"Gated" Antimicrobial Polypeptides For The Treatment Of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease（COPD）associated with long-term smoke exposure leads to persistent lung inflammation and impairment of lung function.In the later stage of the disease,the prolonged suppression of the pulmonary immune system predisposes the lungs to bacterial invasion and persistent stimulation of the pulmonary immune system,resulting in a severe immune imbalance.Antibiotics are commonly used drugs in the clinical treatment of COPD,but their long-term use easily causes bacterial resistance and reduces the efficacy of treatment.Antimicrobial peptides（AMP）are a class of short peptides with antibacterial activity,which can disrupt the cell membrane of bacteria,leading to their death due to leakage of the contents in bacteria,thus avoiding the development of bacterial resistance.Therefore,the combination of antibiotics and AMP addresses the problem of bacterial resistance and improves the effectiveness of treatment.Nebulized drug delivery is the best route of administration for the treatment of COPD,with the advantages of rapid onset of action,high local utilization,and low systemic side effects.However,after nebulized administration,the mucus on the airway surface and the biofilm at the infection site greatly impedes the contact of antibiotics and AMP with bacteria,severely reducing the therapeutic efficacy for COPD.Therefore,the development of AMP/antibiotic nano-delivery systems with mucus/biofilm permeability is essential for antimicrobial therapy in COPD.As analogs of antimicrobial peptides,chemically synthesized cationic polypeptides possess excellent biocompatibility,biodegradability,and unique secondary structure,which can serve not only as effective bactericidal materials but also as ideal drug carriers,attracting much attention in the field of antibacterial therapy and drug delivery.Based on this,we firstly synthesized a series of functionalized polypeptides through multi-accelerated ring-opening polymerization（ROP）,and the kinetics of ROP were further studied.Then,a charge-reversible and secondary structure-regulatable "gated"polypeptide-based drug delivery system was constructed through a post-polymerization modification strategy,which was used for the dual penetration of mucus and biofilm,the mild acid-triggered release of antibiotics,and specific activation of antimicrobial polypeptide after nebulized administration,thus achieving synergistic bactericidal and effective treatment of COPD.The research content of this paper is as follows:In Chapter 1,we firstly provided an overview of COPD and discussed the advantages of antimicrobial polypeptides in antibacterial therapy compared with antimicrobial peptides.Subsequently,we described the various polymerization methodologies for synthetic polypeptides and discussed the factors that influenced the antimicrobial properties of polypeptides.Finally,we analyzed the biological barriers in pulmonary delivery of antimicrobials and summarized strategies to overcome these barriers.In chapter 2,a multi-accelerated polymerization system was constructed based on the α-helix-mediated cooperative acceleration and crown ether-mediated catalytic acceleration.In this system,the amino-modified hollow mesoporous silica nanoparticle（HMSN）was used as initiator and 18-crown-6 as catalyst to initiate POBLG-NCA polymerization in dichloromethane（DCM）or DCM/H2O.The results indicated that the polymerization rate initiated by HMSN was greatly enhanced and the monomer conversion was improved in a low dielectric constant medium such as dichloromethane（DCM）compared to that initiated by n-butylamine.Subsequently,the polymerization kinetics of the HMSN initiator in the DCM/H2O biphase system was further explored.Compared with DCM,the polymerization rate in DCM/H2O was further improved,which was mainly attributed to the reduction of chain termination reaction caused by impurities during the polymerization.Moreover,the introduction of an 18-crown-6 catalyst in the system could further increase the polymerization rate by enhancing the interaction between the NCA monomer and initiation site,thus achieving rapid and controlled polymerization in three ways.The results exhibited that the polymerization initiated by HMSN could be completed within 16 min,which was significantly