HAMA and PLMA/HAMA preparation
HAMA solutions were created by dissolving different ratios of lyophilized HAMA (50 mg/mL, Engineering for Life, EFL, China) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 25 mg/mL, EFL, China) in α-MEM. PLMA solutions were obtained by solubilizing different ratios of lyophilized PLMA (EFL, China) and LAP (25 mg/mL) in PBS. PLMA solution was subsequently mixed with the HAMA solution to obtain antibacterial hybrid hydrogel with final concentrations of 5% (w/v) HAMA, 5% (w/v) HAMA + 2% (w/v) PLMA, 5% (w/v) HAMA + 4% (w/v) PLMA, 5% (w/v) HAMA + 6% (w/v) PLMA.
Characterization of HAMA and PLMA/HAMA
Assessment of rheological properties
To analyze the rheological behavior of the HAMA and PLMA/HAMA hydrogel, a viscoelasticity analysis was conducted using an HAAKE Viscotester iQ Air (Thermo Scientific). A C35 1°/Ti cone rotator with a truncation gap distance of 1 mm was employed for the experiments. The shear viscosity of the HAMA and PLMA/HAMA hydrogel was determined by performing shear rate sweeps, varying the applied shear rate from 0.1 to 100 s− 1 at 25 °C. The dynamic modulus of the hydrogel was determined by frequency sweeps. A value of 0.01 was obtained for the applied strain constant across 0.1–10 Hz at 25 °C.
Assessment of antimicrobial properties
To assess the antibacterial characteristics of various ratios of antibacterial hybrid hydrogel, a bacterial suspension with a concentration of approximately 106 CFU/mL was incubated for 24 h with four types of antibacterial hybrid hydrogel after light curing, using a hand-held blue light torch at 405 nm. Following incubation, the bacterial suspension was appropriately diluted, and the inhibition rate was estimated by measurement of the optical density (OD). Additionally, the bacterial suspension, diluted 104 times, was inoculated onto solid nutrient medium surfaces and cultured for 24 h at 37 °C. The Petri dishes were subsequently retrieved, and colony counting was accomplished using the plate counting method to determine antibacterial efficacy. The experiments were conducted in triplicate.
ApoEVs-AT extraction and labeling
ApoEVs-AT was extracted as reported before [11]. In summary, inguinal adipose tissue was extracted from 4-weel-old Sprague-Dawley rats and sliced into 1–2 cm³ pieces, which were then placed into a suspension culture flask (Wheaton, USA). Apoptosis was induced in the isolated adipose tissue by the addition of serum-free minimum essential medium α (α-MEM, HyClone, USA) with staurosporine (250 nM, STS, Beyotime, China). The mixture was allowed to culture for 2 days at 37 °C in a 5% CO2-95% air atmosphere with shaking at 200 rpm. Apoptosis in the adipose tissue was examined using hematoxylin and eosin (H&E) and immunofluorescence (IF) staining with TdT-mediated dUTP nick-end labeling (TUNEL) assays, as previously described [11]. The tissue pieces were then gently removed using sterile gauze, followed by the careful collection of the supernatant. Large extracellular vesicles and tissue debris were removed via centrifugation at 800×g for 10 min at 4 ℃, followed by 2000×g for 15 min at 4 ℃. Further centrifugation of the supernatant at 16,000×g for 30 min at 4 ℃ yielded ApoEVs-AT which were suspended in PBS for subsequent testing. For labeling, 100 µg of ApoEVs-AT suspended in 1 mL of α-MEM were incubated for 30 min with the membrane-labeling dye DiO (1 µg, Life Tech, V22886) at 37 ℃. The labeled ApoEVs-AT were then re-purified by 30 min centrifugation at 16,000×g at 4 ℃.
ApoEVs-AT identifcation
ZetaView analysis system (Particle Metrix, Germany) was employed to measure the size distribution. To examine the morphological characteristics, ApoEVs-AT were placed on formvar carbon-coated grids, subjected to negative staining with aqueous phosphotungstic acid for 60 s at room temperature, and subsequently imaged using a transmission electron microscope (TEM, Tecnai G2 F20 S-Twin, USA). For detection of phosphatidylserine (PtdSer), 100 µL PBS was used to suspend ApoEVs-AT, 5 µL FITC Annexin V (BD, USA) was added, and the system was allowed to incubate at room temperature for 15 min. A confocal laser scanning microscope (CLSM, Olympus, FV1200, Japan) was used to capture the images. Apoptosis-specific marker proteins were identified using Western blotting. ApoEVs-AT (50 µg) were combined with a 4× loading buffer (Solarbio, China) and heated to boiling for 10 min. SDS-PAGE gel electrophoresis (10% or 15%, 120 V, 90 min) was employed to separate the proteins which were then transferred onto a nitrocellulose membrane. The membrane was subsequently subjected to overnight incubation with primary antibodies, caveolin-1 (1:1000, Sangon Biotech, D161423), Histone H3 (1:1000, Invitrogen, PA5-16,183), cleaved caspase-3 (1:1000, Cell Signaling Technology, #9664) at 4 ℃. Horseradish peroxidase (HRP) conjugated secondary antibodies were subjected to incubation for 2 h at ambient temperature. Highsig ECL Western Blotting Substrate (Tanon, China) was employed for the detection of protein signals by the ImageQuant LAS 4000 mini machine (GE Healthcare, USA). All experiments were conducted at least three times.
