International Journal of
Molecular Sciences Article
Lysozyme Associated Liposomal Gentamicin Inhibits Bacterial Biofilm Yilin Hou, Zhaojie Wang, Peng Zhang, Hu Bai, Yuelin Sun, Jinyou Duan and Haibo Mu * Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China; [email protected] (Y.H.); [email protected] (Z.W.); [email protected] (P.Z.); [email protected] (H.B.); [email protected] (Y.S.); [email protected] (J.D.) * Correspondence: [email protected]; Tel.: +86-29-8709-2226 Academic Editors: Antonella Piozzi and Iolanda Francolini Received: 9 February 2017; Accepted: 4 April 2017; Published: 9 April 2017
Abstract: Bacteria on living or inert surfaces usually form biofilms which make them highly resistant to antibiotics and immune clearance. Herein, we develop a simple approach to overcome the above conundrum through lysozyme-associated liposomal gentamicin (LLG). The association of lysozyme to the surface of liposomes can effectively reduce the fusion of liposomes and undesirable payload release in regular storage or physiological environments. The LLG was more effective at damaging established biofilms and inhibiting biofilm formation of pathogens including Gram-positive and Gram-negative bacteria than gentamicin alone. This strategy may provide a novel approach to treat infections due to bacterial biofilm. Keywords: liposome; lysozyme; gentamicin; biofilm
1. Introduction Biofilms are matrix-enclosed, complex and differentiated communities of bacteria that are adherent to inert or biological surfaces [1]. Upon adhesion, the bacterial cells start producing an extracellular matrix and group together in densely-packed bacterial clusters. From the mature biofilm, individual cells or biofilm fragments are released and can colonize new surfaces [2]. Biofilm formation causes corrosion and biofouling of industrial equipment [3], and is also associated with many illnesses and infections in humans, including oral diseases, native valve endocarditis, and a number of nosocomial infections [2]. Unlike planktonic bacteria, biofilm bacteria have been known to be highly resistant to adverse environmental conditions such as antibiotics, detergents or biocides [4]. Therefore, new efforts for biofilm growth inhibition, biofilm damage, or biofilm eradication are being sought. The biggest challenge in treating biofilm infections is overcoming the resistance and tolerance to antimicrobial agents. Successful therapy requires innovative ways to deliver antimicrobial substances in a sufficiently high concentration to the biofilm bacteria [5]. Liposome is an attractive candidate for drug delivery to biofilms due to its versatility and biocompatibility. It has been shown that liposome-encapsulation improves the efficacy of various antibacterial and antifungal drugs against a broad range of pathogens in vitro and in vivo [6–9]. One advantage of liposomes being used as drug delivery vehicles is their potential to fuse with phospholipid membranes. However, the applications of liposomes, particularly those with sizes below 100 nm, are often hindered by their poor stability due to spontaneous fusion, resulting in payload loss and increase in vesicle size [9,10]. Several strategies have been employed to overcome this problem including optimizing liposome composition [11], polyethylene glycol (PEG)ylation [12] and nanoparticulate stabilizing [13–15]. Herein, we introduce a novel liposome formulation stabilized by lysozyme. The cationic lysozyme is ready to associate with the negatively charged liposome through electrostatic attraction. This strategy Int. J. Mol. Sci. 2017, 18, 784; doi:10.3390/ijms18040784
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Herein, we introduce aa novel novel liposome liposome formulation formulation stabilized stabilized by by lysozyme. lysozyme. The The cationic cationic 2 of 8 lysozyme is ready to associate with the negatively charged liposome through electrostatic attraction. lysozyme is ready to associate with the negatively charged liposome through electrostatic attraction. This strategy strategy stabilizes stabilizes liposomes liposomes against against fusion fusion and and avoids avoids undesirable undesirable leakage leakage of of liposomal liposomal drugs. drugs. This stabilizes liposomes against fusion and avoids undesirable leakage of liposomal drugs. The stabilized The stabilized liposomes with positive charge are hypothesized to easily bind to the negatively The stabilized liposomes with positive charge are hypothesized to easily bind to the negatively liposomes with of positive charge are hypothesized to easily bind to the negatively charged matrix charged matrix matrix of bacterial bacterial biofilm, inhibit bacterial bacterial biofilm formation and damage damage established charged biofilm, inhibit biofilm formation and established of bacterial inhibit bacterial biofilm formation and damage established biofilm built by biofilm builtbiofilm, by Gram-positive Gram-positive or -negative -negative organisms. biofilm built by or organisms. Gram-positive or -negative organisms. 2. Results Results and and Discussions Discussions 2. 2. Results and Discussions 2.1. Preparation and and Characterization of of Lysozyme-Associated Liposomal Liposomal Gentamicin 2.1. 2.1. Preparation Preparation and Characterization Characterization of Lysozyme-Associated Lysozyme-Associated Liposomal Gentamicin Gentamicin Liposomal gentamicin (LG) was prepared by vesicle extrusion technique (Figure (Figure 1a) 1a) [16]. [16]. The The Liposomal by by vesicle extrusion technique Liposomal gentamicin gentamicin(LG) (LG)was wasprepared prepared vesicle extrusion technique (Figure 1a) [16]. size and surface zeta potential of LG were 99 nm and −54.5 mV, respectively (Figure 1b,c). Lysozymesize zeta potential of LG were 99 were nm and mV,− respectively (Figure 1b,c). LysozymeThe and sizesurface and surface zeta potential of LG 99−54.5 nm and 54.5 mV, respectively (Figure 1b,c). associated liposomal liposomal gentamicin gentamicin (LLG) (LLG) were were obtained obtained by by mixing mixing LG LG and and lysozyme. lysozyme. The The size size and associated Lysozyme-associated liposomal gentamicin (LLG) were obtained by mixing LG and lysozyme. The and size surface zeta zeta potential potential of of the the resulting resulting LLG LLG were were measured measured by by dynamic dynamic light light scattering scattering (DLS). (DLS). The The surface and surface zeta potential of the resulting LLG were measured by dynamic light scattering (DLS). size of LLG was slightly larger than that of LG, suggesting the adsorption of lysozyme onto the size LLG was was slightly larger thanthan that that of LG, suggesting the adsorption of lysozyme ontoonto the The of size of LLG slightly larger of LG, suggesting the adsorption of lysozyme liposomal surface. The surface zeta potential changed from −54.5 to 17.5 mV (Figure 1c), which liposomal surface. The surface zeta potential changed from −54.5 17.5 to mV (Figure 1c), which the liposomal surface. The surface zeta potential changed from to − 54.5 17.5 mV (Figure 1c), confirmed the association of positively-charged lysozyme to the negatively-charged liposomes confirmed the association of positively-charged lysozyme to the negatively-charged liposomes which confirmed the association of positively-charged lysozyme to the negatively-charged liposomes through electrostatic attraction. attraction. through through electrostatic electrostatic attraction. we 18, introduce Int. J. Herein, Mol. Sci. 2017, 784
Figure 1.1. 1.(a)(a) (a) Schematic structure of lysozyme-associated lysozyme-associated liposomal gentamycin gentamycin (LLG); (b) (b) Figure Schematic structure of lysozyme-associated liposomal liposomal gentamycin (LLG); (b) hydrodynamic Figure Schematic structure of (LLG); hydrodynamic size; and (c) surface zeta potential of liposome (without Lysozyme) and LLG. size; and (c) surface zeta potential of liposome (without Lysozyme) and LLG. hydrodynamic size; and (c) surface zeta potential of liposome (without Lysozyme) and LLG.
