Antimicrobial Activities Of Multiple Stimuli-Responsive Nanoparticles

Despite the great success in antibiotic treatment of infectious diseases,bacterial infection remains one of the most important global health issues.Conventional antibiotic therapy suffers from poor selectivity and bioavailability,increasing drug resistance,low biofilm permeability,ineffective intracellular bacterial clearance,systemic toxicity and so on.Recently,biodegradable polymeric nanoparticles have shown great potential in overcoming these problems.They can protect the structural integrity and antibacterial activity of antibiotics during circulation,regulate the pharmacokinetics and biodistribution of antibiotics,target specific tissues,cells and even bacteria and release rapidly in response to specific stimuli.Consequently,the selective accumulation and controlled release increase the concentration of the bioactive drug at the infection site,lower the dosage and frequency of administration,thus improving the therapeutic effect and reducing the drug side effects.In this paper,we designed drug-loaded nanoparticles with multiple stimuli-responsiveness to address the key problems in bacterial targeting,bacterial capsular,intracellular bacteria,drug-resistant bacteria and non-antibiotic treatment.The polymer-based carriers have been developed,and their stimuli-response mechanisms and therapeutic effects have also been explored.To enhance the bacteria-targeting efficiency,a drug-loaded micelle with vancomycin as a targeting group was constructed.The lipase-sensitive amphiphilic copolymer poly?ethylene glycol?-polycaprolactone(PEG-PCL,PECL_2k)was synthesized by ring-opening polymerization,and vancomycin?VAN?was grafted to hydrophilic end by hydrazine bond.Subsequently,ciprofloxacin?CIP?was encapsulated to obtain Van-hyd-PECL/Cip micelles with an average size of 76.9 nm and a CIP loading amount of 4.5%.The micelles were enriched in the bacterial infection site by passive targeting?the enhanced permeation and retention?EPR?effect?and active targeting,and could release CIP promptly by biochemical stimuli?H⁺and lipase?at the site of infection.Compared to free drugs,Van-hyd-PECL2k/Cip micelles improved the antibacterial effect with reduced antibiotic dosage and prolonged the lifespan of the mice.To improve biofilm penetration and antimicrobial sensitization,pH/enzyme-sensitive drug-loaded micelles were constructed.The acid-sensitive amphiphilic copolymer PEG-PAsp?DIP?was synthesized by N,N-diisopropylethylenediamine?DIP?-catalysed aminolysis of poly?ethylene glycol?-polyaspartic acid benzyl ester?PEG-PBLA?.Carboxylated azithromycin?Az-sa?via uccinic anhydride esterification of azithromycin was grafted onto the side chain of octadecyl-polyaspartic acid-diethylenetriamine?OA-PAsp?DET??to obtain the lipase-responsive polymeric prodrug OA-PAsp?DET/DET-sa-Az?.The mixture of PEG-PAsp?DIP?and OA-PAsp?DET/DET-sa-Az?could self-assemble into hybrid micelles?DOEAz?at a suitable mass ratio.The biofilm-dispersion agent cis-aconitic anhydride-modified D-tyrosine?CA-Tyr?was then loaded into the hybrid micelles via electrostatic adsorption.The size of heterozygous micelle DOEAz@Tyr was about 107 nm and the loading amounts of Az and CA-Tyr were about 19.1%and 1%,respectively.The micellar size was reduced under acidic pH in the biofilm,owing to the change in hydrophilicity of PEG-PAsp?DIP?induced by the protonation of DIP.At the same time,the biofilm-dispersion factor D-Tyr was released after the cleavage of aconitate amide bond,resulting in a negative-to-positive charge reversal.The micelle was disintegrated in response to lipase and Az was released.Compared to free drugs,DOEAz@Tyr hybrid micelles could accumulate at the biofilm infection site by the EPR effect and permeate into deeper layers of biofilm through surface pores.In an acidic biofilm community,the size of micelles decreased and the surface of the micelles became positively charged.The released D-Tyr would destroy the integrity of the biofilm and allow the small-sized and positively charged micelles to diffuse into the inner layers of the biofilm.Then,with the aid of lipase,Az was released to kill the bacteria in biofilm.The preformed biofilms on the surface of the implanted device could be eradicated by two differnt anti-biofilm mechanisms and eventually the biofilm infection in the body could be cured.Dual·drug loaded micelles with pH/reactive oxygen?ROS?responsiveness were constructed to overcome antibiotic resistance by enzymatic deactivation.The antibiotic ampicillin?Amp?was conjugated to?-lactamase?Bla?inhibitors phenylboronic acid?PBA?modified tetraphenylstyrene?TPE?via a ROS-sensitive group?TK?.Subsequently,the conjugate was coupled to?-cyclodextrin??-CD?via an acid-sensitive boronate linkage to form the pH/ROS-sensitive prodrug molecules?