A self-adhesive hierarchical nanofiber patch for dynamic and multistage management of full-thickness cutaneous wounds | Journal of Nanobiotechnology

Materials

Recrystallized anhydrous trimethylene carbonate (TMC) and DL-lactide (LA) monomers were obtained from Daigang Biomaterial. Benzyl alcohol (BnOH, 99%, with water content ≤ 50 ppm) was sourced from Meryer. Stannous octoate (SnOct₂, 95%), lipase from Aspergillus oryzae (≥ 100,000 U/g solution), and lipopolysaccharides (LPS) from Escherichia coli O111:B4 were purchased from Sigma-Aldrich. Gelatin, derived from porcine skin, was purchased from Beyotime. Methacrylic anhydride (94%) was purchased from Aladdin. Chitosan (CS, 90% deacetylation, 150,000 Da) and collagenase (≥ 125 U/mg) were supplied by Yuanye Bio-Technology. Glycidyl trimethyl ammonium chloride (GTMAC, 98%) was obtained from Haohong Bio-Pharmaceutical Technology. Acetic acid (AC, 99.5%), 2,2,2-trifluoroethanol (TFEA, 99.5%) and lysozyme (≥ 5000 U/mg) were purchased from Macklin. The flavonoid (FN) was kindly provided by Chenguang Biotech Group and is a natural extract from camellia fruit, mainly including afzelin, isoquercitrin, astragalin, isoorientin, quercitrin, etc. Cell lines including Murine macrophage RAW 264.7, murine fibroblast NIH 3T3, human dermal fibroblast BJ, and human umbilical vein endothelial cells‌ (HUVECs) were obtained from the National Collection of Authenticated Cell Cultures. Additional chemical solvents were purchased from General-reagent and Aladdin.

Synthesis and characterization of PLTC

The synthesis of PLTC was conducted as our previously reported [33, 34]. Briefly, LA and TMC monomers (20 g in total, in various ratios), SnOct₂ catalyst (0.1 wt% of monomers), and BnOH initiator (0.1 wt% of monomers) were charged into a heavy-wall pressure vessel equipped with a gas dispersion/vacuum joint and connected to a Schlenk line. The system was degassed through three nitrogen purge-fills and then heated in an oil bath at 130 °C with vigorous stirring until complete melting, followed by a 12-h reaction. The product was dissolved in dichloromethane and purified by precipitation from anhydrous ethanol. The resulting precipitate was dried under vacuum at 40 °C for 48 h.

The molecular weight of polymers was determined using gel permeation chromatography (GPC, Breeze2, Waters). Tetrahydrofuran in chromatographic grade was used as the eluent at a flow rate of 1 mL/min with a column temperature of 35 °C, and polystyrene standards were used for molecular weight calibration. The chemical structure and composition of polymers were characterized by ¹H nuclear magnetic resonance (NMR) spectroscopy using the NMR spectrometer (AVANCE III HD, 400 MHz, Bruker) with deuterated chloroform (CDCl₃) as the solvent.

Synthesis and characterization of GelMA

Gelatin (2 g) was placed in a flask and dissolved in 20 mL of phosphate-buffered saline (PBS, pH 7.4) at 600 rpm and 60 °C. Methacrylic anhydride (1 mL) was added dropwise at a rate of 0.5 mL/min, and the reaction was carried out in a 50 °C water bath for 3 h [35]. The reaction was terminated by adding 100 mL of PBS. The solution was then dialyzed in a dialysis bag with a molecular weight cut-off (MWCO) of 8000–14000 Da) and lyophilized to obtain purified GelMA. The chemical structure of GelMA was confirmed using ¹H NMR spectroscopy with deuteroxide (D₂O) as the solvent.

Synthesis and characterization of QCS

CS (5 g) was placed in a flask and dissolved in 180 mL of deionized water with 0.9 mL of glacial acetic acid at 300 rpm and 50 °C for 30 min. 5 mL of GTMAC aqueous solution (1.2 g/mL) was added dropwise, and the reaction was carried out at 55 °C for 18 h [36]. The crude QCS solution was collected, centrifuged at 4500 rpm for 20 min, and then dialyzed in a dialysis bag (MWCO 3500 Da). The product was lyophilized to obtain purified QCS. The chemical structure of QCS was confirmed using ¹H NMR spectroscopy with D₂O as the solvent.