2.2. Stability Stability of of Lysozyme-Associated Lysozyme-Associated Liposomal Liposomal Gentamicin Gentamicin 2.2. Stability of Lysozyme-Associated 2.2. The stability stability of of LG LG and and LLG LLG were were evaluated evaluated over over time time in in deionized deionized water water (Figure (Figure 2a). 2a). LG LG was was The stability of LG and LLG evaluated over time deionized water (Figure LG was The gradually aggregated while LLG was relatively stable in water. Gentamicin in LG was released more gradually aggregated aggregated while while LLG LLG was was relatively relatively stable stable in in water. water. Gentamicin Gentamicin in in LG LG was was released released more more gradually quickly than in LLG (Figure 2b). These data suggested that the association of lysozyme improved the quickly than in LLG LLG (Figure (Figure 2b). These These data data suggested suggested that that the the association association of of lysozyme lysozyme improved improved the the quickly stability of liposome and prevented the release of gentamicin. stability of of liposome liposome and prevented the release of gentamicin. stability
◦
Figure 2. (a) Size measurements of liposome and LLG over the course of 48 at 25 C in deionized Figure 2. 2. (a) (a) Size Size measurements measurements of of liposome liposome and and LLG LLG over over the the course course of of 48 48 hhh at at 25 25 °C °C in deionized deionized Figure in water; (b) cumulative release profile of gentamicin-loaded liposome and LLG over the course of of 72 72 h at water; (b) (b) cumulative cumulative release release profile profile of of gentamicin-loaded gentamicin-loaded liposome liposome and and LLG LLG over over the the course course water; of 72 hh ◦ C in deionized 25 water. at 25 °C in deionized water. at 25 °C in deionized water.
2.3. Antibiofilm Activities of Lysozyme-Associated Liposomal Gentamicin 2.3. Antibiofilm Antibiofilm Activities Activities of of Lysozyme-Associated Lysozyme-Associated Liposomal Liposomal Gentamicin Gentamicin 2.3. Pseudomonas aeruginosa human pathogen, which is Pseudomonasaeruginosa aeruginosa(P. (P.aeruginosa) aeruginosa)isis isaaaGram-negative Gram-negativeopportunistic opportunistic human pathogen, which Pseudomonas (P. aeruginosa) Gram-negative opportunistic human pathogen, which known for causing chronic pulmonary infections in cysticinfibrosis (CF) patients and patientsand suffering of is known for causing chronic pulmonary infections cystic fibrosis (CF) patients patients is known for causing chronic pulmonary infections in cystic fibrosis (CF) patients and patients non-CF bronchiectasis, and generally employed as a model organism for investigation of biofilms [17].
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biofilms [17]. Gentamicin or lysozyme alone had a mild effect on biomass and live cells of P. aeruginosa suffering of non-CF bronchiectasis, and generally employed as a model organism for investigation of biofilms after 24 h treatment compared to blank control (Figure 3a,b). LLG treatment markedly biofilms [17]. Gentamicin or lysozyme alone had a mild effect on biomass and live cells of P. aeruginosa Gentamicin or biofilm lysozyme alone a mild on To biomass and live cells of P. aeruginosa biofilms after reduced both mass andhad viable celleffect counts. see whether LLG was able to eliminate bacterial biofilms after 24 h treatment compared to blank control (Figure 3a,b). LLG treatment markedly 24 h treatment compared to blank control (Figure 3a,b). LLG treatment markedly reduced both biofilm biofilms built by a Gram-positive organism, Staphylococcus aureus (S. aureus), which can cause lifereduced both biofilm mass and viable cell counts. To see whether LLG was able to eliminate bacterial mass and viable cell counts. To see and whether LLG was able to eliminate bacterial biofilms by threatening infections in humans the nosocomial (hospital) environment [18], wasbuilt tested. biofilms built by a Gram-positive organism, Staphylococcus aureus (S. aureus), which can cause lifeaQuantification Gram-positiveoforganism, Staphylococcus aureus (S. aureus), which can cause life-threatening infections biofilm biomass and cell viability demonstrated that LLG had a more pronounced threatening infections in humans and the nosocomial (hospital) environment [18], was tested. in humans and the nosocomial (hospital) environment effect than gentamicin or lysozyme alone (Figure 3c,d). [18], was tested. Quantification of biofilm Quantification of biofilm biomass and cell viability demonstrated that LLG had a more pronounced biomass and cell viability demonstrated that LLG had a more pronounced effect than gentamicin or effect than gentamicin or lysozyme alone (Figure 3c,d). lysozyme alone (Figure 3c,d).
Figure 3. Crystal violet assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess the antibiofilm activity of LLG against P. aeruginosa biofilm (a,b) and S. aureus assay andand 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Figure 3.3.Crystal Crystalviolet violet assay 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (c,d). assay toassay assess antibiofilm activityactivity of LLG of against P. aeruginosa biofilm (a,b) and(a,b) S. aureus (MTT) to the assess the antibiofilm LLG against P. aeruginosa biofilm and S.(c,d). aureus (c,d).
Fluorescence microscopy imaging of P. aeruginosa (Figure 4a) and S. aureus (Figure 4b) biofilms Fluorescence microscopy imaging of P. aeruginosa (Figure 4a) and S. aureus (Figure 4b) biofilms was pursued to further evaluate the antibiofilm potential of LLG. The blank control biofilms were Fluorescence microscopy imaging of P. aeruginosa (Figure 4a) and aureus (Figure 4b) biofilms was pursued to further evaluate the antibiofilm potential of LLG. TheS.blank control biofilms were densely colonized with hierarchically and three-dimensionally structured formations. Biofilms was pursued to further evaluate the and antibiofilm potential of LLG. The blank controlBiofilms biofilmstreated were densely colonized with hierarchically three-dimensionally structured formations. treated with LLG exhibited only a few isolated bacterial colonies instead of a recognizable biofilm densely and three-dimensionally formations. Biofilms with LLGcolonized exhibited with only ahierarchically few isolated bacterial colonies instead of structured a recognizable biofilm structure. structure. treated with LLG exhibited only a few isolated bacterial colonies instead of a recognizable biofilm structure.
Figure Figure 4. 4. Fluorescence Fluorescence microscopy microscopy of of P. P. aeruginosa aeruginosa (a) (a) and andS. S.aureus aureus(b) (b)biofilm. biofilm. Biofilms Biofilms incubated incubated with withtryptic trypticsoy soybroth broth(TSB) (TSB)are areused usedas ascontrol. control.Scale Scalebars barswere were10 10µm. μm. Figure 4. Fluorescence microscopy of P. aeruginosa (a) and S. aureus (b) biofilm. Biofilms incubated with tryptic soy broth (TSB) are used as control. Scale bars were 10 μm.
