Rational collaborative ablation of bacterial biofilms ignited by physical cavitation and concurrent deep antibiotic release
Introduction
Bacterial infections, especially those caused by drug-resistant bacteria, are among the most prominent underlying causes of many severe diseases and have increasingly raised medical and public concerns across the world [[1], [2], [3]]. Due to the abuse of antibiotics, bacteria have developed increasingly serious resistance towards antibiotics due to bacteria structural transformation and mutation as well as biofilm formation [4]. Bacterial biofilm has extracellular polymeric substances to protect bacteria from threatening external environments [[5], [6], [7]]. Bacterial biofilm can result in human diseases, especially in patients with indwelling devices for continuous medical treatments [8]. The biofilm matrix not only provides a microenvironment for microbial growth, catalysis, and communication but also protects its inhabitants against environmental challenges, such as UV exposure, acids or antibiotics [9,10]. Therefore, it is imperative to develop novel antibacterial systems that can efficiently solve the problem of bacterial infection with serious biofilm formation [11,12].
Several antibiofilm systems have been designed to eliminate or inhibit biofilm, such as antibacterial coatings [[13], [14], [15], [16], [17]] and inorganic materials [[18], [19], [20]]. However, antibacterial coatings are not appropriate for preformed biofilms on implants and some special substrates. Additionally, inorganic antibacterial materials have challenging concerns due to their probable toxicity for biological applications [21,22]. Thus, new antibiofilm systems that adopt organic biomaterials with potential biocompatibility are expecting [23,24]. Herein organic antibacterial systems are attracting more and more attention because of their multifunctional properties, biosafety and less possibility to develop drug resistance [25]. More importantly, a stubborn challenge for thorough elimination of bacterial biofilm is limited penetration of antibacterial materials or antibiotics towards biofilm, resulting in compromised treating efficiency [7]. Complicated and thick extracellular polymeric substances can hinder antibiotic diffusion into deep biofilm [26,27].
To solve the problem of limited biofilm penetration, many strategies have been applied to deeply penetrate bacterial biofilms for efficient biofilm elimination, such as PEO-containing CIP-loaded dual corona vesicles [28], self-adaptive triclosan-loaded micelles [5], acid-environment responsive gold nanoparticles [6] and other smart systems [7,29,30]. Antibiofilm platforms that can deeply penetrate biofilm, efficiently accumulate and spatiotemporally control antibiotic release are promising. Moreover, there have been great developments in smart drug delivery systems in recent years [31,32]. Controllable drug release can be achieved under different stimuli via polymeric nanocarriers [[33], [34], [35], [36]]. For bacterial infection, the pH state decreases in biofilm microenvironment due to the presence of low oxygen fermentation and generated acidic species [37]. Inflammation is known to exacerbate acidity due to the increased level of acidic products [[38], [39], [40]]. Moreover, due to the protection of extracellular polymeric substances, biofilm microenvironment lacks oxygen, also resulting in low pH values compared with healthy tissues [5,6]. Herein, biofilm infected sites possess similar enhanced permeation and retention effect (EPR) like tumors, blood vessels are permeable in inflammation regions due to the mediation of various inflammatory factors, leading to passive targeting of nanoparticles in bacteria-infected regions [1,37,41,42]. Thus, polymeric therapeutic nanoparticles are promising to efficiently accumulate in the biofilm microenvironment [43]. In addition, photoacoustic (PA) technique has been increasingly focused in biomedicine due to its high spatial resolution and deep tissue penetration [44,45]. However, it is rare to examine PA technique in the elimination of bacterial biofilm. PA cavitation probably disrupts the protective matrix of biofilm, thus potentially allowing antibiotics to deeply diffuse into the biofilm [46]. Furthermore, pathogen inhibition systems with imaging/detection potency are new demand and trend for antibacterial materials [[47], [48], [49], [50]].
In this work, gasifiable polymeric nanodroplets are developed to deeply penetrate into biofilm and ablate the biofilm through PA-triggered cavitation, gas shockwave, and deep antibiotic release at the bacteria-infected sites (Scheme 1). Perfluorohexane (PFH) and rifampicin (Rif)-loaded nanodroplets (PFH/Rif@NDs) are fabricated by a two-step emulsion process. Near infrared (NIR) light-absorbable amphiphilic diblock copolymers, PDMAEMA-b-P(BMA-co-CSMA) are employed as stabilizing matrix for the nanodroplets, in which cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) coronas endow the nanodroplets with protonation capacity in acidic condition, and hydrophobic NIR light-absorbing fluorogen locates in the hydrophobic segment. Therefore, PFH/Rif@NDs have the capacity to target, accumulate and adhere to biofilm due to the EPR effect and acid-adaptive performance at the infected sites. In vitro and in vivo assays demonstrate the specific targeting and accumulation of PFH/Rif@NDs in S. aureus biofilm. More importantly, the nanodroplets can effectively eradicate biofilm by PA-triggered shockwave and concurrent deep release of antibiotic to achieve synergistic physical damage and chemotherapeutic treatment.
Section snippets
Materials
2-(Dimethylamino) ethyl methacrylate (DMAEMA, 99%, Aldrich) was dried over calcium hydride, vacuum-distilled, and stored at −20 °C prior to use. Butyl methacrylate (BMA, 99.5%, Aldrich) was vacuum-distilled and stored at −4 °C prior to use. 2-Hydroxyethyl methacrylate (HEMA, 99.5%, J&K), dimethylaminopyridine (DMAP, Meryer), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, Meryer), perfluorohexane (PFH, 97%, Energy Chemical) and all other reagents were used as received. 2,
Polymer synthesis and nanodroplet fabrication
Well-defined polymers were synthesized via facile reversible addition-fragmentation chain transfer (RAFT) polymerization [59]. A Changsha NIR dye was facilely esterified with 2-hydroxyethyl methacrylate and labelled as CSMA (Fig. S1 and Fig. S2). PDMAEMA-b-P(BMA-co-CSMA) diblock copolymers were fabricated from the copolymerization of CSMA and butyl methacrylate (BMA) in the presence of PDMAEMA39-based RAFT agent (Fig. S1 and Fig. S3). Finally, PFH and a representative antibiotic, rifampicin,
Conclusion
In summary, gasifiable nanodroplets loaded with PFH and Rif, PFH/Rif@NDs, were successfully formulated. Upon NIR light irradiation, the nanodroplets could generate significant PFH gasification and gasification-enhanced PA cavitation, which could damage bacterial biofilm and concurrently promote antibiotic efficiently diffuse into deep biofilms for bacteria eradication. In vitro and in vivo assays demonstrated the high antibiofilm capability, which benefited from the synergistic PA-triggered
CRediT authorship contribution statement
Bing Cao: Conceptualization, Investigation, Writing - original draft, Writing - review & editing. Xiaoming Lyu: Investigation, Writing - review & editing. Congyu Wang: Methodology. Siyu Lu: Methodology. Da Xing: Methodology, Resources, Supervision. Xianglong Hu: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Scientific Foundation of China (51973071), the Science and Technology Program of Guangzhou (2019050001), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2016A030306013), the Pearl River Young Talents Program of Science and Technology in Guangzhou (201906010047) and the National Key Research and Development Program of China (2018YFA0209800).
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These authors contributed equally to this work.