Proton-mediated burst of dual-drug loaded liposomes for biofilm dispersal and bacterial killing

bromelain and ciprofloxacin. Simultaneous release of bromelain and ciprofloxacin enhanced bacterial killing in Staphylococcus aureus biofilms as compared with free bromelain and/or ciprofloxacin. After tail-vein injection in mice, liposomes accumulated inside intra-abdominal staphylococcal biofilms. Subsequent liposome-burst and simultaneous release of bromelain and ciprofloxacin yielded degradation of the biofilm matrix by bromelain and higher bacterial killing without inducing septic symptoms as obtained by injection of free bromelain and ciprofloxacin. This shows the advantage of simultaneous release from liposomes of bromelain and ciprofloxacin inside a biofilm.


Introduction
Dispersal of infectious biofilms is frequently considered as a new approach for treating recalcitrant bacterial infections [1].A major problem associated with dispersal approaches is the sudden presence of high concentrations of bacteria dispersed from a biofilm in the blood circulation [2,3], at a rate that is higher than the immune system can deal with [2].In order to prevent septic complications resulting from the dispersal of infectious biofilm, it is imperative that that dispersed bacteria are killed immediately after their dispersal [4].Bromelain is a pineapple extract composed of a variety of proteolytic enzymes, phosphatases, glucosidases, peroxidases, cellulases and glycoproteins [5][6][7] and has recently been demonstrated [8] to enhance antibiotic efficacy through degradation of biofilm matrices yielding dispersal of biofilm bacteria.Clinically however, effective, simultaneous delivery of bromelain into an infectious biofilm together with an antibiotic is extremely difficult, due to the short half-life time of bromelain in blood [9,10].
Nanocarriers like nano-capsules [11,12], micelles [13,14] and liposomes [15,16] are frequently considered nowadays for encapsulation and delivery of enzymes and antibiotics.Release of therapeutic cargo from nanocarriers from the blood circulation must preferentially be confined to a target site, such as inside an infectious biofilm or tumor.Controlled cargo release can be achieved through "internal" stimuli such as pH, hypoxia or temperature or alternatively through "external" stimuli like light irradiation or ultrasound application [17].Unlike micelles that possess a hydrophobic core, liposomes can be loaded with a hydrophobic (in-membrane) and hydrophilic (in-core) cargo.Moreover, several liposome-based medicines have been approved by the FDA [18].However, confining cargo release from liposomes to a target site requires conjugation of stimuli-responsive functionalities with membrane lipids that may disrupt the physico-chemical balance in the lipid membrane of liposomes [17,19].Recently, we synthesized a lipid named DCPA (2-(4-((1,5-bis(octadecenoyl)1,5-dioxopentan-2-yl)carbamoyl) pyridin-1-ium-1-yl)acetate) that complexes with water through hydrogen bonding (DCPA-H 2 O) [20].Water molecules in DCPA-H 2 O liposomes provide a stealth functionality that allows long-time blood circulation, while becoming protonated below pH 6.8 allowing fast selftargeting to infectious biofilms after tail-vein injection in mice.
Based on the hypothesis that simultaneous release of a dispersant and an antibiotic inside an infectious biofilm can prevent septic symptoms, the aim of this paper was to investigate whether self-targeting DCPA-H 2 O liposomes can be dual-loaded with a dispersant and an antibiotic for simultaneous release inside an infectious biofilm.To this end, for the first time, we explored the possibility of in-membrane and in-core, dualloading of self-targeting DCPA-H 2 O liposomal nanocarriers for the controlled and simultaneous release of bromelain and a clinically applied, broad-spectrum antibiotic (ciprofloxacin) inside a biofilm.DCPA-H 2 O liposomes were core-loaded with bromelain.Ciprofloxacin was made amphiphilic by PEGylation so that PEGylated ciprofloxacin could become integrated as a membrane component in the lipid membrane of the DCPA-H 2 O liposomes (see Scheme 1a).Controlled release was hypothesized to be proton-mediated in the acidic environment of an infectious biofilm, purposely disrupting the physico-chemical balance in the lipid membrane of the liposomes to yield their burst and therewith cargo release (see Scheme 1b).

Bacterial strain, culturing and harvesting
Green-fluorescent Staphylococcus aureus ATCC12600 GFP was cultured, as described before [21].Briefly, S. aureus ATCC12600 GFP was cultured from a frozen stock onto tryptone soy agar (TSB) plates supplemented with 10 μg/mL tetracycline at 37 • C, For experiments, one colony was transferred to inoculate 10 mL of TSB, also supplemented with 10 μg/mL tetracycline for S. aureus ATCC12600 GFP at 37  h.This pre-culture was diluted 1:20 in 100 mL TSB and grown statically for 12 h at 37 • C. Cultures were harvested by centrifugation for 5 min at 5000g at 4 • C, washed three times with fresh phosphate buffered saline (PBS, 1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 137 mM NaCl and 2.7 mM KCl), sonicated for 3 × 10 s (Vibra cell model 375, Sonics and Material Inc., Danbury, CT) while cooling in an ice/water bath to break bacterial aggregates.Finally, suspensions were diluted in PBS to concentrations required in the respective experiments, as determined OD 600 using Microdrop.