-CD-PBA-TPE-TK-Amp?CPTTA?.The amphiphilic copolymers PEG-PCL-Ad?PECLA?were synthesized by modifying the hydrophobic terminal of poly?ethylene glycol?-polycaprolactone?PEG-PCL?with adamantane?Ad?.The PECLA and CPTTA formed host-guest complexes and simultaneously self-assembled into pH/ROS double-sensitive micelles in aqueous solution.The particle size of PECLA@CPTTA micelles was about 113.6 nm and Amp-loading amount was about 8.3%.The multi-stimuli-responsive micelles could release the conjugate of Bla inhibitor and antibiotic?PBA-TK-Amp?in the presence of acidic pH and lipase.Under incandescent illumination,TPE generated ROS to break the TK bond and release antibiotic Amp.Compared with free drugs,PECLA@CPTTA micelles could transport Bla inhibitors and antibiotics to the infected site at the same time and be enriched at the site of infection by EPR effect.The released PBA-TK-Amp could inhibit the activity of Bla which was secreted by methicillin-resistant Staphylococcus aureus?MRSA?,thus avoiding the hydrolysis of?-lactam antibiotic Amp and restoring the sensitivity of MRSA to Amp.When stimulated by light,TPE produced ROS to break TK bonds and antibiotic Amp was release to kill the free or attached MRSA,thereby curing the subcutaneous infection of the drug-resistant bacteria.Double enzyme-sensitive drug-loaded nanoparticles with cell targeting ability were constructed to improve the poor permeability and retention of antibiotics in infected cells.The random copolymer P?EPE-r-TPE?was synthesized by using 4-aminobutyric acid as the initiator and acrylic modified triethyl phosphate?EPE-Ar?and tetrastyrene?TPE-Ar?as monomers via michael addition.Then mannose?Man?was grafted on the side chain of the copolymer through PEG to obatin the macrophage-targeted and double enzyme-sensitive copolymer Man-g-P?EPE-r-TPE?.The sideromycins ciprofloxacin?DFO-Cip,abbreviated DC?was synthesized by michael addition of deferoxamine?DFO?and acrylic acid modified ciprofloxacin?Cip-Ar?.In aqueous phase,the copolymers Man-g-P?EPE-r-TPE?could self-assembled into blue-emitting nanoparticles?MPET?.The drug-loaded nanoparticles MPET@D_FeC were prepared by physically encapsulating DC and ferric ion(Fe³⁺)chelate D_FeC.The size of MPET@D_FeC nanoparticles was about 135 nm and the drug-loading capacity was about 11%.The nanoparticles actively targetrf macrophages and promoted the accumulation of antibiotics in infected cells.Under the action of a large amount of lipase and phospholipase secreted by bacteria in the infected cells,MPET@D_FeC nanoparticles could be rapidly degraded to release the ciprofloxacin antibacterial agent D_FeC for intracellular bacterial killing.The process of drug release could be monitored by the increase of blue fluorescence induced by the aggregation of the degradation products TPE.Compared with free drugs,MPET@D_FeC nanoparticles exhibited higher bacterial clearance efficiency in infected cells in vivo and effectively extended the lifespan of mice.To circumvent the limitations of conventional antibiotics?i.e.toxicity and drug resistance?,a black phosphorus quantum dot?BPQDs?-based antibacterial system was developed for combined photothermal/nitrogen monoxide?NO?therapy through the mechanism different from antimicrobial small molecules.The amphiphilic antibacterial polymer mPEG-b-P?LL-r-LLIR??abbreviated as PELI?was synthesized by modifying the side chain of Poly?ethylene glycol?-block-Poly?L-lysine??mPEG-b-PLL?with isovaleric acid.The grafting rate of 50%of PELI was screened for subsequent experiments by antibacterial test and hemolysis experiment.A heat-sensitive nitrosothiol NO donor?CysamNO?was synthesized by reacting mercaptoethylamine with tert-butyl nitrite.BPQDs electrostatically adsorbed CysamNO to form BPQD-SNO,and then electrostatically interacted with PELI to prepare the nanoparticles PELI@BPQD-SNO,which was loaded NO donor and photothermal agent BPQDs.The particle size was about 125.7 nm.The carrying capacity of BPQDs was about 10%and the binding amount of NO donor was 0.034?mol/mg.The nanoparticles improved the stability of BPQDs in vitro.After 10 minutes of NIR irradiation,a large amount of heat was generated within the nanoparticle suspensions to promote the release of NO by the heat-induced S-NO bond cleavage.PELI@BPQD-SNO with broad-spectrum antibacterial activity could kill bacteria by destroying the cell membrane of Gram-negative bacteria and positive bacteria under the synergy effect of heat and NO.Compared with the small-sized particle BPQDs,PELI@BPQD-SNO nanoparticles could avoid kidney clearance and be accumulated at the infection site by EPR effect.Under external NIR light,the temperature of the infection site was increased and NO was release simultaneously.The bacteria in the abscess were killed by the synergistic effect of heat and NO,promoting healing of the injury.