Scanning electron microscopy (SEM) was also applied to evaluate the surface morphology changes of P. aeruginosa (rod shaped pathogen, Figure 5a) and S. aureus (round-shaped pathogen, Figure 5b)
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Scanning electron microscopy (SEM) was also applied to evaluate the surface morphology 4 of 8 was Figure also applied toS.evaluate the surface morphology changes of P. aeruginosamicroscopy (rod shaped(SEM) pathogen, 5a) and aureus (round-shaped pathogen, changes5b) ofbiofilms P. aeruginosa (rod shaped pathogen, Figure 5a) and aureus (round-shaped pathogen, Figure treated with LLG, gentamicin or lysozyme in S. tryptic soy broth (TSB). The control Figure 5b) biofilms treated with LLG, gentamicin or lysozyme in tryptic soy broth (TSB). The control showed a highly organized and well-defined architecture. In LLG-treated biofilms, the cell walls biofilms treated with LLG, gentamicin or lysozyme in tryptic soy broth (TSB). The control showed showed a highly organized and well-defined architecture. In LLG-treated biofilms, the cell became and damaged, the shape and sizeIn ofLLG-treated cells changed dramatically, onlybecame awalls few a highlywrinkled organized and well-defined architecture. biofilms, the celland walls became wrinkled and damaged, the shape and size of cells changed dramatically, and only a few scattered bacterial cells were noted. Overall, these results clearly indicated that LLG had an wrinkled and damaged, the shape and size of cells changed dramatically, and only a few scattered scattered bacterial cells were noted. Overall, these results clearly indicated that LLG had an advantage in disrupting existing biofilms Gram-negative bacterial cells were noted. Overall, theseformed resultsby clearly indicated and that -positive LLG hadorganisms. an advantage in advantage in disrupting existing biofilms formed by Gram-negative and -positive organisms. disrupting existing biofilms formed by Gram-negative and -positive organisms. Int. J. Scanning Mol. Sci. 2017, 18, 784 electron
Figure 5. Scanning electron microscopy of P. aeruginosa (a) and S. aureus (b) biofilm. Biofilms Figure 5. electron microscopy of P.ofaeruginosa (a) and(a)S. and aureusS.(b) biofilm. Figure 5. Scanning Scanning electron P. aeruginosa aureus (b) Biofilms biofilm.incubated Biofilms incubated with TSB are used asmicroscopy control. Scale bars were 1 μm. with TSB are used as control. Scale bars were 1 µm. incubated with TSB are used as control. Scale bars were 1 μm.
To explore the underlying mechanism by which LLG disrupted bacterial biofilms above, To explore by which LLG disrupted biofilms above, the underlying mechanism disrupted bacterial biofilms above, liposomal rhodamine B (LR) or lysozyme associated liposomal rhodaminebacterial B (LLR) were generated. liposomal B or lysozyme associated liposomal rhodamine BB (LLR) were generated. liposomal rhodamine rhodamine B (LR) (LR) lysozyme associated liposomal rhodamine (LLR) were generated. Compared with LR, LLR elicited a much stronger binding to S. aureus biofilm (Figure 6). This might Compared with LR, LLR elicited a much stronger binding to S. aureus This might biofilm (Figure 6). be due to the electrostatic attraction between positive lysozyme on LLR and biofilm matrix, such as be due to the electrostatic attraction between positive lysozyme on LLR and biofilm matrix, such as lysozyme alginate, which usually possesses a negative charge. alginate, which usually possesses a negative charge.
Figure 6. Binding ability of lysozyme liposome to S. aureus biofilm. Lysozyme associated liposomal Figure 6. Binding ability of lysozyme liposome to S. aureus biofilm. Lysozyme associated liposomal rhodamine B (LLR) or liposomal rhodamine B (LR) was incubated with preformed S. aureus biofilm Figure 6. Binding of lysozyme liposome to S.was aureus biofilm.with Lysozyme associated liposomal rhodamine B (LLR)ability or liposomal rhodamine B (LR) incubated preformed S. aureus biofilm for 10 min.B After incubation, the biofilm was collected and quantified for fluorescence intensity. rhodamine (LLR) or liposomal rhodamine (LR) was incubated with S. aureus biofilm for 10 min. After incubation, the biofilm was B collected and quantified forpreformed fluorescence intensity. The The10 biofilm without incubating with anywas liposome formulations was tested in parallel serving as the for min. After incubation, the biofilm collected and quantified for fluorescence intensity. biofilm without incubating with any liposome formulations was tested in parallel serving as The the background signal. biofilm without incubating with any liposome formulations was tested in parallel serving as the background signal. background signal.
Biofilm formation formationwas wasexamined examinedininthe thecase caseofofplanktonic planktonic aeruginosa (Figure 7a,b) exposed Biofilm P. P. aeruginosa (Figure 7a,b) exposed to Biofilm formation was examined in the case of planktonic P. aeruginosa (Figure 7a,b) exposed to to LLG, gentamicin lysozyme Lysozymeshowed showedno noeffects effectson on biofilm biofilm formation formation as as LLG, gentamicin or or lysozyme forfor2424h.h.Lysozyme LLG, gentamicin or lysozyme for 24 h. Lysozyme showed effects on formation as compared with blank blank control. Gentamicin suppressed biofilmno formation andbiofilm decreased live cells cells compared with control. Gentamicin suppressed biofilm formation and decreased live compared with blank control. Gentamicin suppressed biofilm formation and decreased live cells generally whereas LLG facilitated this suppression and reduction significantly. Similar findings generally whereas LLG facilitated this suppression and reduction significantly. Similar findings were generally facilitated suppression andofreduction significantly. wereobserved also whereas observed in the of this S. by aureus by quantification of biomass biofilm biomass (Figure 7c) viability andwere cell also in theLLG case ofcase S. aureus quantification biofilm (FigureSimilar 7c) andfindings cell also observed in the case of S. aureus by quantification of biofilm biomass (Figure 7c) and cell viability viability (Figure 7d). These results suggested that LLG had the potential to prevent planktonic cells of (Figure 7d). These results suggested that LLG had the potential to prevent planktonic cells of Gram(Figure 7d). These results suggested that LLG had the potential to prevent planktonic cells of GramGram-negative or -positive organisms from biofilm formation. negative or -positive organisms from biofilm formation. negative or -positive organisms from biofilm formation.
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Figure 7. The The inhibitory inhibitory effects effects of LLG on P. aeruginosa (a,b) and S. aureus Figure aureus (c,d) (c,d) biofilm biofilm formation. formation.