Animals
Seven to eight weeks-old, healthy female BALB/c nude mice were purchased from SLAC Laboratory Animal Co., Ltd.(Shanghai, China).Upon purchase, the average weight of the mice amounted 16.4 ± 0.1 g.Male Spraque Dawley rats were purchased from Vital River Laboratories (Beijing, China).Rats weighed 287 ± 6 g upon purchase and were randomly divided into four groups of three animals.All animals were housed in the on-site animal facility of Nankai University and experimental procedures were approved by the Institutional Animal Care and User Committee of Nankai University, Tianjin, China (approval number: 2021-SYDMLL-000454).

Lipid synthesis
DCPA was prepared as previously described [20] and the chemical structure was identified by 1 H NMR. PEG 2K -ciprofloxacin was prepared according to the following procedure.Briefly, ciprofloxacin (1.0 g, 3 mmol) was dissolved in 20 mL dioxane:water (1:1).Then, 4.5 mL 1.0 M NaOH and di-tertbutylcarbonate (1.0 g, 4.5 mmol) was added and the mixture was stirred at ambient temperature for 2 h.Thin layer chromatography was applied to monitor whether the reaction was completed.Acetone (20 mL) was added to precipitate the ciprofloxacin-Boc and the solid product was filtered and washed with acetone.The solid was dried under vacuum to obtain 1.21 g ciprofloxacin-Boc as white powder (93% yield).
PEG 2K -OH (2.78 g, 1.38 mmol) was added to a mixture of ciprofloxacin-Boc (200 mg, 0.46 mmol), HBTU (439.9 mg, 1.16 mmol) and DMAP (10.91 mg, 0.09 mmol) in dry DCM (50 mL) under N 2 protection.After stirring at ambient temperature for 10 h, the solution was washed twice with saturated NaCl solution (50 mL) and the resulting organic layer was collected, dried with anhydrous Na 2 SO 4 and Na 2 SO 4 was removed by filtration.Next, the solution was distilled under vacuum to remove DCM and obtain crude PEG 2K -ciprofloxacin-Boc.PEG 2K -ciprofloxacin-Boc was purified by column chromatography with silica gel and a gradient of DCM:MeOH (50:1) yielding 860.7 mg white powder (81% yield).
Removal of the Boc-protector from PEG 2K -ciprofloxacin-Boc (500 mg, 0.21 mmol) was performed by dropwise addition of 5 mL TFA in 5 mL DCM solution under N 2 protection at 0 • C to obtain PEG 2K -ciprofloxacin trifluoroacetate.The mixture was stirred for 30 min at room temperature.Rotary evaporation under reduced pressure yielded a white yellow solid film and 5 mL DMSO was added to dissolve the solid film.Subsequently, the solution was transferred to a dialysis bag (molecular weight cutoff (MWCO) 1 kDa) and dialyzed against deionized water (pH 7.4, adjusting with 1 M NaOH) for 24 h to remove DMSO and trifluoroacetate.Finally, PEG 2K -ciprofloxacin, as light-yellow oil (418 mg, yield 86%), was obtained by lyophilization.

Self-assembly of liposomes, bromelain and additional ciprofloxacin loading
For the synthesis of bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes (B-PEG-cipro/DCPA-H 2 O), DCPA (10 mg) and PEG 2K -ciprofloxacin (1 mg) were dissolved in 1 mL THF.This facilitated self-assembly of liposomes with amphiphilic PEGylated ciprofloxacin as an in-membrane component.For in-core loading of bromelain, this solution was injected in 10 mg bromelain dissolved in 10 mL deionized water under vigorous stirring with a magnetic bar (600 rpm) in a water bath kept at 50 • C.After 5 min, the solution was transferred to a dialysis bag (MWCO 50 kDa) and dialyzed against deionized water for 24 h.Deionized water was refreshed 3 times per day to remove organic solvent.For additional, inmembrane loading of ciprofloxacin, bromelain-loaded B/C-PEG-cipro/ DCPA-H 2 O liposomes, DCPA (10 mg), ciprofloxacin (1 mg) and PEG 2Kciprofloxacin (1 mg) were dissolved in 1 mL organic solvent (THF: DMSO = 4:1) at 50 • C, similar as described for B-PEG-cipro/DCPA-H 2 O.After dialysis, the resulting liposome suspension was filtered using a syringe with 0.45 μm filtration membrane (Merck, America) in order to remove liposomes >0.45 μm.Finally, the liposome suspension was concentrated by low-speed ultrafiltration (15 min, ×1247 g) using a filter with a nominal molecular weight limit of 10 kDa (Merck Millipore, Massachusetts, USA).Liposome concentrations were determined by measuring the weight of liposome powder obtained by lyophilizing 200 μL of a liposome suspension.Before each experiment, the liposome suspension was filter-sterilized (MILLEX®GP 0.22 μm).

