Novel lipophosphonoxin-loaded polycaprolactone electrospun nanofiber dressing reduces Staphylococcus aureus induced wound infection in mice

LPPO

The second generation LPPO DR-6180 (Fig. S4) was synthesized in multigram quantities according to the procedure reported previously⁶.

Nanofibrous materials preparation and loading with LPPO

Nanofibrous wound dressings were prepared by blending electrospinning technology^18,40. Four nanofibrous (polycaprolactone—PCL) materials with different concentrations of LPPO were prepared (0, 2, 5, and 10 wt%). All concentrations were calculated for dry matter (excluded solvent). Firstly, LPPO was dissolved in chloroform/ethanol (Penta s.r.o., Praha, Czech Republic) solvent mixture at a weight ratio of 8:2 (solution for preparation of NANO supplemented with LPPO 2% was prepared by dissolving 0.32 g of LPPO in 84 g of solvent mixture; solution for preparation of PCL supplemented with 5% LPPO was prepared by dissolving 0.8 g of LPPO in 84 g of solvent mixture; solution for preparation of PCL supplemented with 10% LPPO was prepared by dissolving 1.6 g of LPPO in 84 g of solvent mixture). The ultrasound (40 kHz, 50 W) was then applied for 30 min to enhance the solubilization of LPPOs and ensure homogeneity of the spinning solutions using an ultrasonic bath (Shesto, Watford, UK). Then 16 g of polycaprolactone (Merck, Praha, Czech Republic) with an average molecular weight M_n 45,000 was dissolved in each solution. The polymer concentration used for obtaining continuous fibrous meshes was 16 wt% (LPPO is not included in this ratio). All materials were electrospun using Nanospider NS 1WS500U (Elmarco a.s., Liberec, Czech Republic) on polypropylene spunbond microfiber nonwoven (Pegatex S; 20 g/m²; fiber diameter 20 µm; PFNonwovens Czech, Czech Republic). For electrospinning, a positive voltage was applied to the wire (i.e., the spinning electrode) and a negative voltage was applied to the collector; the potential difference was 40 kV. The distance between the spinning electrode and the collector was 160 mm. The temperature during the experiments was 22 ± 5 °C, with a relative humidity of 40 ± 5%. Produced samples were finally sterilized in the flow box using a UV light for 10 min for each side.

Scanning electron microscopy (SEM) and morphological analysis of PCL

Morphological analysis was performed by scanning electron microscopy (Vega Tescan 3, Tescan, Brno, Czech Republic) after 7 nm of gold sputter coating (Quorum Q50ES, Quorum Technologies, Laughton, Great Britain). Samples were analyzed at an accelerating voltage of 10 kV. The average fiber diameter and the diameter distribution were evaluated using NIS Elements (Laboratory imaging s.r.o., Prague, Czech Republic) using 300 randomly selected and measured fields per material. Histograms were used to summarize data from fiber diameter/distribution measurement.

Degradation analysis of PCL-LPPO materials and measurement of LPPO release kinetics

Samples (n = 3) were cut from prepared electrospun planar materials to final weight of 50 ± 0.05 mg. Individual samples were placed in 5 mL tubes with 5 mL of degradation solution composed of phosphate buffered saline (PBS) buffer (pH = 7.4), 0.02% sodium azide and either lipase (Lipase from Pseudomonas cepacia, Merck, Darmstadt, Germany) in a concentration of 5 U/mL or without lipase (Table 1). The enzymatic degradation of the samples was performed at 37 °C for 5 days, without shaking. Samples of the solution were taken for subsequent HPLC analysis after 1, 2, 4, 8, and 12 h during the first day and then every 24 h. The buffer/enzyme solution was replaced at the same time periods, thus the enzyme activity remained constant throughout the entire experiment. Samples of solutions were analyzed for the LPPO content by HPLC (see below). Remaining nanomaterial samples were then washed in distilled water and dried at room temperature and further analyzed for weight loss and morphology by scanning electron microscopy (SEM).