Fabrication of ApoEVs-AT@MNP
The HAMA solution was mixed with ApoEVs-AT to create an active hydrogel solution with a final concentration of 500 µg/mL. Subsequently, 400 µL of the ApoEVs-AT-loaded HAMA hydrogel was transferred to a polydimethylsiloxane (PDMS) mold and placed in a vacuum defoaming machine to remove any foam. The sample was then oven-dried at 35 °C for 5 h. This process was carried out twice to ensure consistency. Next, 300 µL of antibacterial HAMA/PLMA hydrogel solution was added to the PDMS mold. The system was then dried overnight in an oven at 35 °C. Following the drying process, the system was subjected to photocuring using a hand-held blue light torch at 405 nm. The ApoEVs-AT@MNP was then carefully peeled off the mold for further use. The resulting MNP was a square measuring 2 cm on each side and consisted of 20 × 20 microneedle arrays, each with a height of 500 μm and substrate diameters of 200 μm. The base of the microneedle tips was coated with a 5% (w/v) HAMA and 4% (w/v) PLMA mixture, forming the substrate part of the ApoEVs-AT@MNP. Each ApoEVs-AT@MNP contains 400 µg of ApoEVs-AT, the effective dose identified in previous studies.
Characterization of MNP
Morphology observation
The MNP’s bright-field images and overall morphology were examined using a stereomicroscope (Olympus, Japan), and its surface morphological features were observed using SEM (Phenom, Netherlands). Fluorescent MNP, prepared by mixing HAMA with green fluorescent dyes, were visualized using CLSM (Olympus, FV1200, Japan).
Mechanical property
The MNP was evaluated for its mechanical strength using a displacement-force test station (Hengyi, China) equipped with a 50 kg load sensor. Microneedle tips were positioned vertically on a rigid stainless-steel platform, and the sensor was lowered at a rate of 0.1 mm/s. Initially, the sensor and the microneedle tips were 1 cm apart. Measurement of displacement and force commenced when the sensor made contact with the microneedle tips, with the speed adjusted to 0.01 mm/s, and continued until the sensor had traveled 800 μm.
In vitro ApoEVs-AT release profile test
Each ApoEVs-AT@MNP sample was immersed in 5 mL of PBS (pH 7.4) at 37 °C. At specified time points (1, 2, 3, 4, 5, 6, 7 and 8 days), 20 µL of the release medium was collected. The experiment was performed in a water bath at 37 °C. Using a BCA protein assay kit (Thermo Scientific), the total protein concentration released from the ApoEVs-AT@MNP was measured according to the manufacturer’s protocol. Absorbance was recorded with a microplate spectrophotometer (Tecan, Switzerland) at 562 nm. The experiments were repeated at least thrice.
Biological effects of ApoEVs-AT@MNP
The conditioned medium was obtained by incubating ApoEVs-AT@MNP in PBS for 72 h.
Cellular uptake
For cellular uptake studies, 5 × 104 fibroblasts or ECs were initially plated in a confocal culture dish (NEST, China). The cells were allowed to incubate with a conditioned medium for 24 h. Following incubation, cells were fixed at room temperature with neutral paraformaldehyde (4%, Biosharp, USA) for 10 min and permeabilized with Triton X-100 (0.05%, Sigma-Aldrich, USA) for 15 min at ambient temperature. Alexa Fluor 555 Phalloidin (1:200, Invitrogen, A34055-300U) was used to stain the cytoskeleton for 20 min at room temperature, and DAPI (1:1000, Solarbio, C0050) was used to stain the nuclei for 5 min at room temperature. Images were acquired using CLSM (Olympus, FV1200, Japan). All procedures were carried out in triplicate.
CCK-8 assay
Fibroblasts proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Fibroblasts were plated at a density of 1 × 103 cells per well in 96-well plates and left to incubate at 37 °C overnight. The cells were subsequently cultured with a conditioned medium and α-MEM as a blank control. At days 1 through 7, 10 µL of CCK-8 solution was added to individual wells and incubated at 37 °C for 90 min. Absorbance values were recorded using a Multiskan Go Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Similarly, the proliferation of ECs was measured using the same protocol, with observations taken on days 1 through 5. All experiments were performed in triplicate.
Transwell assay
Fibroblasts migration was assessed using Transwell assays. The upper compartment of a Chemotaxicell Chamber (8 μm, Osaka, Japan) was inoculated with 1 × 104 cells while the conditioned medium was added to the lower compartment. α-MEM without ApoEVs-AT served as the control. Following the passage of 24 h, the chamber was washed with PBS. Using a cotton swab, the non-migrated cells on the upper side of the membrane were removed, while 0.5% crystal violet was employed to fix and stain the remaining cells for 10 min. Cells were counted in the central, top, bottom, left, and right fields of view per filter and averaged to determine the number of migrated cells. The same procedure was used to measure ECs migration. All experiments were performed in triplicate.