faster than that of n-butylamine initiator（24 min）.Next,the multi-accelerated effect was extended to the random copolymerization,copolypeptide MCEBK was thus synthesized by initiating CH-NCA and Boc-lys-NCA with HMSN.Subsequently,zwitterionic"gated" polypeptide MEK^CA nanoparticles were prepared by quaternization,deprotection,and amidation reactions.At the dormant state（pH 7.4）,MEK^CA exhibited a negatively charged（-28 mV）,random-coiled structure with a uniform particle size distribution of about 120 nm due to the electrostatic attraction between the quaternary ammonium and carboxyl groups in the side chain.Once treated with a mild acid（pH 6.5）,however,the polypeptide converted into a positively charged（34 mV）,α-helical structure with a particle size of about 140 nm due to the departure of the carboxyl group,achieving mild acid-triggered charge reversal and secondary structure transition.In Chapter 3,a "gated" amphiphilic polypeptide-based drug delivery nanoparticles(MEK^CA/CAZ)were prepared by encapsulating ceftazidime（CAZ）in MEK^CA synthesized in Chapter 2,which could enhance the mucus/biofilm dual penetration after nebulized administration and realize selective release of antibiotics and specific activation of antibacterial polypeptide,thus achieving synergistic sterilization,anti-inflammation,and effective COPD treatment.Under physiological conditions（pH 7.4）,MEK^CA presented a negatively charged and random-coiled structure.The HMSN surface was coated by a flexible polypeptide,and CAZ could not be released from the pore.In the mildly acidic environment（pH 6.5）,the polypeptide transformed into a positively charged,rigid α-helical structure,resulting in the opening of HMSN pores and the release of CAZ.At the same time,the antibacterial function of the polypeptide was activated,realizing a synergistic antibacterial efficacy and down-regulation of multiple pro-inflammatory factors.The mucus penetration ability of the nanoparticle was evaluated by the Calu-3 cell monolayer penetration assay.Compared to the positively charged MEK nanoparticles,the negatively charged MEK^CA nanoparticles exhibited a 5-fold increase in mucus penetration efficiency.Besides,the penetration and aggregation of MEK^CA in the biofilm were also significantly enhanced,indicating that the interaction between nanoparticles and biofilm was enhanced after charge reversal.It was worth mentioning that the introduction of positively charged,long,hydrophobic chains conferred excellent antibacterial properties to the polypeptide.The cationic polypeptide nanoparticles MEK possessed high activity against Pseudomonas aeruginosa,Escherichia coli,and Staphylococcus aureus.Especially in combination with CAZ,the inhibition of bacteria and biofilm exceeded 90%.Subsequently,the in vivo therapeutic efficacy of MEK^CA/CAZ nanoparticles was verified via a COPD mouse model.The results indicated that the number of bacteria in the lung was significantly reduced after MEK^CA/CAZ nanoparticles treatment,and the levels of inflammatory factors（e.g.,IL-6,IL-8,IL-1β,and TNF-α）,pH,partial pressure of O2 and CO2 in arterial blood were restored to normal levels.In addition,the number of neutrophils,T cells,and macrophages in lung tissue was significantly reduced after treatment with MEK^CA/CAZ nanoparticles,and macrophages were polarized from a pro-inflammatory phenotype（Ml）to an anti-inflammatory phenotype（M2）,indicating that MEK^CA/CAZ nanoparticles could effectively alleviate lung inflammation and restore lung function.In chapter 4,we summarized the work and provided an outlook on future research directions.In conclusion,this work has developed a "gated" polypeptide-based drug delivery system based on multi-accelerated polymerization.After nebulized administration,the drug-loaded nanoparticles have the ability to dual penetration of mucus and biofilm,thereby achieving synergistic sterilization,anti-inflammation,and effective COPD treatment.Based on the rational design of responsive polypeptide nanoparticles,the controllable release of antibiotic drugs as well as the selective bactericidal effect of the antibacterial polypeptide is achieved,which solves a series of key problems such as low drug accumulation at the lesion site and nonspecific cytotoxicity to normal cells,thus providing a new insight for the design of smart drug delivery carriers.