| Background:Periprosthetic joint infection（PJI）following arthroplasty presents disastrous consequences to patients’health.Although PJI occurs only at a lower rate of around 1.0%in primary arthroplasies,the number of PJI is constantly increasing with the extensive application of arthroplasty.Current strategies against PJI are typically removal of the compromised implant,thorough debridement,cement spacer retention,revision and prolonged exposure to sensitive antibiotics.Patients may suffer long-term huge physiological,psychological and economic distress,but they cannot expect intended and definite cure of the infection after demanding treatment.Such situation poses serious challenges to the present treatment paradigm,giving doctors’motivation to explore more effective but less agonizing strategy against PJI.Research shows that bacterial biofilm formed on prostheses or abiotic tissue is one of the major causes of refractoriness in PJI.Bacterial biofilms are composed of planktonic bacteria and extracellular matrix produced by the cells,which is inherently characterized by enhanced resistance against antimicrobial agents and reduced susceptibility to host immune defences.Therefore,much attention should be paid to how to kill bacteria entrenched in the biofilms.In the light of ultrasound（US）-induced cavitation effects,the synergy of acoustic microbubbles（MBs）and sensitive antibiotics has been proposed as an alternative for the eradication of biofilm-enclosed bacteria.MBs create cavitation effects triggered by US energy,which imposes physical and biological effects to extracellular matrix structure,thereby restoring susceptibility of biofilm-encapsulated cells to antibiotic action and facilitating easier transport of antibiotics through biofilms for killing bacteria.However,acoustic MBs are subject to spontaneous collapse,thus the poor stability might restrict the extensive therapeutic use in vivo.More recently,acoustic nanodroplets（NDs）,typically including a liquid perfluorocarbon core,have indicated stronger stability.NDs have shown great promise as an original ultrasound contrast agent in clinical sonography.Under right stimulation from acoustic or thermal energy,NDs would undergo a phase transition into gaseous bubbles.NDs can cause mechanical bioeffects to tissues or cells after phase change and cavitation.But the phase change threshold needed for NDs’excitation increases due to enhanced stability the liquid core confers on NDs,requirin g higher acoustic energy like focused ultrasound or high thermal energy which are the current common stimulation mode But high energy accumulated on the surface of implants could inevitably damage implants themselves and cause coagulative necrosis of surrounding tissue,which would touch the safe baseline of in vivo PJI research.In consequence,this situation heightens the need for novel and safer stimulation modes for achieving ultimate cavitaiton in vivo of NDs to fight against biofilms.Staphylococcus aureus,widely distributed in nature,can colonize on human skin or other organs at a higher rate,which is more vulnerable to surgical or haematogenous infection.Staphylococcus aureus can excrete various toxin,causing several system or organ infections as well as prolonged infection due to biofilms formed on prostheses or abiotic tissue.Biofilms transform biological characteristics of bacteria living therein,enhancing antibiotic tolerance,which renders the treatment in more difficulty.Recently,the extensive use of antibiotics leads annually growing number of drug-resistant strains,especially methicillin-resistant Staphylococcus aureus（MRSA）which has been recognized to be one of the most common pathogenic organisms and more resistant to killing.MRSA accounted for 24%of PJI in one recent study.Symptoms caused by MRSA become severer than that in methicillin-sensitive Staphylococcus aureus infections.Fortunately,vancomycin has been demonstrated an excellent bactericidal effect against MRSA infections and vancomycin-resistant MRSA strains have not yet been reported.However,vancomycin-sensitive MRSA planktonic bacteria colonized on the prosthetic surfaces could gradually turn to be vancomycin-tolerant MRSA biofilm bacteria,which could highly limit the antimicrobial effect of vancomycin alone and prompt the need for agonizing operations again.Consequently,this subject plan to explore an original and safe stimulating mode for NDs.Based on this new stimulating mode,NDs’antimicrobial effect and its possible mechanism against MRSA biofilms in vitro either alone or as an enhancer of vancomycin will be evaluated.Objectives:1.Establishment of a new phase-shift and cavitational mode for NDs could be safely applicable to PJI study.2.To evaluate NDs’independent bactericidal effect through new stimulation mode against MRSA biofilms in vitro and its mechanism.3.To evaluate NDs’synergistic effect for vancomycin against MRSA biofilms through new stimulation mode.Methods:1.Lipid NDs incorporating perfluoropentane（PFP）were fabricated through rotary evaporation while lipid MBs encasing perfluoropropane（C3F8）were synthesized by vigorous shaking method.Morphology and structure of both particles were analyzed by macroscopic observation and light microscopy.Size distribution and Zeta potential of both particles were determined by Malvern laser particle size analyzer system.NDs were divided into 4 groups to receive individual stimulation for phase change and cavitation:US