Fabrication of SAHN patches

A TFEA solution of PLTC (7% w/v) and a dilute acid solution of CS (3.5% w/v, H₂O:AC = 9.8:0.2 v/v) were prepared and mixed at room temperature to form the working solution for the outer barrier. A GelMA solution (15% w/v, H₂O:TFEA = 1:1 v/v) containing the photoinitiator phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP, previously synthesized in-house [37]) at a concentration of 0.15% w/v was prepared. An aqueous solution of QCS (3% w/v) was mixed with the GelMA solution at room temperature to form the base working solution for the inner scaffold, to which FN at varying loading rates (5%, 10%, 20% w/w) was added.

The electrospinning working solutions were loaded into a 2.5 mL disposable syringe fitted with a 24G stainless steel needle. Electrospinning was conducted on an electrospinning system (Elite, Beijing Yongkangleye) at a chamber temperature of 40 °C and a humidity of 30–50% RH. A positive voltage was applied to the stainless-steel needle, while a negative voltage was applied to the stainless-steel flat collector covered with aluminium foil. The distance from the needle tip to the collector was maintained at 15 cm.

For the fabrication of the outer barrier, a positive voltage of + 16 kV and a negative voltage of − 6 kV were applied, with an infusion rate of 1 mL/h for 1 h using an optimized PLTC:CS solution (5:5, v/v). Subsequently, the solution was switched to the inner scaffold working solution, which had an optimized GelMA:QCS ratio of 8:2 (v/v). The electrospinning parameters were adjusted to a positive voltage of + 12 kV, a negative voltage of − 6 kV, and an infusion rate of 1 mL/h for 1 h. The aluminium foil was then removed, and the constructs were exposed to 405 nm ultraviolet (UV) light (5 W) for 3 min for curing, followed by vacuum drying at 40 °C for 2 h. The constructs were then cut to size according to the application requirements to obtain the SAHN patches.

Morphological characterization

The immediate nanofiber morphology post-electrospinning was observed and recorded using an inverted fluorescence microscope (ECLIPSE Ts2R, Nikon). The surface morphology of each layer of the SAHN patches was examined using a scanning electron microscope (SEM, Phenom Pharos, Thermo Scientific). The SAHN patches were sectioned with a blade to observe their cross-sectional morphology. ImageJ software was utilized to quantify nanofiber and micropore sizes.

Fourier-transform infrared spectroscopy (FT-IR) analysis

A Fourier-transform infrared spectrometer (Nexus 670, Nicolet) was employed to detect the chemical composition of the SAHN patches and their components. The nanofiber patches were ground into powder, mixed with potassium bromide at a ratio of 1:100 (w/w), and compressed into pellets for scanning in the range of 400–4000 cm⁻¹.

The interlayer hydrogen bonding of the SAHN patch was also characterized by FT-IR. GelMA and GelMA/QCS nanofiber membranes were prepared separately. An interwoven membrane of PLTC/CS and GelMA/QCS was fabricated using the built-in dual-channel syringe pump of the electrospinning system. After vacuum drying, the infrared spectra of the samples were characterized by attenuated total reflection (ATR).

Zeta potential measurements

The interlayer electrostatic interactions of the SAHN patch were characterized by Zeta potential measurements. Although the surface charge of membrane samples can often be assessed with a solid-state Zeta potential analyzer, this approach may not be suitable for analyzing the interlayer charge interactions of the SAHN patch. Alternatively, a Zeta potential characterization method was developed herein by using dispersive nanofibers.

