Construction Of Near-infrared Region Ⅱ Phototherapy System And Its Application In Anti-biofilm Therapy

Drug-resistant bacteria and their biofilms are difficult to eradicate,posing a serious threat to public health.The formation of biofilms leads to a significant increase in bacterial resistance,resulting in a sharp decrease in the efficacy of antibiotics.Currently,phototherapy techniques,including photothermal therapy（PTT）and photodynamic therapy（PDT）,rapidly generate heat radiation or reactive oxygen species（ROS）under laser radiation,effectively killing bacteria and disrupting biofilm in a short time.However,the development of phototherapy techniques in the anti-biofilm has some limitations:the dense extracellular polymeric substances（EPS）of biofilm hinder the penetration of phototherapy agents（PAs）;the internal microenvironment of biofilm exhibit chemical gradient characteristics,such as low p H,hypoxia,high concentrations glutathione（GSH）,which can consume the ROS generated by PAs;bacteria that settle in different cubicles of biofilms can evade the killing of ROS and thermal irradiation generated by the PAs.These obstacles greatly weaken the efficacy of phototherapy,resulting in unsatisfactory anti-biofilm performances.Additionally,phototherapy techniques are limited by tissue depth,as they can only treat superficial skin layers and are ineffective in treating lesions and infections deeper than 1 cm.Therefore,these are important to create novel PAs,expanding their application in the field of biofilm therapy.In this paper,based on the unique physicochemical properties of biofilm,various biodegradable near-infrared region-Ⅱ（NIR-Ⅱ）PAs and amphiphilic polymers were designed and synthesized.These PAs and polymers form nanoparticles by self-assembly.These particles can penetrate EPS,disrupt the microenvironment,and target bacteria within the biofilm,achieving efficient clearance of biofilm and internal persistent bacteria.The specific details are as follows:（1）Three types of NIR-Ⅱ PAs are synthesized.The excitation-emission wavelengths and photothermal-photodynamic properties of these PAs are successfully modulated through electron acceptor planarization,extensionπ-conjugation,and modulation two-electron acceptor.Validation results indicate that planarized organic structures triggerπ-πstacking,thereby exhibiting excellent photothermal performance.Adding alkane chains to the organic structure can effectively reduce intermolecularπ-πstacking,allowing the photothermal performance of PA to transition to photodynamic performance.Therefore,polymer PT exhibits excellent photothermal,polymer DBP demonstrates photothermal-photodynamic,and polymer PNIR-Ⅱ shows relatively good photodynamic performance without photothermal effects.The emission wavelengths of PT,DBP,and PNIR-Ⅱ are 1147,1121,and 1137 nm,respectively.Additionally,nanoparticles based on three PAs are constructed,which can be applied to biofilm treatment in different scenarios.The research provides an effective theoretical basis and design method for the construction of phototherapeutic polymers.（2）The dual-responsive nanoparticles（NPs₁）for biofilm therapy are developed.NPs₁are obtained through self-assembly of GSH-responsive degradable photothermal polymer PT and thermosensitive polymer P0.Under 1064 nm laser irradiation,PT exhibits excellent photothermal performance,reaching a maximum temperature of 87.6°C.NPs₁encountered high concentrations of GSH,leading to the rupture of PT’s disulfide structure upon reaction with GSH after entering the biofilm.Subsequently,the heat generated by PT rapidly killed bacteria while disrupting the NPs₁.This research provides a new validation for the development of NIR-Ⅱ light-activated efficient photothermal polymers for biofilm clearance.（3）The design of unique core-shell-shaped nanoparticles（DA₃-NPs）that eradicate biofilm by precise in-situ phototherapy,is reported.DA₃-NPs are fabricated by encapsulating a GSH degradable polymer DBP,which provides ROS and thermal irradiation,into an amphiphilic thermosensitive azo polymer P3 that functions bacterial targeting via perfectly matched charge interaction.The obtained electroneutral DA₃-NPs effectively penetrate EPS and precisely target bacteria within the biofilm,as evidenced by the in-situ/ex-situ co-localization using confocal and flow cytometry.Thereafter,in-situ phototherapy can be implemented by generating ROS and heat on the site of the internal bacteria.Moreover,the azo and disulfide bonds in DA₃-NPs could be broken by laser irradiation and GSH,triggering the responses of shell detachment and core disintegration in a sequential manner.GSH consumption and carbon radical production of the DA₃-NPs in this process further augment the anti-biofilm performance.Compared with the non-targeted electronegative nanoparticles,DA₃-NPs perform 61 times superior efficiency of biofilm removal.Therefore,this study provides a precise in-situ phototherapy strategy for nonantibiotic treatment of biofilm infections.（4）A treatment strategy for biofilm infections in deep tissue and constructed nitric oxide（NO）-enhanced PDT nanoparticles（NPs₃）,is designed.Based on the NIR-Ⅱ light penetration properties,NPs₃maintain high ROS production rates even beyond 1 cm tissue barriers.NPs₃are prepared by self-assembly of GSH-triggered NO-donor polymer and photosensitive polymer PNIR-Ⅱ.After entering the biofilm and encountering the GSH in the microenvironment,NPs₃precisely released NO.Under 1064 nm laser irradiation,NPs₃rapidly produced ROS.The reaction between ROS and NO generated more potent bactericidal reactive nitrogen species（RNS）in a short time,thereby orderly disrupting the biofilm.This study provides new materials for clearing biofilm infections in deep tissue.In summary,this thesis addresses specific problems encountered by phototherapy techniques in the application of biofilm therapy:1)short excitation laser of PAs;2)EPS hindering the penetration of PAs;3)the ROS is consumed by GSH in the biofilm microenvironment;4)the biofilm in deep tissues is difficult to remove.Three phototherapeutic polymers and various amphiphilic degradable polymers are synthesized and assembled into three nanoparticles NPs₁,DA₃-NPs,and NPs₃.These were successfully applied to the treatment of drug-resistant bacterial biofilms,clearance of persistent bacteria within biofilm,and clearance of deep tissue biofilm infections.Subsequently,the killing and clearance effects of nanoparticles on biofilms and their internal persistent bacteria are systematically studied.These research contents provide theoretical basis and feasibility methods for the treatment of biofilm infections and related clinical applications,providing strong reference for future research and clinical practice.