Controlled drug drug delivery delivery by by lipid lipid nanoparticles nanoparticles have Controlled have attracted attracted much much attention. attention. More More recently, recently, Harker et al. utilized an electrohydrodynamic technique to prepare core-shell lipid nanoparticles Harker et al. utilized an electrohydrodynamic technique to prepare core-shell lipid nanoparticles with a tunable size and highingredient active ingredient loading encapsulation capacity, encapsulation efficiency and awith tunable size and high active loading capacity, efficiency and controlled controlled release [19]. Many nano materials, for example, the metal-loaded nanofibers [20–22], have release [19]. Many nano materials, for example, the metal-loaded nanofibers [20–22], have shown high shown high antibacterial activity against or biofilm bacteria the current study, antibacterial activity against planktonic orplanktonic biofilm bacteria [23,24]. In the [23,24]. current In study, we developed we developed a platform to deliver antibiotic to treat bacterial biofilms through lysosome associated a platform to deliver antibiotic to treat bacterial biofilms through lysosome associated liposomes. liposomes. Thismade approach made liposomes easier to to biofilms; attach to abiofilms; a universal This approach liposomes more stablemore and stable easierand to attach universal survival survival for lifestyle for microbes lifestyle microbes in nature.in nature. 3. Materials and Methods 3.1. Materials 3.1. Materials 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphor1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) (DPPC) and 1,2-dipalmitoyl-sn-glycero-30 -rac-glycerol) (DPPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Gentamicin (1 phosphor-(1′-rac-glycerol) (DPPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Sulfate was Sulfate purchased Solarbio (Beijing, China). Rhodamine B was purchased from Aladdin Gentamicin wasfrom purchased from Solarbio (Beijing, China). Rhodamine B was purchased from (Shanghai, China). AllChina). reagentsAll were of analytical and usedgrade as received without further purifying. Aladdin (Shanghai, reagents were grade of analytical and used as received without Pseudomonas further purifying. aeruginosa (PAO1) and Staphylococcus aureus (ATCC 29213) were generous gifts received from Xiaodong Xia (College Food Science and aureus Engineering, A&F University). Pseudomonas aeruginosa (PAO1) of and Staphylococcus (ATCCNorthwest 29213) were generous gifts
received from Xiaodong Xia (College of Food Science and Engineering, Northwest A&F University). 3.2. Preparation and Characterization of LLG 3.2. Preparation Characterization of LLGa previously described extrusion method [16]. Briefly, 9 mg Liposomesand were prepared following of lipid (DPPC/DPPG = 9/1, molar ratio)awere dissolved in 1 mL chloroform, and[16]. thenBriefly, the organic Liposomes were prepared following previously described extrusion method 9 mg solvent was evaporated to form a dried lipid film. The lipid film was rehydrated with mL of of lipid (DPPC/DPPG = 9/1, molar ratio) were dissolved in 1 mL chloroform, and then the3organic deionized water, or 2 mM B (RhB), or 20The mM gentamicin, by with vortexing solvent was evaporated to rhodamine form a dried lipid film. lipid film was followed rehydrated 3 mLfor of 1deionized min and sonicating for 5 min to produce multilamellar vesicles (MLVs). The solution was extruded water, or 2 mM rhodamine B (RhB), or 20 mM gentamicin, followed by vortexing for 1 min through a 100 nmfor pore-sized membrane for 10 times to form narrowly distributed small and sonicating 5 min topolycarbonate produce multilamellar vesicles (MLVs). The solution was extruded unilamellar vesicles (SUVs). Particles were purified by washing withtowater times using 10 kDa through a 100 nm pore-sized polycarbonate membrane for 10 times form 3narrowly distributed MWCO Amicon centrifugal filters (EMD Millipore, Billerica, CA, USA) to remove unencapsulated small unilamellar vesicles (SUVs). Particles were purified by washing with water 3 times using 10 drugs. To prepare LLG, the pH of both lysozyme andMillipore, liposome solutions using kDa MWCO Amicon centrifugal filters (EMD Billerica, was CA,adjusted USA) to to 6.5 remove unencapsulated drugs. To prepare LLG, the pH of both lysozyme and liposome solutions was
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HCl. Then the liposomes and lysozyme at 1:100 (molar ratio) were mixed together, followed by 10 min bath sonication. The hydrodynamic size and surface zeta potential of LLG were measured by dynamic light scattering (DLS) measurements (Malvern Zetasizer NANO-ZS90, Malvern, UK). The gentamicin content was determined by sodium phosphotungstate precipitation method. 3.3. Stability Studies Stability of LLG or bare liposome was analyzed in deionized water. 2 mL freshly prepared li posome samples at 1 mg·mL−1 were incubated at 25 ◦ C for 48 h. The size change of the liposome samples in deionized water were measured by DLS. 3.4. Release Behaviors The kinetics of gentamicin release was studied from the prepared LLG. The 1 mL fresh prepared liposome solution (2 mg·mL−1 ) was initially incubated at 25 ◦ C in tube. Liposomes were taken at regular time intervals, centrifuged using 10 kDa MWCO Amicon centrifugal filters (EMD Millipore, Billerica, CA, USA) and the filtrate was obtained for gentamicin measurement. 3.5. Binding Ability of Lysozyme Liposome to Biofilm Lysozyme associated liposomal RhB (LLR) and liposomal RhB (LR) were prepared as described in Section 3.2. S. aureus (~109 colony forming units (CFU)) were grown in 6-well plates at 37 ◦ C for 24 h supplemented with 2 mL of TSB to allow biofilm formation. The non-adhered cells were removed with pipette and the plate was washed three times using 0.9% (w/v) NaCl. Then existing biofilms were incubated at 37 ◦ C in 1.8 mL TSB supplemented with 0.2 mL LLR or LR for 10 min. After that, the medium was removed and the biofilm was washed, collected using cell scraper in phosphate buffered saline (PBS), vortexed and subjected to fluorescence detection on a RF-5301 fluorescence spectrometer (Shimadzu, Kyoto, Japan) at excitation and emission wavelengths of 509 nm and 526 nm, respectively. Relative fluorescence intensity was expressed as percentage, and biofilm treated with LLG was used as 100% fluorescence intensity. 3.6. Antibiofilm Activity As described previously [25], 100 µL bacterial TSB solutions (~108 CFU) were seeded into 96-well polystyrene microtitre plates (Corning, NY, USA) at 37 ◦ C for 24 h to allow biofilm formation. The non-adhered cells were removed with pipette and the plate was washed three times using 100 µL 0.9% (w/v) NaCl. Then existing biofilms were incubated at 37 ◦ C in 90 µL TSB supplemented with 10 µL LLG, equivalent gentamicin or lysozyme for 24 h. Each treatment included 6 parallel wells. Biofilms incubated with TSB only were used as blank. Biofilm mass (crystal violet staining assay) and viable cells (MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay) were evaluated as previous [26]. All experiments were performed 3 times. Error bars represent standard deviation (SD). For biofilm inhibition assay, 100 µL of bacteria in TSB (~108 CFU) were seeded into individual wells of microtiter plates in the presence of compounds for 24 h. Biofilm mass were evaluated as described [26]. For fluorescence microscopy, S. aureus or P. aeruginosa (~108 CFU) was grown on glass coverslips at 37 ◦ C for 24 h in 24-well plates supplemented with 1 mL of TSB to allow biofilm formation. The coverslips were washed to remove unattached cells and were treated with liposomes for 24 h at 37 ◦ C. Existing biofilms were treated and imaged as previous [25]. SEM was conducted as described previously [27].
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3.7. Statistical Analysis All graphical evaluations were made using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Analysis of variance (ANOVA) was used to evaluate significant differences. 4. Conclusions In this study, we applied positively-charged lysozyme to stabilize the negatively-charged liposomes through electrostatic attraction. The lysozyme-associated liposomal gentamycin (LLG) was more effective at disrupting the preformed biofilms built by Gram-positive and -negative pathogenic bacteria than lysozyme or gentamycin. Further study demonstrated that lysozyme associated liposomes could attach to the biofilm matrix, such as alginate, which usually possessed a negative charge. Meanwhile, LLG was shown to prevent planktonic bacterial cells from biofilm formation. This strategy provided a novel platform for antibiotic delivery and might be useful to develop new therapeutics for treatment of chronic and stubborn infections related to microbial biofilm. Acknowledgments: This work was supported by the Scientific Research Fund of Northwest A&F University (Z109021636). Author Contributions: Yilin Hou and Haibo Mu designed research; Yilin Hou, Zhaojie Wang and Hu Bai performed research; Peng Zhang performed statistical analysis; Yilin Hou, Yuelin Sun and Haibo Mu wrote and reviewed the manuscript; Jinyou Duan improved the English language of the text. Conflicts of Interest: The authors declare no conflict of interest.