GelMA/QCS nanofiber membranes (10 mg/mL) were immersed in PBS (pH 7.4) and truncated using an ultrasonic disperser (SCIENTZ-750F, NingBo Scientz Biotechnology) in an ice bath at 750 W for 5 min [38]. PLTC and PLTC/CS nanofiber membranes, due to their higher mechanical strength, were immersed in PBS (10 mg/mL) and placed in an ice bath before being treated with a mechanical stirrer (RW 20 digital, IKA) at 1200 rpm for 15 min. Post-treatment, centrifugation (1000 rpm, 3 min) was performed to eliminate precipitates, yielding a dispersion of short nanofibers. The Zeta potential of the nanofiber dispersion was measured with a Zeta potential measurement system (Zetasizer Nano ZS90, Malvern Panalytical). A mixed dispersion of two types of nanofibers was generated by combining them in a 1:1 volume ratio.

Mechanical properties

The tensile properties of the raw polymers and the patches were determined using a universal testing machine (HZ-1007E, Dongguan LiXian Instrument Technology). Samples were cut into rectangles of 2 × 0.5 cm and tested at a stretching rate of 1 mm/min to analyze their tensile strength and elongation at break.

Water absorption

The initial mass of the SAHN patches and their respective components was measured, after which they were immersed in PBS (pH 7.4) and incubated in a shaking bed (100 rpm, 37 °C, THZ-100, Shanghai Yiheng Technology Instrument) for 1 h. The samples were then removed, surface liquid was blotted with filter paper, and the mass was remeasured. The water absorption was calculated using the following formula:

where \({M}_{0}\) is the initial mass and \({M}_{1}\) is the mass after water absorption.

Degradation

The mass of the SAHN patches was measured and placed in sample vials, to which 2 mL of PBS (pH 7.4), lysozyme, collagenase, and lysozyme/collagenase solutions were added, and incubated in the shaking bed (100 rpm, 37 °C). Additionally, the degradation of the outer barrier was carried out in PBS (pH 7.4), lysozyme, lipase, and lysozyme/lipase solutions. At specific time points, the samples were removed, surface liquid was blotted with filter paper, and the samples were vacuum dried at room temperature for 2 h, after which they were weighed. The mass loss of the samples was calculated using the following formula:

where \({M}_{0}\) is the initial mass and \({M}_{d}\) is the mass after degradation.

The outer barrier and inner scaffold were placed in PBS and incubated in the shaking bed (100 rpm, 37 °C). At specific time points, the samples were taken out, rinsed with deionized (DI) water, blotted to remove surface liquid using filter paper, and vacuum-dried at room temperature for 2 h. The surface morphology of each SAHN patch layer after water absorption and degradation was then examined using SEM.

In vitro FN release

The SAHN patches were weighted and placed in sample vials, to which 2 mL of PBS (pH 7.4) was added, and incubated in the shaking bed (100 rpm, 37 °C). At specific time points, 1 mL of the solution was taken out, and the same volume of fresh PBS was added. The absorbance at 283 nm was measured using a UV–Vis spectrophotometer (V-770, JASCO) to determine the cumulative release of FN.

Interlayer and tissue adhesion of the SAHN patch

To evaluate the SAHN patch’s interlayer adhesion, lap-shear and peel-off tests were conducted. PLTC/CS and GelMA/QCS (or FN@GelMA/QCS) were sequentially electrospun onto aluminum foil. The samples were then cut, masked with another aluminum foil layer to expose 5 mm-wide strips, and electrospun with PLTC/CS (or PLTC). After drying, the samples were cut into inner-outer layer lapped specimens (5 × 5 mm overlap area). For the lap-shear test, the sample’s ends were gripped by the universal testing machine and stretched at 1 mm/min until failure. Shear strength was calculated by dividing the maximum load at failure by the overlap area.

For the peel-off test, sample preparation was similar, but with different cutting directions. The sample was peeled at a constant rate of 1 mm/min at a 90° angle. Peel strength was determined by measuring the force to separate the layers divided by the sample width.

To test the SAHN patch’s adhesion to skin, it was cut into 5 × 15 mm strips. Fresh porcine skin (from Nanjing Gulou Market) was used to simulate human skin. The SAHN patch was applied to the skin (5 × 5 mm adhesion area) and stretched at 1 mm/min.

