Curcumin-loaded PLGA microparticles integrated with ZnO/GelMA hydrogel microneedles for infectious wound healing and reduction of hypertrophic scars | Journal of Nanobiotechnology

Materials

The 2, 2-di(4-tert-octylphenyl)-1-pyridylhydrazine (DPPH) and chloroform were procured from Aladdin Industrial Corporation (Shanghai, China). Curcumin (C₂₁H₂₀O₆) was procured from Sunny Bio-tech Co. Ltd. (Shanghai, China). Poly (d, l-lactide-co-glycolide) (PLGA) with a molecular weight of 10,000 and 50,000, respectively, was procured from Shenzhen Maiqi Biomaterial Co., Ltd. (Shenzhen, China). Gelatin, methacrylic anhydride (94%), and zinc oxide were procured from Sigma-Aldrich (Shanghai, China). The lap and microneedle moulds (PDMS) were obtained from Engineering for Life (Suzhou, China). Phosphate-buffered saline (PBS) and the rhodamine were procured from McClelland Chemical Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC), 4’, 6-diamidino-2-phenylindole (DAPI), chemiluminescence (ECL), the Trypan Blue and dimethyl sulfoxide (DMSO) were procured from Solarbio Science & Technology Co., Ltd. (Shanghai, China). The culture medium (DMEM) and fetal bovine serum (FBS) were procured from Thermo Fisher Scientific (Massachusetts, USA), and the former contained 100 µg/mL streptomycin and 100 U/mL penicillin. The L929 cells and KFB were generously provided by the Affiliated Hospital of Qingdao University (Qingdao, China). The rats and New Zealand female rabbits were obtained from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China).

Preparation and characterization of GelMA hydrogel microneedles

Preparation of microneedles

A solution of 4 wt% PLGA and 1 wt% curcumin was completely dissolved in chloroform, resulting in a mixed solution, which was then placed in a syringe and attached to a push pump. The distance between the needle tip and the collection device was maintained at 14 cm, with a flow rate of 1.5 mL/h. Using an electrospray technique, Cur/PLGA microparticles (referred to as C/P) were stably obtained at room temperature (23–28 °C) and stored under vacuum at room temperature. According to the method proposed by Van Den Bulcke et al., [27] gelatin was dissolved in PBS solution under water bath heating conditions to obtain a 10% w/v gelatin solution. Methyl acrylate (MA) was added to the continuously stirred gelatin solution and the reaction was maintained at 60 °C for 1 h. The resulting mixed solution was filtered and placed in a dialysis bag for one week. The dialysis solution was then filtered and lyophilized to obtain lyophilized GelMA. The synthesised GelMA was dissolved in LAP initiator to produce a GelMA hydrogel precursor solution. After mixing the C/P with the GelMA hydrogel precursor solution under ultrasound, the mixture was placed in a microneedle mould. The mixture was subjected to repeated vacuum extraction at 50 °C to remove bubbles and ensure that the solution filled the needle tips. After several rounds of concentration in an oven at 30–35 °C, the preliminary structure of the microneedles was formed. ZnO NPs were mixed with the GelMA precursor solution under ultrasound and deposited onto the microneedle substrate. After photopolymerisation, the mixture was dried at 30–35 °C to ensure complete solidification of the hydrogel, resulting in the final MN-C/P-Z structure. According to the method described for preparing MN-C/P-Z, prepare pure GelMA hydrogel microneedles without C/P and ZnO NPs, referred to as MN; GelMA hydrogel microneedles loaded only with C/P, referred to as MN-C/P; and GelMA hydrogel microneedles loaded only with ZnO NPs, referred to as MN-Z group, and a blank group as an experimental control group.