Bromelain and ciprofloxacin loading efficiencies
For the measurement of Rh-bromelain and in-membrane ciprofloxacin loading efficiencies, 1 mL of a liposome suspension in PBS (5.0 mg/ mL, pH 7.4) was transferred into a dialysis bag (MWCO 10 kDa) and subsequently immersed in 20 mL of PBS (pH 5.0) at 37 • C.After gently shaking 24 h, an aliquot (1 mL) of the dialysis solution was taken and fluorescence emission spectroscopy (F-700, Hitachi, Tokyo, Japan) was applied to measure the bromelain concentration.For calibration, redfluorescence intensity was measured between 560 and 650 nm (excitation wavelength 545 nm) as a function of the concentration of Rhbromelain in PBS (pH 5.0).UV-vis absorption spectroscopy (Shimadzu, Shanghai, China) was applied to determine the in-membrane ciprofloxacin loading efficiency.To this end, a calibration curve was prepared of the UV-vis absorption at 277 nm as a function of ciprofloxacin concentration.Loading efficiencies were subsequently calculated according to

LE% weight of loaded cargo total weight of lipid and loaded cargo
× 100% (1)

Release of bromelain and ciprofloxacin
Bromelain and additional, in-membrane ciprofloxacin release was essentially measured as above described using a dialysis bag, but as a function of time up to 24 h and at pH 7.4, 6.5 and 5.0, using fluorescence emission spectroscopy and UV-vis absorption.Possible release of PEG 2Kciprofloxacin as a liposome membrane component during dialysis was done by comparing the 1 H NMR spectra of freeze-dried PEG-cipro/ DCPA-H 2 O liposomes before and after dialysis (see above).Dialysis solutions were freeze-dried, after which freeze-dried powders were dissolved in CDCl 3 for 1 H NMR spectroscopy.The ratio of peak area at 4.12 ppm of DCPA and PEGylated ciprofloxacin was used to determine the amount of PEG 2K -ciprofloxacin released as a membrane component during dialysis.

Characterization of liposomes
Hydrodynamic diameters and polymer dispersity indices (PDIs) of all liposomes (0.05 mg/mL) dispersed in PBS were measured by dynamic light scattering using a Malvern Zetasizer Nano-ZS90 apparatus (25 • C).Zeta potentials of liposomes (0.05 mg/mL) dispersed in PBS with different pH were also measured using a Zetasizer (Malvern Nano-ZS90).Measurements were carried out in triplicate with separately prepared liposome suspensions.
Transmission Electron Microscopic (TEM) samples were prepared on 200 mesh carbon film supported copper grids.One drop of a liposome solution (5 μL, 0.1 mg/mL) was placed on the grid and allowed to sediment for 60 s.Filter paper was then used to remove the residual solution.The copper grids were treated with phosphotungstic acid for 30 s and dried in vacuum at 37 • C for 48 h.The resulting samples were imaged using Talos TM F200C TEM at 200 kV.

Dispersal of bacterial biofilm in vitro
1 mL S. aureus ATCC 12600 GFP in suspension (10 8 bacteria/mL) in PBS was added to a confocal culture dish at 37 • C for 1 h to allow bacteria to sediment and adhere to the well surface.Subsequently, the bacterial suspension was removed, the well was washed 2 times with 1 mL of PBS to remove non-adhering bacteria and 1 mL TSB medium was added to the dishes.Culture dishes were incubated for 48 h at 37 • C and medium was refreshed after 24 h.After 48 h, biofilms were rinsed three times with PBS to remove non-adhering bacteria and exposed to PBS, ciprofloxacin at its minimal bactericidal concentration (10 μg/mL, see Table S1) in PBS, bromelain (10 μg/mL) in PBS, suspensions of B-PEGcipro/DCPA-H 2 O (loaded bromelain 10 μg/mL) or B/C-PEG-cipro/ DCPA-H 2 O (loaded 10 μg/mL ciprofloxacin and10 μg/mL bromelain) for 1 h.After exposure, the liquid was removed and the biofilm was washed twice with fresh PBS.Subsequently, these biofilms were stained with red-fluorescent concanavalin A for 30 min in the dark to visualize biofilm matrix, the biofilms were washed two times with fresh PBS before imaging using a laser scanning confocal microscopy (CLSM; TCS SP8, Leica, Wetzlar, Germany).An argon ion laser (488 nm) and a green He/ Ne laser (561 nm) were used to excite the GFP and rhodamine labeled concanavalin A, respectively.Fluorescence was collected at 500-535 nm (GFP) and 580-610 nm (rhodamine).All data were acquired and analyzed using Leica software, version 2.0 to yield biofilm thickness prior to and after exposure to liposomes as well as relative biomass.
For scanning electron microscopic (SEM) analysis, biofilms were cultured on glass slides in 24-well plates, as described above.After exposure to PBS or bromelain (10 μg/mL), ciprofloxacin (10 μg/mL) or B/C-PEG-cipro/DCPA-H 2 O (loaded 10 μg/mL ciprofloxacin and 10 μg/ mL bromelain) for 1 h, the biofilms were washed twice carefully with PBS and fixed with 2.5% glutaraldehyde for 2 h at 4 • C.After fixation, samples were dehydrated with a series of graded ethanol solutions.Finally, samples were gold-coated by sputtering and imaged using Quanta 200 SEM (FEI, Hillsboro, OR, USA).