To test the tissue adhesion of the SAHN patch after hydration and degradation, it was placed in PBS and incubated in the shaking bed (100 rpm, 37 °C). At specific intervals, the SAHN patch was taken out, rinsed with DI water, blotted with filter paper to remove surface liquid, and vacuum-dried at room temperature for 2 h. After cutting, it was attached to porcine skin for the peel-off test.

Antibacterial properties

Bacterial penetration experiments were designed on Transwell Permeable Supports, featuring polycarbonate (PC) microporous membranes with 8-μm pores that allow free passage of representative Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria Staphylococcus aureus (S. aureus), to assess the ability of SAHN patches to resist bacterial invasion [39, 40]. Commercial wound management products including gauze (Cofoe Medical Technology), Band-Aid (Johnson & Johnson), Tegaderm hydrocolloid dressing (Minnesota Mining and Manufacturing), and Tegaderm hydrogel (Minnesota Mining and Manufacturing) were used for comparison. Membrane samples, such as the SAHN patches, were punched into circular discs (1.5 cm in diameter) and embedded into the Transwell inserts using a glass rod (0.6 cm in diameter) to cover the microporous membranes. Tegaderm hydrogel (50 μL) was evenly distributed into the inserts with a 1 mL disposable syringe. In the 24-well plate, 0.5 mL of PBS (pH 7.4) was added to each well, and the inserts were placed. E. coli or S. aureus suspension (50 μL, 1 × 10⁶ CFU/mL) was added to the inserts and incubated at 37 °C.

At 3, 12, and 24 h, bacteria that penetrated through the membrane were detected using the LIVE/DEAD Bacterial Staining Kit (Beyotime) with N,N-dimethylaniline N-oxide (DMAO) and propidium iodine (PI).

Parallelly, after a 24-h incubation, the Transwell inserts were removed, allowing the wells to continue incubating for an additional 24 h. Biofilms that formed were stained with crystal violet (CV, 0.1%, Aladdin) and photographed using a digital camera and the inverted fluorescence microscope.

The antibacterial activity of the SAHN patches was further investigated using the spread plate method. A mixture of 50 μL E. coli or S. aureus suspension (1 × 10⁵ CFU/mL), 100 μL PBS (pH 7.4), and 3 μL Luria–Bertani (LB) medium (Yuanye Bio-Technology) with the patches (0.6 cm in diameter) was placed in a 96-well plate and incubated in the shaking bed (100 rpm, 37 °C) for 24 h. The co-incubated bacterial suspension was diluted 1000-fold, and 30 μL was spread onto agar plates for further 24-h incubation. The antibacterial effect was evaluated by observing the number of grown colonies.

Parallelly, the bacterial suspension co-incubated with the patches was serially diluted (10, 100, 1000, 10,000, and 100,000 times), and 10 μL of each dilution was spotted onto agar plates and incubated for 24 h to count the grown colonies.

Hemostatic performance

The initial mass of SAHN patches and commercial wound management products was measured. Samples were immersed in citrated whole blood from SD rats (male, 250 g, purchased from Nanjing Qinglongshan Animal Breeding Institute) and incubated in the shaking bed (100 rpm, 37 °C) for 5 min. The samples were then removed, excess surface liquid was blotted with filter paper, and the mass was remeasured. The blood absorption of the samples was calculated using the aforementioned water absorption formula.

50 μL of rat recalcified (CaCl₂, 10 mM) blood were dropped onto the surface of 10 mg of the samples and incubated in the shaking bed (100 rpm, 37 °C) for 5 min. Then, 10 mL of deionized water was added and further incubated for 10 min. The optical density (OD) of the supernatant of each sample at 540 nm was measured using a Microplate Photometer (Multiskan FC, Thermo Scientific). The group without samples served as the negative control. The Blood Clotting Index (BCI) was calculated using the following formula:

50 μL of citrated whole blood were dropped onto the surface of 10 mg of the samples and incubated in the shaking bed (100 rpm, 37 °C) for 5 min. Free red blood cells (RBCs) were washed off with PBS (pH 7.4). After adding 10 mL of deionized water, the samples were further incubated for 10 min to lyse the adherent RBCs. The OD of the supernatant of each sample at 540 nm was measured. The group with 50 µL of blood added directly to 10 mL of deionized water served as the negative control. RBC adhesion was calculated using the following formula:

