-term antibacterial effect of hydrogel.Biofilm disruption experiments confirmed the biofilm disruption effect of hydrogel and the inhibition of biofilm formation,and some results such as ROS,NOX and MDA confirmed the short-and long-term antibacterial effects of hydrogel by affecting the intracellular metabolism of bacteria.(3)The results of cytotoxicity experiments showed the biosafety of hydrogel(cell survival rate >95%),and the scratch test showed that the hydrogel could promote mouse fibroblast migration to some extent.(4)The results of animal tests showed that CCP hydrogel can kill the planktonic bacteria in the middle ear and destroy the bacterial biofilm in the middle ear after treating chronic otitis media in mice for 14 days without recurrence.Conclusion:This experiment successfully prepared a responsive hydrogel drug delivery system with rapid p H and temperature response characteristics,excellent shortterm and long-term antibacterial effects,biofilm disruption effect and better biocompatibility,which has therapeutic effect on chronic inflammation caused by biofilm and has some potential application as a drug delivery system in the treatment of chronic infections in clinical biofilms.