The HPLC analyses were performed using Dionex Ultimate 3000 HPLC system equipped with a DAD 3000 UV–VIS diode array detector (Dionex, Sunnyvale, California, USA) controlled by Chromeleon 6.80 SR12 software on Phenomenex Kinetex Hilic column (particle size 2.6 µm, column dimensions 150 mm × 4.6 mm) at 40 °C, at a flow rate of 1.4 mL/min. Samples were diluted 1/4 with pure acetonitrile, vortexed for approximately 30 s, and consecutively filtered (nylon syringe filters, 0.22 µm, Chromservis, Prague, Czech Republic). The injection volume was 20 µL. Wavelengths of 200, 207, 262 and 280 nm were used for the detection of LPPO. The gradient elution program is shown in Table S4. LPPO was quantified by comparison with external standards of LPPO DR-6180. The calibration levels used were 50, 100, 200, 500 and 1000 mg/L.

Wettability of PCL

The wettability of PCL electrospun nanofibrous materials (supplemented with or without LPPO) was tested by microtensiometer Krüss K121 (Krüss GmbH, Germany). Dynamic wetting was tested by the Washburn adsorption test. Similar surface densities of these samples were used: (1) PCL (NANO) and density of 27.1 ± 1.3 gm² and (2) PCL supplemented with 10 wt% of LPPO (NANO-LPPO10%) and density of 28.3 ± 1.8 gm². The sample of 30 mm width and 40 mm length was cut and placed into the holder for foils. The holder with nanofibrous sample was then inserted into the clip, with the bottom sample edge arranged in a horizontal position and parallel orientation of the sample edge during immersion in the test liquid (HPLC water, VWR Int., Stříbrná Skalice, Czech Republic). The ambient conditions were temperature of 22 °C and 58% relative humidity. The wicking, weight of water wicked into the fibrous system in time, into NANO and NANO-LPPO10% was measured five times for each condition. The spontaneous wicking of porous materials even of textiles or nanofibrous layers can be described by the Washburn equation²⁰. The results were expressed in the form of average squared mass gain over time at the beginning of wicking represented by the direction of the regression curve of the wicking rate (Fig. S1).

In vitro study in human dermal fibroblasts (HDF) and human keratinocyte cell line (HaCaT)

HDFs were isolated from skin specimens, as previously described⁴¹, obtained from two healthy donors during aesthetic surgery with the informed consent of patients (in complete agreement with the Helsinki Declaration after approval by the Ethical Committee of the University Hospital Královské Vinohrady) at the Department of Aesthetic Surgery (Third Faculty of Medicine, Charles University, Prague, Czech Republic). HDF cultures were expanded in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and penicillin/streptomycin (Biochrom). They were further culturing at 37 °C and 5% CO₂. Cells at passages 7–8 were used in all experiments.

The HaCaT cell line⁴², used in the present study was obtained from Cell Lines Service (Eppelheim, Germany). Cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (streptomycin and penicillin) (all from Biochrom, Berlin, Germany).

In all in vitro experiments, TGF-β1 (PeproTech, London, UK) at 30 ng/mL⁴³ was used as a positive control to evaluate the ability of HDFs to undergo myofibroblast differentiation. We examined whether LPPO modulates TGF-β1 signaling (both canonical and non-canonical). In parallel, LPPO and/or TGF-β1 effect was also studied in HaCaT cells. A standard cultivation medium was used as control.

MTS assay

Cell viability and proliferation were determined using colorimetric microculture assay with MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye (Promega, Madison, WI, USA)⁴⁴. Cells were seeded (HDFs—3000 cells/well; HaCaT—10,000 cells/well) into 96-well-plates in culture medium (10% FBS). After 24 h the medium was replaced with a medium containing LPPO (0.1, 1, 5, 10, 25, 50, and 100 mg/L) in the presence or absence of TGF-β1 (PeproTech). Following the next 72 h MTS (at a final concentration of 0.21 mg/mL) was added into the culture medium. After an additional 3 h, cell proliferation was evaluated by measuring the absorbance at wavelength 490 nm using the automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). The absorbance of control wells was taken as 100%, and the results were expressed as a percentage of the untreated control. The experiments were performed in technical triplicates and repeated three times.