Citrated whole blood was centrifuged (3000 rpm, 10 min, 4 °C) to obtain platelet-rich plasma (PRP). 50 μL of PRP were dropped onto the surface of 10 mg of the samples and incubated in the shaking bed (100 rpm, 37 °C) for 5 min. Free platelets were washed off with PBS (pH 7.4). 1 mL of Triton X-100 (1%, Beyotime) was added and further incubated for 10 min to lyse the adherent platelets. Platelet adhesion was measured using a Lactate Dehydrogenase (LDH) Assay Kit (Nanjing Jiancheng Bioengineering Institute) at an OD of 450 nm. The group with 50 µL of PRP added directly to 1 mL of Triton X-100 (1%) served as the negative control. Platelet adhesion was calculated using the following formula:

A rat liver prick injury model was used to evaluate the in vivo hemostatic properties of the SAHN patch. The standard hemostatic gelatin sponge (Jinling Pharmaceutical) served as the control. SD rats (male, 200 g) were anesthetized using isoflurane. An abdominal incision was made to expose the liver, and a pre-weighed filter paper was placed beneath it. A 1 mL syringe was used to quickly stab the liver surface, and a 1-cm diameter circular sample was immediately applied to the wound until bleeding ceased. Blood loss was determined by weighing the filter paper before and after blood absorption.

Hemocompatibility

Citrated whole blood was centrifuged (1500 rpm, 10 min, 4 °C) to collect RBCs, which were then washed with PBS (pH 7.4) and resuspended to form an RBC suspension. Samples were incubated with the RBC suspension in the shaking bed (100 rpm, 37 °C) for 1 h, followed by centrifugation, and the OD of the supernatant at 540 nm was measured. The mixture of RBCs and DI water served as the positive control, while the mixture of RBCs and PBS (pH 7.4) served as the negative control. The hemolysis was calculated using the following formula:

In vitro antioxidant activity

5 mg of the SAHN patches were placed in 1 mL of PBS and incubated in the shaking bed (100 rpm, 37 °C) for 1 h, then centrifuged (3000 rpm, 5 min, 25 °C). 40 μL of the supernatant was mixed with 60 μL of 1,1-diphenyl-2-picrylhydrazyl (DPPH, Aladdin) and incubated at 37 °C in the dark for 30 min, after which the OD at 510 nm was measured. The control group was set up with 80% methanol solution instead of DPPH reagent solution. The DPPH radical scavenging was calculated using the following formula:

where \({OD}_{Sample}\) is the OD of the sample group, \({OD}_{Control}\) is the OD of the control group, and \({OD}_{Blank}\) is the OD of the DPPH solution without the sample.

The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) working solution was prepared according to the ABTS Free Radical Scavenging Ability Assay Kit (Boxbio) instructions and mixed with the aforementioned supernatant, then incubated at 37 °C in the dark for 6 min, and the OD at 405 nm was measured. The ABTS radical scavenging was calculated following the manufacturer’s instruction.

In vitro anti-inflammatory activity

RAW 264.7 macrophages (2 × 10⁵/well) were seeded in a 24-well plate and incubated for 8 h. The medium was replaced with medium containing LPS (5 μg/mL) except for the negative control group (i.e., the Normal group). Then, extracts of the SAHN patches, prepared by incubating the samples in the medium (0.1 g/mL) for 24 h, were added to the wells. The cells were further incubated for 24 h in the presence of the extracts. The supernatant was collected and centrifuged (5000 rpm, 10 min, 4 °C). The secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) was measured using the corresponding enzyme-linked immunosorbent assay (ELISA) kits (Beyotime). Cells treated only with LPS served as the positive control, while cells untreated with LPS served as the negative control.