Characterization of microneedles

The microstructure of MN-C/P-Z and C/P was observed using a light microscope (Nikon, Japan) and a scanning electron microscope (SEM, Phenom G2, Netherlands). The contact angle of MN-C/P-Z was measured to evaluate their hydrophilic properties. The MN-C/P-Z array was brought into contact with pig skin, and a certain pressure was applied to ensure complete contact between the microneedles and the pig skin surface. The adhesion of the microneedles to the pig skin was then observed and recorded by gently bending the pig skin surface, with corresponding photographs being taken for further analysis. In the compression test, which was conducted to evaluate the mechanical properties and stability of the MN-C/P-Z, a microneedle sample was fixed on a stainless-steel platform with adhesive, ensuring the needle tips were facing up. The sensor probe was set to approach the microneedles at a constant speed of 5 mm/min, with an initial distance of 1 cm between the probe and the needle tip. For evaluating the actual application effect of MN-C/P-Z and their interaction with biological tissues, a porcine skin tissue puncture test was performed. The tips of the MN-C/P-Z were stained with trypan blue dye and punctured directly into porcine skin tissue. Afterward, the surface dye was removed with alcohol before observation and photography. The MN-C/P-Z were stained with rhodamine dye and punctured into the porcine skin tissue. Following the removal of the microneedles, the porcine skin was embedded, sectioned with a cryostat, and observed under a fluorescence microscope. ImageJ software was utilized for analysis, and the diameter of C/P was measured.

To determine the release of curcumin from C/P and MN-C/P-Z, the amount of curcumin released at various time points was measured. The standard curve was calculated and plotted using Origin software. The cumulative release rate at each time point is the amount of curcumin released divided by the total amount released.

The mass of the MN-C/P-Z was weighed and recorded as W₀. The microneedles were then soaked in PBS buffer solution at room temperature and removed at 1 min, 3 min, 10 min, 30 min, and 24 h, respectively. The wet mass of the microneedles was recorded as W₁, and the swelling ratio S was calculated.

In vitro cytocompatibility of GelMA hydrogel microneedles

Live/dead staining was used to assess the growth and survival of L929 cells seeded with different GelMA hydrogel microneedles. The specific procedure was as follows: L929 cells suspension at a density of 1.0 × 10⁴ cells/mL was seeded into a 24-well plate, and the extraction fluid collected from control, MN, MN-C/P, MN-Z, and MN-C/P-Z was added. Cells were then cultured in a 37 °C incubator with 5% CO₂. Live/dead staining was performed on the 1st, 3rd, and 5th days of culture. Before the assay, the live/dead cell staining solution was prepared in advance. The cells were first stained with the live cell staining solution for 20 min, followed by staining with the dead cell staining solution for 5 min. The distribution of live and dead cells was observed and photographed under a fluorescence microscope. Simultaneously, the bioactivity of L929 cells seeded with the extraction fluid from different microneedles was evaluated using the CCK-8 assay. Briefly, L929 cells were seeded in a 96-well plate and co-cultured with the extraction fluid from each group for 1, 3, and 5 days. The culture medium was then harvested. At the same time points, complete culture medium containing 10% CCK-8 was added and incubated at 37 °C for 2 h. Absorbance at 450 nm was measured using a microplate reader.

The scratch assay is commonly used to evaluate cells’ migration ability. The procedure follows: L929 cells were seeded in a 12-well plate and cultured until they formed a monolayer, then starved overnight. A vertical scratch was made on the surface of the cell monolayer using the tip of a 200 µL sterile pipette. The cells were then washed with PBS to remove cell debris and co-cultured with the extraction fluid from each group at 37 °C. Observations and photographs were taken at 0, 12, 24, and 48 h using an Olympus inverted microscope. The degree of scratch healing was quantitatively calculated and evaluated using ImageJ software. The effect of cell migration was quantitatively analyzed by comparing the cell coverage in the scratched area.

Blood compatibility was evaluated using fresh rabbit whole blood. A red blood cell suspension with a volume fraction of 2% was prepared by centrifuging the whole blood. Microneedles from different groups were soaked in PBS for 48 h to prepare the extraction solutions. Subsequently, 200 µL of each extraction solution (experimental group, A_t), 200 µL of PBS (negative control, A_n), and 200 µL of Triton X-100 (positive control, A_p) were mixed with 200 µL of the red blood cell suspension in 1.5 mL centrifuge tubes. After incubation for 1 h at room temperature, the mixtures were centrifuged at 1500 rpm for 5 min, and the supernatants were collected. The absorbance of the supernatants was measured at 545 nm. The hemolysis rate was calculated using the following formula:

Antimicrobial experiments & DPPH

Antimicrobial activity of microneedles by surface contact method

The antimicrobial activity of the microneedles was tested using a surface contact method [28]. Each side of the microneedles was subjected to ultraviolet (UV) light exposure for 30 min and then placed in a 48-well plate. A 10 µL aliquot of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) suspensions (10⁷ CFU/mL for each strain) was inoculated independently onto designated microneedle surfaces. After a 12-hour incubation at 37 °C, the samples were diluted with 100 µL of PBS and then used for further analysis. The plate colony counting method was employed to quantify bacterial populations. The bacterial suspension was diluted 100-fold, and 50 µL of the solution was plated onto LB agar plates. The number of colonies was enumerated after a 24-hour incubation period at 37 °C. The bacterial viability was determined using a Live/Dead BacLight viability kit. Live bacteria were identified by green fluorescence (SYTO 9 stain), while dead or membrane-compromised bacteria exhibited red fluorescence (propidium iodide stain). Their viability was observed under an inverted fluorescence microscope. The morphological changes of bacteria before and after treatment were investigated using SEM. The bacteria were collected by centrifugation and washed, then fixed in 1.25% glutaraldehyde and 4% paraformaldehyde in PBS containing 4% sucrose at pH 7.4, overnight at 4 °C. The samples were washed and subjected to an ascending series of dehydration in ethanol (50, 70, 90, 95, and 100%) for 10 min each. The samples were then chemically dried using hexamethyldisilazane (HMDS), drop-casted on glass coverslips, sputter-coated with 1 nm of platinum, and examined using SEM.

Bacterial biofilm assay

The activated bacteria were inoculated into a 48-well plate, with 100 µL of bacterial culture medium and 100 µL of each group of MNs extract solution added to each well. Subsequently, the culture plate was transferred to a constant-temperature incubator set at 37 °C for 36 h to facilitate biofilm formation. Following the designated culture period, the culture medium was removed, and each well was washed three times with sterile PBS buffer to remove any unattached bacteria. Subsequently, the biofilm was stained with a 1% crystal violet solution for 30 min, after which photography was conducted. Following the staining procedure, the excess dye was removed with sterile water, and a 33% glacial acetic acid solution was added to elute the crystal violet from the biofilm. The absorbance was determined by measuring the optical density at 590 nm using a plate reader or spectrophotometer.

DPPH

The DPPH radical scavenging activity of the microneedle extracts was evaluated using a standard DPPH assay. Briefly, 100 µL of each microneedle extract was mixed with 100 µL of a 0.1 mM DPPH methanol solution in a 96-well plate. The mixture was incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a microplate reader. The scavenging rate was calculated using the formula:

Herein, A₀ denotes the absorbance of DPPH free radicals in the absence of any sample, whereas A_S represents the absorbance of DPPH free radicals following the reaction with the added sample.

In vitro anti-scar experiment

The experiment was approved by the Ethics Committee of The Affiliated Hospital of Qingdao University in accordance with the institution’s guidelines (approval number: QYFYWZLL29048). The explant method was employed to extract scar tissue cells cultured for 3–5 passages before subsequent experimentation.

Cytoskeleton

The scar tissue cells were seeded onto culture dishes containing sterile coverslips and co-cultured with various MNs extract solutions for 48 h. The cells were fixed with 4% paraformaldehyde for 15 min and then rinsed three times with PBS. Subsequently, the cells were treated with 0.1% Triton X-100 at room temperature for 10 min, followed by three rinses with PBS. FITC staining was conducted at room temperature for 30 min, followed by 10 min of counterstaining with DAPI at room temperature. Subsequently, the coverslips were sealed, and the fluorescence images of the cytoskeleton were observed under a confocal microscope.

Immunofluorescence

The scar tissue cells were seeded onto culture dishes containing sterile coverslips and co-cultured with various MNs extract solutions for 48 h. The cells were fixed with 4% paraformaldehyde for 15 min and then rinsed thrice with PBS. The cells were treated with 0.1% Triton X-100 at room temperature for 10 min, followed by three rinses with PBS. To reduce non-specific binding, cells were blocked with 5% normal goat serum (NGS) in PBS for 30 min at room temperature. Subsequently, the blocking solution was discarded, and the primary antibody solution, prepared at the appropriate working concentration, was added. The cells were incubated in a humidified chamber at 4 °C overnight. Subsequently, the cells were washed three times for five minutes each with TBST buffer (PBS containing 0.1% Tween 20) to remove unbound primary antibodies. The secondary antibody, conjugated with a fluorophore that corresponds to the species of the primary antibody, was then added and incubated at room temperature in the dark for 1 h. Subsequently, the cells were counterstained with DAPI for 5 min. Subsequently, the coverslips were sealed, and the images were observed and captured under a fluorescence microscope, with all groups being photographed under the same exposure time. The fluorescence intensity and distribution were analyzed quantitatively using dedicated image analysis software.