Migration (wound healing) assay

The migration of HaCaT cells was evaluated using a scratch assay. Briefly, a confluent layer of cells cultured on a 6-well plate was scratched using a pipette tip creating a “wound”. Afterwards, the medium was replaced with a medium containing LPPO at 1, 5 and 10 mg/L in the presence or absence of TGF-β1 (PeproTech). The wounded area was photographed at the beginning (0 h) and in 24 h. The migration distance (gap area) was determined using NIS Elements (Nikon, Tokyo, Japan) and expressed as a percentage of the original gap created at 0 h. The experiments were performed in technical duplicates and repeated twice.

Western blot analysis

Based on the MTS assay we selected only non-toxic concentrations (1, 5, 10 mg/L) of LPPO for further western blot analysis. HDFs and HaCaTs were seeded on Petri dishes at the density of 5000 and 10,000 cells/cm², respectively and cultivated for 7 days with LPPO in the presence or absence of TGF-β1. The set of primary and secondary antibodies applied in the analysis is shown in Table S5. The analysis was performed as described previously⁴⁵. Briefly, cells were washed with cold PBS and collected in Laemmli sample buffer (100 mM TRIS–HCL pH approx. 6.8, 10% glycerol, 2% SDS) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Immediately after collection, cells were disrupted using a sonicator (QSonica, 40% amplitude, 15 s). After boiling (95 °C, 5 min), samples were separated in SDS-PAGE gel (10% Bis–Tris) and transferred to PVDF membrane using iBlot 2 (Thermo Fischer Scientific). Following 1 h of blocking in 5% NFDM/BSA (non-fat dry milk/bovine serum albumin) dissolved in TBS (tris-buffered saline) with 0.1% Tween at room temperature, membranes were incubated with the primary antibody at 4 °C overnight. The next day, the membranes were washed in TBS-Tween (3 × 5 min) and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After incubation, the membranes were again washed in TBS-Tween (3 × 5 min), and protein bands were detected using ECL (SuperSignal West Pico PLUS chemiluminescent Substrate, Thermo Fischer Scientific) and the signal was acquired at MF-ChemiBis 2.0 (DNR Bio-Imaging Systems). β-actin was used as a sample loading control. The chemiluminescent signal of all detected proteins was quantified using the Image Studio (LI-COR) western blot densitometry software and normalized to β-actin.

Imunofluorescence of cultured cells

HDFs and HaCaTs were seeded at a density of 5000 and 10,000 cells/cm², respectively. Both cells were cultivated with LPPO (1, 5 and 10 mg/L) in the presence or absence of TGF-β1 (PeproTech) for 7 days. The set of primary and secondary antibodies applied in the analysis is shown in Table S6. The analysis was performed as described previously⁴⁶. Briefly, tested specimens were fixed with 2% buffered paraformaldehyde (pH = 7.2) for 5 min. and washed with PBS. Cells were permeabilized by exposure to Triton X-100 (Sigma-Aldrich), and sites for antigen-independent binding of antibodies were blocked by incubation with porcine serum albumin. Commercial antibodies were used at concentrations recommended by the suppliers. All specimens were mounted to Vectashield (Vector Laboratories, Eching, Germany) and inspected by using an Eclipse 90i microscope equipped with filter blocks for the three types of dyes (Nikon, Tokyo, Japan) as well as a Hamamatsu CCD camera (Hamamatsu, Shizuoka, Japan) and a computer-assisted image analysis system NIS (Nikon, Tokyo, Japan).

Animal study

The experimental conditions complied with the European rules of animal care and welfare in compliance with the ARRIVE guidelines. The experiment was approved by the Ministry of Health of the Czech Republic (MZDR 20378/201/-4/OVZ).