Cell proliferation and adhesion

The SAHN patches were punched into 6-mm diameter samples and placed in a 96-well plate with the inner scaffold facing up. Each well was seeded with 8 × 10³ NIH 3T3 fibroblasts and incubated for 1, 2, and 3 days. Cells seeded on tissue culture polystyrene (TCPS) served as the control group. Cell viability was assessed using the MTT formazan (Aladdin) assay by measuring the OD at 570 nm. Parallelly, cells were stained using the Calcein/PI Cell Viability/Cytotoxicity Assay Kit (Beyotime) and photographed under the inverted fluorescence microscope.

BJ fibroblasts and HUVECs were similarly seeded on the outer barrier and inner scaffold of the patch. Cells were fixed in 4% paraformaldehyde (Servicebio) and stained using 488-Phalloidin (1:50, Share-Bio) and Hoechst 33342 (10 μg/mL, Aladdin) successively to investigate the cell adhesion on the SAHN patch. The cell number and area were analyzed using ImageJ software to calculate the cell attached rate and nucleo-cytoplasmic ratio.

Treatment of full-thickness cutaneous wounds in mice

Male ICR mice (25 g) were purchased from Nanjing Qinglongshan Animal Breeding Institute and acclimated for 7 days. Hair on the mice’s backs was shaved, and anesthesia was induced with Avertin (2.5%, 250 μL, Aladdin). Full-thickness skin defects were created using a 6-mm biopsy punch [41]. The patches (6 mm) were applied to the wounds, covered with fixed gauze for protection against scratching. 5 mice were used in each group. On days 3, 6, and 12, the gauze and patches were removed, and the wounds were photographed. Wound closure was dynamically analyzed using ImageJ software with the following formula:

where \({A}_{0}\) is the wound area on day 0, and \({A}_{n}\) is the wound area on day \(n\).

At predetermined time points, mice were euthanized via CO₂ inhalation. The wound areas were excised and fixed in 4% paraformaldehyde (Servicebio) for 24 h. The samples were then processed for paraffin embedding and dehydrated by an automatic dehydrator. The embedded tissues were sectioned using a rotary microtome. The sections were mounted on glass slides and subjected to three main types of staining. For hematoxylin–eosin (H&E) staining, the sections were dewaxed in xylene, rehydrated, and stained with hematoxylin (10 min) for nuclear visualization followed by eosin (5 min) for cytoplasmic and extracellular matrix components.

Masson’s trichrome staining was performed to specifically highlight collagen fibers and other connective tissue components. For Masson’s trichrome staining, the sections were stained with Weigert’s iron hematoxylin for 10 min, followed by differentiation to remove excess stain. They were then stained with Biebrich scarlet-acid fuchsin for 5 min. After differentiation with phosphomolybdic acid, collagen fibers were stained blue with aniline blue for 5 min. Finally, sections were dehydrated and mounted.

Immunohistochemical staining was carried out for CD31, TNF-α, IL-6, and IL-1β using standard protocols. The sections were first immersed in citrate buffer (pH 6.0), and boiled. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide in methanol for 10 min. Non-specific binding was blocked with 5% normal goat serum for 30 min at room temperature. The sections were then incubated with primary antibodies (Servicebio) against CD31 (1:200), TNF-α (1:200), IL-6 (1:200), and IL-1β (1:200) overnight at 4°C. After three washes with PBS, the sections were incubated with biotinylated secondary antibodies (1:200) for 30 min at 37°C. The sections were then treated with avidin–biotin-peroxidase complex (ABC) reagent for 30 min at room temperature. Diaminobenzidine (DAB) was used for 2 min as the chromogen, and the sections were counterstained with hematoxylin for 2 min. Finally, the sections were dehydrated, cleared, and mounted with a resinous mounting medium.

The stained sections were imaged under the microscope, and quantitative analysis was performed using ImageJ software to assess the number of microvessels, inflammatory cell infiltration, and collagen deposition.

Statistical analysis

Data were plotted using OriginPro software, presented as mean ± standard deviation (s.d.) with individual data points displayed as scatters. The number of replicates (n) for each experiment is indicated in the figures. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A probability value (P) less than 0.05 was considered to indicate statistically significant differences.