Tube formation assay
ECs were pretreated with a conditioned medium and α-MEM without ApoEVs-AT for 2 days. The cells were then seeded at 104 cells per well into Matrigel-coated 96-well plates. After 6 h, an inverted microscope (Olympus, Japan) was employed to capture phase-contrast images. Image Pro Plus software was employed to determine the number of nodes and the length of the tubular structures in each field. All experiments were repeated at least thrice.
Adipogenic differentiation assay
To examine fibroblasts differentiation induced by ApoEVs-AT released from ApoEVs-AT@MNP, a 24-well plate was seeded with fibroblasts at 1 × 105 cells per well. The fibroblasts were categorized into two groups: a blank group and an ApoEVs-AT@MNP group. To ensure consistent treatment, the culture medium was replaced every 2 days. Following 20 days of incubation, cells were collected. Total RNA was extracted using RNAiso Plus (TaKaRa Biotechnology, Japan) and reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The cDNA was then amplified using SYBR Premix ExTaq (TaKaRa Biotechnology, Japan) on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies, China). The PCR conditions were 95 °C for 2 min, followed by 44 cycles of 95 °C for 5 s and 60 °C for 30 s (n = 3). The expression of PPARγ2, C/EBPα, Adiponectin, and FABP4 was examined to evaluate adipogenesis in fibroblasts. Primer sequences are provided in Supplementary Table S1. All experiments were performed in triplicate.
Infected wound healing model
An intraperitoneal injection of 1% pentobarbital sodium (10 mL/kg) was used to induce general anesthesia on 4-week-old male Sprague-Dawley rats (n = 4) before conducting any surgical procedures. After shaving and disinfecting the dorsal area with 75% ethanol, a pair of circular full-thickness skin wounds, each 2 cm in diameter, were created by resecting along markings drawn with a pen. A concentrated suspension of Staphylococcus aureus was applied to the wound and recorded as day 2. The wound was examined after 48 h, recorded as day 0. If the wound exhibited suppuration, the model was considered successful. The wounds were assigned to two groups: ApoEVs-AT@MNP group (left); and blank group (right). ApoEVs-AT@MNPs were inserted in the relevant group on days 0 and 8, and covered with a transparent dressing (Fig. 1B). The blank group was subcutaneously injected with 100 µL of PBS around the wounds on days 0 and 8. The wounds were photographed digitally on days 0, 4, 8, 12, and 16. ImageJ 1.53a software was used to measure the wound areas. On days 8 and 16, the rats were euthanized (n = 3 per time point) through an overdose of anesthesia, and the tissue specimens were collected for additional analysis.
Histology and IF staining
For histological evaluation, wound tissues collected on days 8 and 16 were subjected to fixation with paraformaldehyde (4%, Biosharp, USA) overnight. A gradient of ethanol was then used to dehydrate the samples, which were cleared in xylene, followed by their embedding in paraffin and cutting into sections of 6 μm thickness. The obtained sections were then subjected to staining with hematoxylin and eosin (H&E) (Solarbio, China) and Masson’s trichrome stain (Baso, China). The stained sections were examined using optical microscopy (Olympus, Japan).
For a further assessment of the specific structure of the specimens, 5% bovine serum albumin (BSA, Sigma-Aldrich, USA) was used to block the sections near the center of the wound at room temperature for 2 h. They were then allowed to incubate overnight with primary antibodies at 4 °C. Primary antibodies, Perilipin A (1:200, Abcam, ab3526) and CD31 (1:200, Abcam, ab24590), were respectively employed to mark adipocytes and blood vessels. Primary antibodies, collagen 1 (Col 1, 1:200, Abcam, ab270993), alpha-smooth muscle actin (α-SMA, 1:200, Abcam, ab5694), and collagen 3 (Col 3, 1:200, Abcam, ab184993), were utilized to mark various kinds of fibers. Subsequently, the secondary antibodies, goat anti-rabbit 555 (1:200, Invitrogen, A21428) and goat anti-mouse 488 (1:200, Invitrogen, A11008), were subjected to incubation for 1 h at 37 ℃. DAPI (1:1000, Solarbio, C0050) was then employed to mark the nuclei at room temperature for 5 min. Images were captured by CLSM (Olympus, FV1200, Japan) and ImageJ 1.53a software was employed for their analysis (n = 3).
Statistical analysis
Each experiment was repeated independently at least three times to ensure the reproducibility of the data. All numerical data are presented as means ± SD. Statistical significance was analyzed using GraphPad Prism 9.0.0 software. Paired and unpaired t-tests and one-way and two-way ANOVA were used to assess significant differences. A value of p < 0.05 was considered statistically significant. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.