Mice

Inbred male Balb/c (Balb) (Charles River Laboratories, Munich, Germany) mice, 8–12 weeks of age weighing 17–22 g, were used in the experiment. Breading of mice and all experimental procedures were conducted under specific-pathogen-free (SPF) conditions in an SPF Animal Facility, National Institute of Public Health, Prague, Czech Republic. Animals were housed under 12 h day/night light cycle and standard environmental conditions (22 °C, 55% relative humidity). Mice had free access to sterile water and commercial ST1 diet (Velaz, Prague, Czech Republic) ad libitum. Prior to the wound healing experiment all mice were housed in groups of three, in sanitized cages on sterile paper bedding, and were provided with environmental enrichment, including in-cage plastic housing.

The allocation of mice (n = 54) in treatment groups is shown in Table 2. All mice were sacrificed under general anesthesia by cervical dislocation 7 days after surgery. Wound samples were removed for histological, bacteriological culture-based and bacteriological quantitative PCR-based evaluations (see below). In addition, plasma and liver were also examined for the presence of LPPO residues.

Table 2 Allocation of mice (n = 54) in treatment groups.

Wound model

Two days before wounding mice received 100 mg/kg of cyclophosphamide via intraperitoneal (i.p.) injection³⁶. On day 0, the day of wounding and inoculation, mice were anesthetized with Isoflurane (Aerrane 100%, Baxter, Lessines, Belgium). Hair was clipped from the cervical to mid-lumbar dorsum, and the skin was scrubbed with iodine solution followed by an ethanol rinse. An 8-mm disposable skin biopsy punch (Kai Medical, Kai Europe, Solingen, Germany) was used to create a full-thickness skin defect overlying the thoracic spinal column and the adjacent musculature. Aliquots of 25 μL containing the inoculum (see below) in a PBS suspension were pipetted into the wound and allowed to absorb. A circular cutout (30 mm in diameter) of transparent dressing (TegadermRoll; 3 M Health Care, St. Paul, MN) was placed over the wound with or without PCL-scaffolds supplemented or not with LPPO at 2, 5, and 10% (NANO, NANO-LPPO2%, NANO-LPPO5%, and NANO-LPPO10%).

Bacterium and inoculum preparation

Staphylococcus aureus strain CNCTC 5480 (= ATCC 29213) obtained from the Czech National Collection of Type Cultures (https://www.szu.cz/cnctc) was used in the present study. To prepare an inoculum for experimental wound infection, 15 mL of Luria–Bertani (LB) medium (Oxoid Ltd, Basingstoke, UK) in a 100-mL Erlenmeyer flask was inoculated with 50 μL of cell suspension with a density of ≈ 10⁸ colony-forming units [CFU] per mL, which was prepared in saline from an overnight CNCTC 5480 culture on a sheep blood agar (BA) plate (Oxoid). The inoculated medium was then cultured at 37 °C with shaking in a thermostatically controlled water bath. After 3.5 h, 1 mL of the mid-exponential growth phase culture was taken and the harvested cells were washed twice and finally diluted in PBS (pH = 7.2). The bacterial quantity was determined by the measurement of the optical density (OD) and based on the known correlation between OD and CFU values the final suspension was adjusted to contain ≈ 2.5 × 10⁴ CFU/mL of PBS. The bacterial density of the final suspension was checked by CFU counting using serial dilution and plating onto LB agar plates.

Histology of open wounds

Skin-wound specimens were removed from sacrificed mice and routinely processed for histological staining (fixation in 4% buffered formaldehyde, dehydration using increasing concentrations of ethanol, paraffin embedding, sectioning, and staining). Deparaffinized Sections (2–3 µm thick) were stained with hematoxylin–eosin (H + E—basic staining), Sirius Red (SR—collagen staining) and Gram staining (to distinguish between gram-positive/negative bacteria).

Culture-based characterization of experimental bacterial wound infection

Inoculated blood agar (BA) plates were cultured aerobically at 37 °C for 20 h. The resulting bacterial growth was visually evaluated and if positive, bacteria were isolated, cultured on BA and checked for their identity with S. aureus CNCTC 5480. Species identification of isolates was based on whole-cell profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS using the Microflex LT instrument and BioTyper software version 3.1 (Bruker Daltonics, Bremen, Germany). Isolates identified as S. aureus were then checked for their identity with CNCTC 5480 at the strain level using SmaI-based macrorestriction analysis based on a previously published protocol⁴⁷.