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Molecular Sciences Article
Lysozyme Associated Liposomal Gentamicin Inhibits Bacterial Biofilm Yilin Hou, Zhaojie Wang, Peng Zhang, Hu Bai, Yuelin Sun, Jinyou Duan and Haibo Mu * Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China; [email protected] (Y.H.); [email protected] (Z.W.); [email protected] (P.Z.); [email protected] (H.B.); [email protected] (Y.S.); [email protected] (J.D.) * Correspondence: [email protected]; Tel.: +86-29-8709-2226 Academic Editors: Antonella Piozzi and Iolanda Francolini Received: 9 February 2017; Accepted: 4 April 2017; Published: 9 April 2017
Abstract: Bacteria on living or inert surfaces usually form biofilms which make them highly resistant to antibiotics and immune clearance. Herein, we develop a simple approach to overcome the above conundrum through lysozyme-associated liposomal gentamicin (LLG). The association of lysozyme to the surface of liposomes can effectively reduce the fusion of liposomes and undesirable payload release in regular storage or physiological environments. The LLG was more effective at damaging established biofilms and inhibiting biofilm formation of pathogens including Gram-positive and Gram-negative bacteria than gentamicin alone. This strategy may provide a novel approach to treat infections due to bacterial biofilm. Keywords: liposome; lysozyme; gentamicin; biofilm
1. Introduction Biofilms are matrix-enclosed, complex and differentiated communities of bacteria that are adherent to inert or biological surfaces [1]. Upon adhesion, the bacterial cells start producing an extracellular matrix and group together in densely-packed bacterial clusters. From the mature biofilm, individual cells or biofilm fragments are released and can colonize new surfaces [2]. Biofilm formation causes corrosion and biofouling of industrial equipment [3], and is also associated with many illnesses and infections in humans, including oral diseases, native valve endocarditis, and a number of nosocomial infections [2]. Unlike planktonic bacteria, biofilm bacteria have been known to be highly resistant to adverse environmental conditions such as antibiotics, detergents or biocides [4]. Therefore, new efforts for biofilm growth inhibition, biofilm damage, or biofilm eradication are being sought. The biggest challenge in treating biofilm infections is overcoming the resistance and tolerance to antimicrobial agents. Successful therapy requires innovative ways to deliver antimicrobial substances in a sufficiently high concentration to the biofilm bacteria [5]. Liposome is an attractive candidate for drug delivery to biofilms due to its versatility and biocompatibility. It has been shown that liposome-encapsulation improves the efficacy of various antibacterial and antifungal drugs against a broad range of pathogens in vitro and in vivo [6–9]. One advantage of liposomes being used as drug delivery vehicles is their potential to fuse with phospholipid membranes. However, the applications of liposomes, particularly those with sizes below 100 nm, are often hindered by their poor stability due to spontaneous fusion, resulting in payload loss and increase in vesicle size [9,10]. Several strategies have been employed to overcome this problem including optimizing liposome composition [11], polyethylene glycol (PEG)ylation [12] and nanoparticulate stabilizing [13–15]. Herein, we introduce a novel liposome formulation stabilized by lysozyme. The cationic lysozyme is ready to associate with the negatively charged liposome through electrostatic attraction. This strategy Int. J. Mol. Sci. 2017, 18, 784; doi:10.3390/ijms18040784
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Herein, we introduce aa novel novel liposome liposome formulation formulation stabilized stabilized by by lysozyme. lysozyme. The The cationic cationic 2 of 8 lysozyme is ready to associate with the negatively charged liposome through electrostatic attraction. lysozyme is ready to associate with the negatively charged liposome through electrostatic attraction. This strategy strategy stabilizes stabilizes liposomes liposomes against against fusion fusion and and avoids avoids undesirable undesirable leakage leakage of of liposomal liposomal drugs. drugs. This stabilizes liposomes against fusion and avoids undesirable leakage of liposomal drugs. The stabilized The stabilized liposomes with positive charge are hypothesized to easily bind to the negatively The stabilized liposomes with positive charge are hypothesized to easily bind to the negatively liposomes with of positive charge are hypothesized to easily bind to the negatively charged matrix charged matrix matrix of bacterial bacterial biofilm, inhibit bacterial bacterial biofilm formation and damage damage established charged biofilm, inhibit biofilm formation and established of bacterial inhibit bacterial biofilm formation and damage established biofilm built by biofilm builtbiofilm, by Gram-positive Gram-positive or -negative -negative organisms. biofilm built by or organisms. Gram-positive or -negative organisms. 2. Results Results and and Discussions Discussions 2. 2. Results and Discussions 2.1. Preparation and and Characterization of of Lysozyme-Associated Liposomal Liposomal Gentamicin 2.1. 2.1. Preparation Preparation and Characterization Characterization of Lysozyme-Associated Lysozyme-Associated Liposomal Gentamicin Gentamicin Liposomal gentamicin (LG) was prepared by vesicle extrusion technique (Figure (Figure 1a) 1a) [16]. [16]. The The Liposomal by by vesicle extrusion technique Liposomal gentamicin gentamicin(LG) (LG)was wasprepared prepared vesicle extrusion technique (Figure 1a) [16]. size and surface zeta potential of LG were 99 nm and −54.5 mV, respectively (Figure 1b,c). Lysozymesize zeta potential of LG were 99 were nm and mV,− respectively (Figure 1b,c). LysozymeThe and sizesurface and surface zeta potential of LG 99−54.5 nm and 54.5 mV, respectively (Figure 1b,c). associated liposomal liposomal gentamicin gentamicin (LLG) (LLG) were were obtained obtained by by mixing mixing LG LG and and lysozyme. lysozyme. The The size size and associated Lysozyme-associated liposomal gentamicin (LLG) were obtained by mixing LG and lysozyme. The and size surface zeta zeta potential potential of of the the resulting resulting LLG LLG were were measured measured by by dynamic dynamic light light scattering scattering (DLS). (DLS). The The surface and surface zeta potential of the resulting LLG were measured by dynamic light scattering (DLS). size of LLG was slightly larger than that of LG, suggesting the adsorption of lysozyme onto the size LLG was was slightly larger thanthan that that of LG, suggesting the adsorption of lysozyme ontoonto the The of size of LLG slightly larger of LG, suggesting the adsorption of lysozyme liposomal surface. The surface zeta potential changed from −54.5 to 17.5 mV (Figure 1c), which liposomal surface. The surface zeta potential changed from −54.5 17.5 to mV (Figure 1c), which the liposomal surface. The surface zeta potential changed from to − 54.5 17.5 mV (Figure 1c), confirmed the association of positively-charged lysozyme to the negatively-charged liposomes confirmed the association of positively-charged lysozyme to the negatively-charged liposomes which confirmed the association of positively-charged lysozyme to the negatively-charged liposomes through electrostatic attraction. attraction. through through electrostatic electrostatic attraction. we 18, introduce Int. J. Herein, Mol. Sci. 2017, 784
Figure 1.1. 1.(a)(a) (a) Schematic structure of lysozyme-associated lysozyme-associated liposomal gentamycin gentamycin (LLG); (b) (b) Figure Schematic structure of lysozyme-associated liposomal liposomal gentamycin (LLG); (b) hydrodynamic Figure Schematic structure of (LLG); hydrodynamic size; and (c) surface zeta potential of liposome (without Lysozyme) and LLG. size; and (c) surface zeta potential of liposome (without Lysozyme) and LLG. hydrodynamic size; and (c) surface zeta potential of liposome (without Lysozyme) and LLG.