Self-targeting and accumulation of liposomes in an infectious biofilm underneath an abdominal imaging window in mice
Self-targeting of liposomes to an infectious biofilm was studied as done before [20].Briefly, mice were anesthetized by intraperitoneal injection of 0.3 wt% pentobarbital sodium (40 mg/kg).Then, the right flanks of the mice were disinfected with 75% (v/v) ethanol and a lateral incision was made through the skin and the abdominal wall and a suture was sewed along the edge of the wound.A sterilized abdominal imaging window, consisting of a circular glass coverslip (12 mm diameter; Thermo Scientific, Waltham, Massachusetts, USA) in a titanium frame, was placed glass side up in the incision.The skin and the abdominal wall were placed in a slot, prepared in the side of the titanium frame.Finally, sutures were tightened to secure the window-frame firmly in the animal.After the surgery, the mice were kept at 37 • C until fully recovered.After full recovery, usually requiring 2 days, mice were anesthetized and injected with 200 μL green fluorescent S. aureus ATCC12600 GFP (10 9   bacteria/mL) underneath the abdominal imaging window to grow a biofilm.After 48 h of biofilm growth, mice were tail-vein injected with (100 μL) Rh-bromelain (0.25 mg/mL) and Rh-bromelain loaded liposome suspensions containing an equivalent Rh-bromelain concentration.After injection, when the images were taken, mice were anesthetized and intravital images were taken in a Caliper IVIS Lumina II (NightOWLIILB983, Berthold Technologies, Germany) as a function of time up to 24 h.All images were acquired with the following parameters: exposure time 0.5 s, emission wavelength 520 nm, excitation wavelength 480 nm.In vivo self-targeting of each group was monitored in three mice.The acquired images were analyzed with Living Image 4.5.1 software (IndiGO, version 2.0.5.0).

In vivo eradication of a staphylococcal infection underneath an abdominal imaging window in mice
After 48 h of biofilm growth underneath the window, treatment consisted of tail-vein injection every other day starting on day 0 with 100 μL of PBS, bromelain (200 μg/mL) in PBS, bromelain and ciprofloxacin in PBS (each 200 μg/mL), 100 μL B/C-PEG-cipro/DCPA-H 2 O liposomes (200 μg/mL ciprofloxacin and bromelain).Injection was carried out at day 0, 2, and 4, and intravital images were acquired each on day 0, 1, 3 and 5 by two-photon laser scanning confocal microscope (Leica, TCS SP8 STED 3×, Germany).After sacrifice at day 5, 1 g of tissue surrounding the wound was excised, and homogenized in PBS.The homogenate was serially diluted and plated on TSB agar plates.After incubation for 24 h at 37 • C, number of colonies forming units (CFUs) were enumerated.During the course of the experiment, activity of the mice was monitored and body weight and temperature measured.

Statistical analysis
All data shown represent means ± standard deviations (SD) and were examined for statistically significant differences using a two-tailed Student's t-test (GraphPad Prism 8).
Amphiphilic PEG 2K -ciprofloxacin was prepared as described in Scheme S1b. 1 H NMR confirmed the chemical structure of intermediate reaction compounds (Fig. S2 and Fig. S3) as well as of PEG 2K -ciprofloxacin (Fig. S4).