Western blot

The scar tissue cells were seeded onto 6-well plates and co-cultured with various MNs extract solutions at 37 °C until the cell density exceeded 90%. Total protein was extracted from cells of each group, lysed, and denatured by heating at 95–100 °C for 10 min. Subsequently, SDS-PAGE electrophoresis was conducted, followed by transfer, blocking, and an overnight incubation period with the primary antibody at 4 °C. Subsequently, the membrane was washed with TBST buffer to remove any unbound primary antibodies. The secondary antibody was incubated at room temperature for one hour. Subsequently, the membrane was rewashed with TBST buffer to remove any unbound secondary antibodies. The detection of signals was conducted using ECL or fluorescent substrates. The density of the immunoblots was subsequently analyzed using the ImageJ software.

In vivo rat experiment

The experiment was approved by the Ethics Committee of Qingdao University for Laboratory Animals according to institutional guidelines (approval number: 20240325SD2020240410043). The infected wound healing experiment was conducted using six-week-old female Sprague-Dawley (SD) rats. Before the commencement of the experiment, the rats were anesthetized by inhalation of 3% isoflurane, and the dorsal hair was removed using a razor and depilatory cream. Four full-thickness circular wounds with a diameter of 10 mm were created using a skin biopsy device. A solution of S. aureus at a concentration of 10⁸ CFU/mL was administered to each wound in a volume of 40 µL. Each MNs was applied to the wounds and secured with 3 M Tegaderm film (3 M Healthcare, USA). The experimental groups were Ctrl, MN, MN-C/P, MN-Z, and MN-C/P-Z. The control group was not treated, whereas the remaining groups were administered the corresponding MNs treatments. Wound photographs were taken at regular intervals. The extent of wound closure was quantified using the ImageJ software, and the formula for calculating this parameter is as follows: The percentage of wound closure was calculated using the following formula:

In this formula, “t” represents the data on day t, and “0” represents the data on day 0. In vivo antibacterial performance tests were conducted on days 0 and 3. On days 7 and 14, wound tissues from each group were collected and processed for histological examination. This involved fixation with paraformaldehyde for H&E staining and Masson’s trichrome staining.

In vivo rabbit experiment

The experiment was approved by the Ethics Committee of Qingdao University for Laboratory Animals according to institutional guidelines (No. 20240305NZR06240410044). Adult male New Zealand White rabbits (2.5–3 kg) were used to establish a hypertrophic scar model. Rabbits were anesthetized by intravenous injection of dexmedetomidine (0.05 mg/kg), chlorpromazine (2.5 mg/kg), and lidocaine (1 mg/kg) [29]. A corneal trephine was used to make four circular wounds, 10 mm in diameter and more than 10 mm apart, on the ventral side of each ear. The perichondrium was carefully removed with a cartilage separator, taking care not to damage the cartilage. The groups were defined as Ctrl, MN, MN-C/P, MN-Z, and MN-C/P-Z. The Ctrl group received no treatment, while the others received the appropriate MNs treatments. Photographs were taken weekly. After 4 weeks, the Scar Elevation Index (SEI) was measured as shown in Fig. 6a, where α + β represents the scar tissue area, and β represents the normal skin area. Ultrasound measurements of scar thickness were also made in each group of rabbit ears. The scar tissues were then collected and fixed with paraformaldehyde. Histological observation and examination were performed using H&E staining, Masson’s staining, Sirius red staining (observed under polarized light), and immunofluorescence staining for Col I, and Col III.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. All experiments were conducted at least three times. The statistical significance between the samples was analyzed by the t-test (two groups) or one-way analysis of variance (ANOVA, multiple groups). The statistically significant difference between groups was expressed as * P < 0.05, **P < 0.01 and ***P < 0.001.