2.2. Stability Stability of of Lysozyme-Associated Lysozyme-Associated Liposomal Liposomal Gentamicin Gentamicin 2.2. Stability of Lysozyme-Associated 2.2. The stability stability of of LG LG and and LLG LLG were were evaluated evaluated over over time time in in deionized deionized water water (Figure (Figure 2a). 2a). LG LG was was The stability of LG and LLG evaluated over time deionized water (Figure LG was The gradually aggregated while LLG was relatively stable in water. Gentamicin in LG was released more gradually aggregated aggregated while while LLG LLG was was relatively relatively stable stable in in water. water. Gentamicin Gentamicin in in LG LG was was released released more more gradually quickly than in LLG (Figure 2b). These data suggested that the association of lysozyme improved the quickly than in LLG LLG (Figure (Figure 2b). These These data data suggested suggested that that the the association association of of lysozyme lysozyme improved improved the the quickly stability of liposome and prevented the release of gentamicin. stability of of liposome liposome and prevented the release of gentamicin. stability
◦
Figure 2. (a) Size measurements of liposome and LLG over the course of 48 at 25 C in deionized Figure 2. 2. (a) (a) Size Size measurements measurements of of liposome liposome and and LLG LLG over over the the course course of of 48 48 hhh at at 25 25 °C °C in deionized deionized Figure in water; (b) cumulative release profile of gentamicin-loaded liposome and LLG over the course of of 72 72 h at water; (b) (b) cumulative cumulative release release profile profile of of gentamicin-loaded gentamicin-loaded liposome liposome and and LLG LLG over over the the course course water; of 72 hh ◦ C in deionized 25 water. at 25 °C in deionized water. at 25 °C in deionized water.
2.3. Antibiofilm Activities of Lysozyme-Associated Liposomal Gentamicin 2.3. Antibiofilm Antibiofilm Activities Activities of of Lysozyme-Associated Lysozyme-Associated Liposomal Liposomal Gentamicin Gentamicin 2.3. Pseudomonas aeruginosa human pathogen, which is Pseudomonasaeruginosa aeruginosa(P. (P.aeruginosa) aeruginosa)isis isaaaGram-negative Gram-negativeopportunistic opportunistic human pathogen, which Pseudomonas (P. aeruginosa) Gram-negative opportunistic human pathogen, which known for causing chronic pulmonary infections in cysticinfibrosis (CF) patients and patientsand suffering of is known for causing chronic pulmonary infections cystic fibrosis (CF) patients patients is known for causing chronic pulmonary infections in cystic fibrosis (CF) patients and patients non-CF bronchiectasis, and generally employed as a model organism for investigation of biofilms [17].
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biofilms [17]. Gentamicin or lysozyme alone had a mild effect on biomass and live cells of P. aeruginosa suffering of non-CF bronchiectasis, and generally employed as a model organism for investigation of biofilms after 24 h treatment compared to blank control (Figure 3a,b). LLG treatment markedly biofilms [17]. Gentamicin or lysozyme alone had a mild effect on biomass and live cells of P. aeruginosa Gentamicin or biofilm lysozyme alone a mild on To biomass and live cells of P. aeruginosa biofilms after reduced both mass andhad viable celleffect counts. see whether LLG was able to eliminate bacterial biofilms after 24 h treatment compared to blank control (Figure 3a,b). LLG treatment markedly 24 h treatment compared to blank control (Figure 3a,b). LLG treatment markedly reduced both biofilm biofilms built by a Gram-positive organism, Staphylococcus aureus (S. aureus), which can cause lifereduced both biofilm mass and viable cell counts. To see whether LLG was able to eliminate bacterial mass and viable cell counts. To see and whether LLG was able to eliminate bacterial biofilms by threatening infections in humans the nosocomial (hospital) environment [18], wasbuilt tested. biofilms built by a Gram-positive organism, Staphylococcus aureus (S. aureus), which can cause lifeaQuantification Gram-positiveoforganism, Staphylococcus aureus (S. aureus), which can cause life-threatening infections biofilm biomass and cell viability demonstrated that LLG had a more pronounced threatening infections in humans and the nosocomial (hospital) environment [18], was tested. in humans and the nosocomial (hospital) environment effect than gentamicin or lysozyme alone (Figure 3c,d). [18], was tested. Quantification of biofilm Quantification of biofilm biomass and cell viability demonstrated that LLG had a more pronounced biomass and cell viability demonstrated that LLG had a more pronounced effect than gentamicin or effect than gentamicin or lysozyme alone (Figure 3c,d). lysozyme alone (Figure 3c,d).
Figure 3. Crystal violet assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess the antibiofilm activity of LLG against P. aeruginosa biofilm (a,b) and S. aureus assay andand 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Figure 3.3.Crystal Crystalviolet violet assay 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (c,d). assay toassay assess antibiofilm activityactivity of LLG of against P. aeruginosa biofilm (a,b) and(a,b) S. aureus (MTT) to the assess the antibiofilm LLG against P. aeruginosa biofilm and S.(c,d). aureus (c,d).
Fluorescence microscopy imaging of P. aeruginosa (Figure 4a) and S. aureus (Figure 4b) biofilms Fluorescence microscopy imaging of P. aeruginosa (Figure 4a) and S. aureus (Figure 4b) biofilms was pursued to further evaluate the antibiofilm potential of LLG. The blank control biofilms were Fluorescence microscopy imaging of P. aeruginosa (Figure 4a) and aureus (Figure 4b) biofilms was pursued to further evaluate the antibiofilm potential of LLG. TheS.blank control biofilms were densely colonized with hierarchically and three-dimensionally structured formations. Biofilms was pursued to further evaluate the and antibiofilm potential of LLG. The blank controlBiofilms biofilmstreated were densely colonized with hierarchically three-dimensionally structured formations. treated with LLG exhibited only a few isolated bacterial colonies instead of a recognizable biofilm densely and three-dimensionally formations. Biofilms with LLGcolonized exhibited with only ahierarchically few isolated bacterial colonies instead of structured a recognizable biofilm structure. structure. treated with LLG exhibited only a few isolated bacterial colonies instead of a recognizable biofilm structure.
Figure Figure 4. 4. Fluorescence Fluorescence microscopy microscopy of of P. P. aeruginosa aeruginosa (a) (a) and andS. S.aureus aureus(b) (b)biofilm. biofilm. Biofilms Biofilms incubated incubated with withtryptic trypticsoy soybroth broth(TSB) (TSB)are areused usedas ascontrol. control.Scale Scalebars barswere were10 10µm. μm. Figure 4. Fluorescence microscopy of P. aeruginosa (a) and S. aureus (b) biofilm. Biofilms incubated with tryptic soy broth (TSB) are used as control. Scale bars were 10 μm.