Self-assembly of liposomes and their characterization
Self-assembly of DCPA and amphiphilic PEG 2K -ciprofloxacin into liposomes is new and required optimization of the concentration ratio of PEG 2K -ciprofloxacin over DCPA.First, DCPA and PEG 2K -ciprofloxacin were mixed in different weight ratios in 1 mL tetrahydrofuran (THF) in absence of bromelain.Then, the solvent was injected into 10 mL bromelain solution in deionized water, yielding in-core loading of bromelain in PEG-cipro/DCPA-H 2 O liposomes.When the ratio of PEGylated ciprofloxacin over DCPA exceeded 0.1, the hydrodynamic diameter and polydispersity indices of the liposomes formed increased rapidly (Fig. 1a) to values considered too high for the prevention of reticulo-endothelial rejection and biofilm penetration [22].Moreover, at higher ratios liposomes became unstable during storage (Fig. 1b).
Therefore, in the remainder of this work, liposomes were formed by mixing 10 mg DCPA and 1 mg PEG 2K -ciprofloxacin in 1 mL THF. 10 mL bromelain in deionized water (1 mg/mL) was added to the mixture to yield bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes.To determine the loading efficiency, bromelain was Rhodamine B labeled (see Fig. S5a for 1 H NMR spectra).Fluorescence emission spectroscopy was subsequently applied (see Fig. S5b for spectra), after preparation of a calibration curve (see Fig. S5c) to yield the loading efficiency of bromelain, amounting 12 ± 2%.Since amphiphilic PEG 2K -ciprofloxacin is only weakly antibacterial as compared with hydrophobic ciprofloxacin (Table S1), the above described self-assembly procedure was also carried out in presence of ciprofloxacin (1 mg) to facilitate in-membrane loading of ciprofloxacin in addition to in-membrane PEG 2K -ciprofloxacin in bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes.Liposomes could be additionally in-membrane loaded with ciprofloxacin with a loading efficiency of 10 ± 1%, as determined using UV-vis absorption spectroscopy (see Fig. S6).Note that during dialysis, a minor loss (6.8%) of PEG 2K -ciprofloxacin as a membrane component occurred (Fig. S7).
Average diameters of bromelain-loaded DCPA-H 2 O, bromelainloaded PEG-cipro/ DCPA-H 2 O and bromelain-loaded PEG-cipro/DCPA-H 2 O with additional ciprofloxacin-loading were all hoovering between 65 and 69 nm, with a PDI of around 0.2 (Fig. 2a).All liposomes demonstrated a spherical morphology (Fig. 2b).The pH-responsiveness of bromelain-loaded DCPA-H 2 O, bromelain-loaded PEG-cipro/DCPA-H 2 O and bromelain-loaded PEG-cipro/DCPA-H 2 O with additional ciprofloxacin-loading was virtually identical, with zeta potentials becoming positive for pH values below 7.0 (Fig. 2c).A negative zeta potential at physiological pH is of major importance for allowing long circulation in blood [23,24], while a reversal to a positive zeta potential is pivotal for self-targeting to acidic tumor or infection sites [20,25].

In vivo blood circulation times of bromelain-loaded PEG-cipro/ DCPA-H 2 O liposomes and self-targeting of an abdominal biofilm
Next, it was evaluated whether blood circulation times of bromelain had indeed benefited from en-capsulation in PEG-cipro/DCPA-H 2 O liposomes by measuring bromelain concentrations in the blood of rats after tail-vein injection.Bromelain freely dissolved in blood was cleared from the blood circulation within 6 h, which is considerably faster than when loaded in any of the liposomes involved in this study (Fig. 3a).Accordingly, the half-life time of free bromelain in blood increased from  around 1 h to between 33 and 35 h upon transport within liposomes (Fig. 3b), which is significantly longer than freely dissolved bromelain in blood or orally administered bromelain in human intestines, that has been described to remain biologically active with a half-life of 6-9 h [9].
Self-targeting to a green-fluorescent staphylococcal biofilm of bromelain and bromelain-loaded liposomes was studied in mice, using an abdominal window model [20] (see Fig. 4a for experimental scheme).The abdominal window uniquely allows for real-time observation of a fluorescent biofilm and its targeting by fluorescent nanocarriers [26][27][28].Quantitative analyses of intra-abdominal images of a S. aureus biofilm underneath an abdominal window in mice after tail-vein injection of bromelain or liposomes (Fig. 4b), demonstrated minor, shortlived accumulation of Rhodamine-labeled bromelain in the biofilm.However, protected in self-targeting liposomes, bromelain accumulation in biofilms continued to increase up to 6 h (Fig. 4c).Notably, the maximum accumulation of bromelain loaded in self-targeting liposomes was 5 times higher 24 h after tail-vein injection than when injected freely dissolved in PBS (Fig. 4d).