alone,US plus incubation at 37℃,incubation at room temperature and incubation at 37℃for24h.US parameters were set:an unfocused ultrasonic transducer with a frequency of 1.0MHz,an acoustic intensity of 3.0 W/cm2 and a duty cycle of 50%.After the first 5 minutes of US exposure,NDs were immediately placed at room temperature or 37℃for further incubation until 24 hours.The morphological changes of NDs in the four groups at initial state,5 minutes,8,16,and 24 hours were recorded under a light microscope.2.MRSA252 biofilms were cultured on coverslip disks in a 24-well plate using static culture method.Biofilm interventions were divided into 8 groups as follows:non-treatment（control）,vancomycin,US,US+MBs,US+vancomycin,US+MBs+vancomycin,US+NDs,US+NDs+vancomycin,followed by incubation at 37℃for 24 hours.The treatment of each group was performed in triplicate.The remaining biofilms,stained with live/dead agents,were observed via confocal laser scanning microscope.The obtaine d images were introduced into Image J software for quantitatively analyzing live cell ratio.MRSA biofilm samples after intervention were coated with gold and imaged with a scanning electron microscope for detailed morphological changes.3.MRSA biofilms cultured in a 96-well plate were grouped as mentioned above.Biofilms were treated with Alamar blue and subjected to microplate reader.According to manufacturer’s equation,percent reduction of Alamar blue was determined for evaluation of metabolic activity of biofilm bacteria.Results:1.Successfully-prepared NDs and MBs exhibited different macroscopic images,which showed that NDs appeared to sink in the bottom whereas MBs floated on top of solution.Microscopic images indicated that MBs were spherical while NDs were nearly spherical.Both of particles were relative homogeneity in diameter and well-dispersed.Initial NDs and MBs exhibited mean diameters of 309 and 1,463 nm,respectively,with size distribution was acceptable,and the zeta-potentials were-16.0 and-6.38 m V,respectively.Compared with US alone,incubation at room temperature and incubation at 37℃,NDs treated with US plus incubation at 37℃suggested a gradually expanded diameter and increasingly larger,and the number of larger NDs was rising.NDs presented in foam-like shape large enough for macroscopic observation at 16 h,which suggested a substantial phase transition to bubbles,and then a continuous coalescence of NDs-converted nanobubbles into larger bubbles.Ultimately at 24 h,most of the coalesced bubbles underwent spontaneous cavitation,which reflected an irreversible and steady progress of phase shift and cavitation.2.Confocal laser scanning microscope images demonstrated that loosened structure and many micropores were observed after treatment with US or US+MBs compared with vancomycin group.Groups form US+vancomycin with or without MBs also exhibited similar loosened morphology with a small number of dead cells.More noticeable destruction in biofilm structure and dead bacteria occurred in US+NDs group.Treatment with US+NDs+vancomycin resulted in a more sparse biofilm distribution than any other groups.ImageJ software analysis indicated that exposure to US+NDs obtained lower ratio of viable bacteria 43%±3%（P<0.05）that other groups（excluding group from US+NDs+vancomycin）.Exposure to US+NDs+vancomycin achieved significantly more bacteria killing than other treatment 23%±5%（P<0.05）.Scanning electron microscope images indicated that US+NDs group showed many residual circular arc margins around biofilm remains,where located the damaged bacteria remained in cluster arrangement.US+NDs+vancomycin group showed no significant such changes but cytolysis.3.The percent reduction of AB in vancomycin,US,US+MBs,US+vancomycin and US+MBs+vancomycin groups had not any significant difference from that in the control.Application of US or US+MBs in combination with vancomycin saw a significant fall of percent reduction to 31.00%±1.28%（P<0.05）and 29.77%±0.83%（P<0.05）,respectively,comparing with that in vancomycin-alone treatment（37.73%±1.02%）,but they were no significant difference from each other.The most significant decline in percent reduction of AB occurred in US+NDs group（16.43%±0.95%）（P<0.05）and US+NDs+vancomycin group（7.43%±0.76%）（P<0.05）,comparing with other treatment without NDs.Moreover,US+NDs+vancomycin treatment exhibited significantly lower percent reduction of AB than US+NDs（P<0.05）.Conclusion:1.Through sequential stimulation towards NDs with short-term,low-intensity US and long-term incubation with physiological temperature,a new mode of phase shift and cavitation in lipid NDs incorporating PFP was established:an initial unfocused pulsed ultrasound for 5 minutes（acoustic intensity 3.0 W/cm2,frequency 1.0 MHz,duty cycle50%）and a subsequent incubation at 37℃into a 24-hour duration.2.Acoustic NDs could exert a significant bactericidal effect against MRSA biofilms in vitro through a new stimulation mode.The ratio of viable bacteria was 43%,the percent reduction of AB was 16.43%.3.Acoustic NDs could significantly enhance anti-biofilm effect by vancomycin against MRSA biofilms in vitro through the new stimulation mode.The ratio of viable bacteria was23%and the percent reduction of AB was 7.43%,which indicated stronger bactericidal effect than ND alone.This anti-biofilm strategy on the basis of NDs could provide original idea and pave the way for further PJI researches. |
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