Scanning electron microscopy (SEM) was also applied to evaluate the surface morphology changes of P. aeruginosa (rod shaped pathogen, Figure 5a) and S. aureus (round-shaped pathogen, Figure 5b)
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Scanning electron microscopy (SEM) was also applied to evaluate the surface morphology 4 of 8 was Figure also applied toS.evaluate the surface morphology changes of P. aeruginosamicroscopy (rod shaped(SEM) pathogen, 5a) and aureus (round-shaped pathogen, changes5b) ofbiofilms P. aeruginosa (rod shaped pathogen, Figure 5a) and aureus (round-shaped pathogen, Figure treated with LLG, gentamicin or lysozyme in S. tryptic soy broth (TSB). The control Figure 5b) biofilms treated with LLG, gentamicin or lysozyme in tryptic soy broth (TSB). The control showed a highly organized and well-defined architecture. In LLG-treated biofilms, the cell walls biofilms treated with LLG, gentamicin or lysozyme in tryptic soy broth (TSB). The control showed showed a highly organized and well-defined architecture. In LLG-treated biofilms, the cell became and damaged, the shape and sizeIn ofLLG-treated cells changed dramatically, onlybecame awalls few a highlywrinkled organized and well-defined architecture. biofilms, the celland walls became wrinkled and damaged, the shape and size of cells changed dramatically, and only a few scattered bacterial cells were noted. Overall, these results clearly indicated that LLG had an wrinkled and damaged, the shape and size of cells changed dramatically, and only a few scattered scattered bacterial cells were noted. Overall, these results clearly indicated that LLG had an advantage in disrupting existing biofilms Gram-negative bacterial cells were noted. Overall, theseformed resultsby clearly indicated and that -positive LLG hadorganisms. an advantage in advantage in disrupting existing biofilms formed by Gram-negative and -positive organisms. disrupting existing biofilms formed by Gram-negative and -positive organisms. Int. J. Scanning Mol. Sci. 2017, 18, 784 electron
Figure 5. Scanning electron microscopy of P. aeruginosa (a) and S. aureus (b) biofilm. Biofilms Figure 5. electron microscopy of P.ofaeruginosa (a) and(a)S. and aureusS.(b) biofilm. Figure 5. Scanning Scanning electron P. aeruginosa aureus (b) Biofilms biofilm.incubated Biofilms incubated with TSB are used asmicroscopy control. Scale bars were 1 μm. with TSB are used as control. Scale bars were 1 µm. incubated with TSB are used as control. Scale bars were 1 μm.
To explore the underlying mechanism by which LLG disrupted bacterial biofilms above, To explore by which LLG disrupted biofilms above, the underlying mechanism disrupted bacterial biofilms above, liposomal rhodamine B (LR) or lysozyme associated liposomal rhodaminebacterial B (LLR) were generated. liposomal B or lysozyme associated liposomal rhodamine BB (LLR) were generated. liposomal rhodamine rhodamine B (LR) (LR) lysozyme associated liposomal rhodamine (LLR) were generated. Compared with LR, LLR elicited a much stronger binding to S. aureus biofilm (Figure 6). This might Compared with LR, LLR elicited a much stronger binding to S. aureus This might biofilm (Figure 6). be due to the electrostatic attraction between positive lysozyme on LLR and biofilm matrix, such as be due to the electrostatic attraction between positive lysozyme on LLR and biofilm matrix, such as lysozyme alginate, which usually possesses a negative charge. alginate, which usually possesses a negative charge.
Figure 6. Binding ability of lysozyme liposome to S. aureus biofilm. Lysozyme associated liposomal Figure 6. Binding ability of lysozyme liposome to S. aureus biofilm. Lysozyme associated liposomal rhodamine B (LLR) or liposomal rhodamine B (LR) was incubated with preformed S. aureus biofilm Figure 6. Binding of lysozyme liposome to S.was aureus biofilm.with Lysozyme associated liposomal rhodamine B (LLR)ability or liposomal rhodamine B (LR) incubated preformed S. aureus biofilm for 10 min.B After incubation, the biofilm was collected and quantified for fluorescence intensity. rhodamine (LLR) or liposomal rhodamine (LR) was incubated with S. aureus biofilm for 10 min. After incubation, the biofilm was B collected and quantified forpreformed fluorescence intensity. The The10 biofilm without incubating with anywas liposome formulations was tested in parallel serving as the for min. After incubation, the biofilm collected and quantified for fluorescence intensity. biofilm without incubating with any liposome formulations was tested in parallel serving as The the background signal. biofilm without incubating with any liposome formulations was tested in parallel serving as the background signal. background signal.
Biofilm formation formationwas wasexamined examinedininthe thecase caseofofplanktonic planktonic aeruginosa (Figure 7a,b) exposed Biofilm P. P. aeruginosa (Figure 7a,b) exposed to Biofilm formation was examined in the case of planktonic P. aeruginosa (Figure 7a,b) exposed to to LLG, gentamicin lysozyme Lysozymeshowed showedno noeffects effectson on biofilm biofilm formation formation as as LLG, gentamicin or or lysozyme forfor2424h.h.Lysozyme LLG, gentamicin or lysozyme for 24 h. Lysozyme showed effects on formation as compared with blank blank control. Gentamicin suppressed biofilmno formation andbiofilm decreased live cells cells compared with control. Gentamicin suppressed biofilm formation and decreased live compared with blank control. Gentamicin suppressed biofilm formation and decreased live cells generally whereas LLG facilitated this suppression and reduction significantly. Similar findings generally whereas LLG facilitated this suppression and reduction significantly. Similar findings were generally facilitated suppression andofreduction significantly. wereobserved also whereas observed in the of this S. by aureus by quantification of biomass biofilm biomass (Figure 7c) viability andwere cell also in theLLG case ofcase S. aureus quantification biofilm (FigureSimilar 7c) andfindings cell also observed in the case of S. aureus by quantification of biofilm biomass (Figure 7c) and cell viability viability (Figure 7d). These results suggested that LLG had the potential to prevent planktonic cells of (Figure 7d). These results suggested that LLG had the potential to prevent planktonic cells of Gram(Figure 7d). These results suggested that LLG had the potential to prevent planktonic cells of GramGram-negative or -positive organisms from biofilm formation. negative or -positive organisms from biofilm formation. negative or -positive organisms from biofilm formation.
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Figure 7. The The inhibitory inhibitory effects effects of LLG on P. aeruginosa (a,b) and S. aureus Figure aureus (c,d) (c,d) biofilm biofilm formation. formation.