Proton-mediated, controlled release of bromelain and ciprofloxacin from PEG-cipro/DCPA-H 2 O liposomes
At pH 7.4, bromelain and in-membrane ciprofloxacin release was similarly small for all types of liposomes included (Fig. 5a and Fig. 5b, respectively).Both bromelain (Fig. 5a) and ciprofloxacin (Fig. 5b) release remained small at more acidic pH down to at least pH 5.0 when released from bromelain-loaded DCPA-H 2 O liposomes.However, when released from PEG-cipro/DCPA-H 2 O liposomes, bromelain release increased to almost 100% of the initial loading within 24 h.Concurrent with increasing cargo release, the diameter of PEG-cipro/DCPA-H 2 O liposomes increased with decreasing pH (Fig. 5c), ultimately leading to a burst and full disintegration of the liposomes (Fig. 5d).Bromelainloaded DCPA-H 2 O liposomes remained fully intact.This confirms our hypothesis that PEGylated ciprofloxacin in the liposomal membrane acts as a proton-sponge and becomes positively charged at low pH, disrupting the charge balance in the membrane and causing burst of the liposomes (see Scheme 1b).This method of creating a burst of the lipid membrane of liposomes yielded simultaneous release of both inmembrane and in-core cargo.Moreover, this proton-mediated cargo release was faster than can be achieved using both pH-responsive liposomes or pH-responsive micelles that typically possess conjugated pHresponsive functionalities, reacting through slower ring opening (topotecan hydrochloride) [29], triggering of phase transitions (methacryloyl sulfadimethoxines) [30], or lipid breaking switch (orthoester [31], vinyl ester [32], hydrazine [33] and Schiff bases [34]. Circular dichroism (CD) spectra of bromelain in PBS and bromelain released from bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes (Fig. 5e), both demonstrated two negative bands at 208 and 222 nm, characteristic for α-helix conformation of bromelain [35].This demonstrates that loading of and release of bromelain did not affect its secondary structure.Accordingly, the enzymatic ability of bromelain was not affected and its ability to degrade gelatin was similar after loading and release as compared with free bromelain (Fig. S9).This is a unique advantage of maintaining the enzymatic activity of bromelain by liposomal encapsulation that could not be achieved by protection of bromelain through chemical modification, yielding 20 to 200 fold lower degradation of gelatin [10,36] than observed here.

Dispersal of staphylococcal biofilms by bromelain-loaded PEG-cipro/ DCPA-H 2 O liposomes
Dispersal of green-fluorescent S. aureus biofilms was evaluated by exposing staphylococcal biofilms to PBS, bromelain dissolved in PBS with and without ciprofloxacin and in bromelain-loaded PEG-cipro/ DCPA-H 2 O liposomes with additional ciprofloxacin-loading.After exposure, biofilms were stained with red-fluorescent concanavalin A in order to visualize the EPS matrix.Confocal laser scanning microscopic (CLSM) images (Fig. S10) demonstrated that prior to exposure (i.e.exposure to PBS), staphylococcal biofilms had a thickness of around 35 μm (Fig. 6a) that was not affected by exposure to ciprofloxacin.Exposure to bromelain with or without ciprofloxacin yielded a decrease in biofilm thickness to around 23 μm.However, bromelain-loaded PEG-cipro/ DCPA-H 2 O liposomes with additional ciprofloxacin-loading yielded the largest dispersal of staphylococcal biofilm, reducing their thickness to around 12 μm.Interestingly, biofilm exposure to ciprofloxacin in solution did not yield a reduction in the number of staphylococcal CFUs, while the reduction in biofilm thickness upon exposure to bromelain in solution was not accompanied by a reduction in the number of CFUs (Fig. 6b).These observations confirm that bromelain is not a bactericidal compound (see also Table S1), and furthermore illustrate that the biofilm mode of growth protects its inhabitants against antibiotic exposure [37,38].However, combined exposure to bromelain and ciprofloxacin yielded reductions in both biofilm thickness and the number of CFUs, that were largest when bromelain was core-loaded in PEG-cipro-DCPA-H 2 O liposomes with additional in-membrane, ciprofloxacin loading (Fig. 6b).Accordingly, the volumetric density of staphylococci in remaining biofilm was lowest after exposure to B/C-PEG-cipro-DCPA-H 2 O liposomes (Fig. 6c), due to the synergy between ciprofloxacin killing of biofilm inhabitants, and matrix degradation and biofilm dispersal achieved by bromelain (compare respective SEM micrographs in Fig. 6d).

In vivo eradication of an abdominal biofilm by bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes
In vivo advantages of B/C-PEG-cipro/DCPA-H 2 O liposomes for the eradication of infectious biofilms were explored using intra-vital imaging of an abdominal biofilm in living mice (see scheme in Fig. 7a).From the on-set of infection, mice were examined for clinical signs of infection and sepsis, including reduced activity or body weight and low temperature [39].Mice became inactive immediately after initiating infection with green-fluorescent S. aureus and tail-vein injection of PBS, while their weight (Fig. 7b) and body temperature (Fig. 7c) started to decrease as compared with uninfected mice.Also after tail-vein injection of bromelain-only inactivity and weight loss were observed (Fig. 7b).Temperature drop was more severe after injection of bromelain than in case of PBS injection (Fig. 7c), indicative of sepsis arising from the bromelain-induced dispersal of staphylococci into the blood circulation.Septic symptoms.i.e. inactivity, weight loss and decreased temperature could be prevented by injection of bromelain and ciprofloxacin combined in solution or in B/C-PEG-cipro/DCPA-H 2 O liposomes.However, as a result of the proton-mediated burst of the liposomes inside the biofilm yielding simultaneous release of bromelain and ciprofloxacin in the close vicinity of the bacteria to be dispersed, septic symptoms were most effectively prevented upon injection of B/C-PEG-cipro/DCPA-H 2 O liposomes.This confirms our hypothesis that simultaneous release inside biofilms is required for effective prevention of sepsis after dispersal, although it must be noted that septic symptoms in humans are different from septic symptoms in rodents.Most notably low temperature (hypoxia) is a symptom of sepsis in rodents, while high fever with occasional shivering is a septic symptom in humans [40].
Whereas arguably clinical signs of infection and sepsis may be the real proof of the pudding, more detailed, microbiological analyses of the infecting biofilm itself were also carried out.Fig. 8a presents intra-vital images of the abdominal biofilm at different points in time after initiating treatment from which biofilm thickness could be derived.Biofilm thickness decreased as a function of time after initiating treatment with bromelain combined with ciprofloxacin or B/C-PEG-cipro/DCPA-H 2 O liposomes.However, treatment with B/C-PEG-cipro/DCPA-H 2 O liposomes yielded significantly better reduction of biofilm thickness than the combination of bromelain and ciprofloxacin in solution (Fig. 8b).
The number of CFUs harvested from tissue surrounding the window site, decreased in parallel with the reductions observed in biofilm thickness and were 3 to 4 log units larger in mice treated with B/C-PEG-cipro/ DCPA-H 2 O liposomes than when treated with PBS (Fig. 8c).Granulocyte counts in blood extracted from mice at sacrifice, were lowest upon treatment with B/C-PEG-cipro/DCPA-H 2 O liposomes (Fig. 8d).Collectively, the data in Fig. 8 confirm the course of clinical symptoms presented in Fig. 7.

In vivo biosafety of bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes
Bromelain acts as a proteolytic agent, hydrolyzing peptide and glycosidic bonds in glycoproteins and hydrolyzing complex carbohydrates [41].This makes bromelain ideal for the degradation of multicomponent biofilm matrices but at the same time creates the need to establish the biosafety of bromelain as a dispersant in vivo.Hence, we performed an extensive blood analysis and histological evaluation of major organ tissue after treatment of a bacterial infection using bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes.At sacrifice, no indications were found of any damage to organ tissues (Fig. S11), while

Data and materials availability
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.Additional data related to this paper may be requested from the authors.

Declaration of Competing Interest
H.J.B. is also director of a consulting company SASA BV.The authors declare no potential conflicts of interest with respect to authorship and/ or publication of this article.Opinions and assertions contained herein are those of the authors and are not construed as necessarily representing views of the funding organization or their respective employer (s).

Data availability
Data will be made available on request.

C for 24 Scheme 1 .
Scheme 1. Preparation of bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes with proton-mediated controlled release of bromelain and PEGylated ciprofloxacin.(a) DCPA and PEGylated ciprofloxacin were allowed to self-assemble in the presence of bromelain to yield bromelain-loaded liposomes with PEGylated ciprofloxacin incorporated in the lipid membrane of the liposomes.(b) In the acidic environment of an infectious biofilm, protonation of water and PEGylated ciprofloxacin causes a hydrophilicity and charge disbalance in the lipid membrane of the liposomes, yielding burst of the liposomes and therewith release of core-loaded bromelain and PEGylated ciprofloxacin as a membrane component.