Controlled drug drug delivery delivery by by lipid lipid nanoparticles nanoparticles have Controlled have attracted attracted much much attention. attention. More More recently, recently, Harker et al. utilized an electrohydrodynamic technique to prepare core-shell lipid nanoparticles Harker et al. utilized an electrohydrodynamic technique to prepare core-shell lipid nanoparticles with a tunable size and highingredient active ingredient loading encapsulation capacity, encapsulation efficiency and awith tunable size and high active loading capacity, efficiency and controlled controlled release [19]. Many nano materials, for example, the metal-loaded nanofibers [20–22], have release [19]. Many nano materials, for example, the metal-loaded nanofibers [20–22], have shown high shown high antibacterial activity against or biofilm bacteria the current study, antibacterial activity against planktonic orplanktonic biofilm bacteria [23,24]. In the [23,24]. current In study, we developed we developed a platform to deliver antibiotic to treat bacterial biofilms through lysosome associated a platform to deliver antibiotic to treat bacterial biofilms through lysosome associated liposomes. liposomes. Thismade approach made liposomes easier to to biofilms; attach to abiofilms; a universal This approach liposomes more stablemore and stable easierand to attach universal survival survival for lifestyle for microbes lifestyle microbes in nature.in nature. 3. Materials and Methods 3.1. Materials 3.1. Materials 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphor1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) (DPPC) and 1,2-dipalmitoyl-sn-glycero-30 -rac-glycerol) (DPPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Gentamicin (1 phosphor-(1′-rac-glycerol) (DPPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Sulfate was Sulfate purchased Solarbio (Beijing, China). Rhodamine B was purchased from Aladdin Gentamicin wasfrom purchased from Solarbio (Beijing, China). Rhodamine B was purchased from (Shanghai, China). AllChina). reagentsAll were of analytical and usedgrade as received without further purifying. Aladdin (Shanghai, reagents were grade of analytical and used as received without Pseudomonas further purifying. aeruginosa (PAO1) and Staphylococcus aureus (ATCC 29213) were generous gifts received from Xiaodong Xia (College Food Science and aureus Engineering, A&F University). Pseudomonas aeruginosa (PAO1) of and Staphylococcus (ATCCNorthwest 29213) were generous gifts
received from Xiaodong Xia (College of Food Science and Engineering, Northwest A&F University). 3.2. Preparation and Characterization of LLG 3.2. Preparation Characterization of LLGa previously described extrusion method [16]. Briefly, 9 mg Liposomesand were prepared following of lipid (DPPC/DPPG = 9/1, molar ratio)awere dissolved in 1 mL chloroform, and[16]. thenBriefly, the organic Liposomes were prepared following previously described extrusion method 9 mg solvent was evaporated to form a dried lipid film. The lipid film was rehydrated with mL of of lipid (DPPC/DPPG = 9/1, molar ratio) were dissolved in 1 mL chloroform, and then the3organic deionized water, or 2 mM B (RhB), or 20The mM gentamicin, by with vortexing solvent was evaporated to rhodamine form a dried lipid film. lipid film was followed rehydrated 3 mLfor of 1deionized min and sonicating for 5 min to produce multilamellar vesicles (MLVs). The solution was extruded water, or 2 mM rhodamine B (RhB), or 20 mM gentamicin, followed by vortexing for 1 min through a 100 nmfor pore-sized membrane for 10 times to form narrowly distributed small and sonicating 5 min topolycarbonate produce multilamellar vesicles (MLVs). The solution was extruded unilamellar vesicles (SUVs). Particles were purified by washing withtowater times using 10 kDa through a 100 nm pore-sized polycarbonate membrane for 10 times form 3narrowly distributed MWCO Amicon centrifugal filters (EMD Millipore, Billerica, CA, USA) to remove unencapsulated small unilamellar vesicles (SUVs). Particles were purified by washing with water 3 times using 10 drugs. To prepare LLG, the pH of both lysozyme andMillipore, liposome solutions using kDa MWCO Amicon centrifugal filters (EMD Billerica, was CA,adjusted USA) to to 6.5 remove unencapsulated drugs. To prepare LLG, the pH of both lysozyme and liposome solutions was
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HCl. Then the liposomes and lysozyme at 1:100 (molar ratio) were mixed together, followed by 10 min bath sonication. The hydrodynamic size and surface zeta potential of LLG were measured by dynamic light scattering (DLS) measurements (Malvern Zetasizer NANO-ZS90, Malvern, UK). The gentamicin content was determined by sodium phosphotungstate precipitation method. 3.3. Stability Studies Stability of LLG or bare liposome was analyzed in deionized water. 2 mL freshly prepared li posome samples at 1 mg·mL−1 were incubated at 25 ◦ C for 48 h. The size change of the liposome samples in deionized water were measured by DLS. 3.4. Release Behaviors The kinetics of gentamicin release was studied from the prepared LLG. The 1 mL fresh prepared liposome solution (2 mg·mL−1 ) was initially incubated at 25 ◦ C in tube. Liposomes were taken at regular time intervals, centrifuged using 10 kDa MWCO Amicon centrifugal filters (EMD Millipore, Billerica, CA, USA) and the filtrate was obtained for gentamicin measurement. 3.5. Binding Ability of Lysozyme Liposome to Biofilm Lysozyme associated liposomal RhB (LLR) and liposomal RhB (LR) were prepared as described in Section 3.2. S. aureus (~109 colony forming units (CFU)) were grown in 6-well plates at 37 ◦ C for 24 h supplemented with 2 mL of TSB to allow biofilm formation. The non-adhered cells were removed with pipette and the plate was washed three times using 0.9% (w/v) NaCl. Then existing biofilms were incubated at 37 ◦ C in 1.8 mL TSB supplemented with 0.2 mL LLR or LR for 10 min. After that, the medium was removed and the biofilm was washed, collected using cell scraper in phosphate buffered saline (PBS), vortexed and subjected to fluorescence detection on a RF-5301 fluorescence spectrometer (Shimadzu, Kyoto, Japan) at excitation and emission wavelengths of 509 nm and 526 nm, respectively. Relative fluorescence intensity was expressed as percentage, and biofilm treated with LLG was used as 100% fluorescence intensity. 3.6. Antibiofilm Activity As described previously [25], 100 µL bacterial TSB solutions (~108 CFU) were seeded into 96-well polystyrene microtitre plates (Corning, NY, USA) at 37 ◦ C for 24 h to allow biofilm formation. The non-adhered cells were removed with pipette and the plate was washed three times using 100 µL 0.9% (w/v) NaCl. Then existing biofilms were incubated at 37 ◦ C in 90 µL TSB supplemented with 10 µL LLG, equivalent gentamicin or lysozyme for 24 h. Each treatment included 6 parallel wells. Biofilms incubated with TSB only were used as blank. Biofilm mass (crystal violet staining assay) and viable cells (MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay) were evaluated as previous [26]. All experiments were performed 3 times. Error bars represent standard deviation (SD). For biofilm inhibition assay, 100 µL of bacteria in TSB (~108 CFU) were seeded into individual wells of microtiter plates in the presence of compounds for 24 h. Biofilm mass were evaluated as described [26]. For fluorescence microscopy, S. aureus or P. aeruginosa (~108 CFU) was grown on glass coverslips at 37 ◦ C for 24 h in 24-well plates supplemented with 1 mL of TSB to allow biofilm formation. The coverslips were washed to remove unattached cells and were treated with liposomes for 24 h at 37 ◦ C. Existing biofilms were treated and imaged as previous [25]. SEM was conducted as described previously [27].
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3.7. Statistical Analysis All graphical evaluations were made using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Analysis of variance (ANOVA) was used to evaluate significant differences. 4. Conclusions In this study, we applied positively-charged lysozyme to stabilize the negatively-charged liposomes through electrostatic attraction. The lysozyme-associated liposomal gentamycin (LLG) was more effective at disrupting the preformed biofilms built by Gram-positive and -negative pathogenic bacteria than lysozyme or gentamycin. Further study demonstrated that lysozyme associated liposomes could attach to the biofilm matrix, such as alginate, which usually possessed a negative charge. Meanwhile, LLG was shown to prevent planktonic bacterial cells from biofilm formation. This strategy provided a novel platform for antibiotic delivery and might be useful to develop new therapeutics for treatment of chronic and stubborn infections related to microbial biofilm. Acknowledgments: This work was supported by the Scientific Research Fund of Northwest A&F University (Z109021636). Author Contributions: Yilin Hou and Haibo Mu designed research; Yilin Hou, Zhaojie Wang and Hu Bai performed research; Peng Zhang performed statistical analysis; Yilin Hou, Yuelin Sun and Haibo Mu wrote and reviewed the manuscript; Jinyou Duan improved the English language of the text. Conflicts of Interest: The authors declare no conflict of interest.
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