Fig. 1 .
Fig. 1.Diameter and storage stability of bromelain-loaded PEG-cipro/DCPA-H 2 O liposomes containing different weight ratios of amphiphilic PEG 2K -ciprofloxacin to DCPA.(a) Average diameter and polydispersity indices (PDI) of liposomes as a function of the weight ratio of PEG 2K -ciprofloxacin (PEG-cipro) to DCPA suspended in deionized water and measured using dynamic light scattering.(b) Average diameters of liposomes containing different weight ratios of PEG 2K -ciprofloxacin to DCPA as a function of time during suspension (50 μg/mL) in phosphate buffered saline.Note that data for weight ratios of 0.00, 0.05, 0.10 and 0.20 are overlapping.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 2 .
Fig. 2. Diameters, micrographs and zeta potentials of bromelain-loaded DCPA-H 2 O (B-DCPA-H 2 O), PEGcipro/DCPA-H 2 O (B-PEG-cipro/DCPA-H 2 O) and B/C-PEG-cipro/DCPA-H 2 O liposomes with additional ciprofloxacin-loading.(a) Diameter distributions and PDI of liposomes suspended in PBS measured using dynamic light scattering.(b) Transmission electron micrographs of liposomes.(c) Zeta potentials of liposomes as a function of pH in a PBS with pH adjusted with HCl or NaOH.Zeta potentials were measured immediately (within 2 min) after suspending in PBS with a specific pH.All error bars denote standard deviations over triplicate experiments with separately prepared batches of liposomes.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 3 .
Fig. 3. Blood circulation times of Rhodamine-labeled bromelain in B-DCPA-H 2 O, B-PEG-cipro/DCPA-H 2 O and B/C-PEG-cipro/DCPA-H 2 O liposomes with additional ciprofloxacin-loading after tail-vein injection in rats.(a) Percentage of red-fluorescent Rh-bromelain or Rh-bromelain loaded liposomes as a function of time after tail-vein injection, expressed as a percentage of the injected dose (ID).The injected dose amounted 1.0 mg/kg (0.5 mL at a bromelain concentration of 600 μg/mL.Bromelain and liposome concentrations were measured employing fluorescence emission spectroscopy (see Fig. S8 for calibration curves in rat plasma).(b) Blood circulation halflife times of bromelain, calculated from the data in panel (a).All error bars denote standard deviations over three rats in each group.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 4 .
Fig. 4. Self-targeting and accumulation of Rhodamine-labeled bromelain and Rh-bromelain loaded in B-DCPA-H 2 O, B-PEG-cipro/DCPA-H 2 O and B/C-PEG-cipro/ DCPA-H 2 O liposomes with additional ciprofloxacin-loading after tail-vein injection in mice.(a) Schematics of intravital imaging of tail-vein injected bromelain and bromelain-loaded liposomes towards a S. aureus ATCC12600 GFP biofilm underneath an abdominal imaging window.(b) Fluorescence images taken at different times after tail-vein injection of bromelain or bromelain-loaded liposomes.Fluorescence represents Rhodamine-labeled bromelain on a pseudo-colour scale reflecting the fluorescence intensity.(c) Self-targeting of bromelain and bromelain loaded liposomes into infectious biofilm as a function of time after tail-vein injection of liposomes, expressed as total number of photons per second per square centimeter (p/s/cm 2 /sr).(d) Percentage accumulation bromelain in infectious biofilms, 24 h after tail-vein injection, expressed with respect to maximum accumulation of B/C-PEG-cipro/DCPA-H 2 O liposomes (panel c).All error bars denote SD over three mice in each group.Significance levels (Students' test) for the comparisons are indicated by asterisks: ***P < 0.001.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 5 .
Fig. 5. Controlled release of bromelain and in-membrane ciprofloxacin from B-DCPA-H 2 O, B-PEG-cipro/DCPA-H 2 O and B/C-PEG-cipro/DCPA-H 2 O liposomes with additional ciprofloxacin-loading.(a) Percentage cumulative bromelain release as a function of time at 37 • C from different liposomes, suspended (5 mg/mL) in PBS, with pH adjusted through addition of HCl or NaOH.Release of bromelain was measured using fluorescence emission spectroscopy (see Fig. S5c for a calibration curve) and expressed relative to the total amount of core-loaded bromelain.Error bars denote the standard deviations over triplicate experiments with separately prepared batches of liposomes.(b) Same as panel a, now for ciprofloxacin release from liposome using UV-vis absorption spectroscopy (see Fig. S6b for a calibration curve).(c) Average diameter of bromelain-loaded liposomes obtained using dynamic light scattering after 30 min exposure to PBS at different pH.Error bars denote the standard deviations over triplicate experiments with separately prepared batches of liposomes.(d) Same as panel c, now presenting transmission electron micrographs.(e) CD spectra of bromelain dissolved in PBS and bromelain released from bromelain-loaded liposomes in PBS.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 7 .
Fig. 7. Clinical signs of infection and sepsis due to dispersal of a S. aureus ATCC12600 GFP biofilm underneath an abdominal imaging window in mice.(a) Experimental time-line: Growth of an infectious biofilm was initiated by injection of green-fluorescent staphylococci underneath the abdominal window.Two days after initiating infection, treatment was started by tail-vein injection of 0.1 mL PBS, bromelain (200 μg/mL), bromelain and ciprofloxacin (200 μg/mL of each) or suspension of bromelain-loaded PEG-cipro/DCPA-H 2 O with additional ciprofloxacin-loading (loaded 200 μg/mL ciprofloxacin and 200 μg/mL bromelain).(b) Body weight of mice in different treatment groups as a function of time after initiating biofilm growth and treatment.Body weight upon entry in the study prior to window implantation amounted 16.4 ± 0.1 g, as averaged over 15 mice.(c) Same as panel b, now for the body temperature of the mice.All error bars denote standard deviations over three mice in each group.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8 .
Fig. 8.In vivo treatment results of an infectious, S. aureus ATCC12600 GFP biofilm underneath an abdominal imaging window in mice.(a).Reconstructed 3D intravital images of green-fluorescent S. aureus biofilms at different days after starting treatment up to sacrifice at day 5.(b) Thickness of staphylococcal biofilms underneath an abdominal imaging window as a function of time after initiating treatment, derived from COMSTAT analyses [28] of intravital images.(c) Number of S. aureus CFUs per g tissue excised from infection site around the abdominal imaging window after sacrifice at day 5.(d) Granulocyte counts in blood of mice in different treatment groups at sacrifice (day 5).All error bars denote standard deviations over three mice in each group.Asterisks indicate statistical significance over comparisons indicated by the spanning bars at *p < 0.05, **p < 0.01 and *** p < 0.001 (Students' t-test).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).