Gold nanoclusters encapsulated microneedle patches with antibacterial and self‐monitoring capacities for wound management

The management of infected wounds is always of great significance and urgency in clinical and biomedical fields. Recent efforts in this area are focusing on the development of functional wound patches with effective antibacterial, drug delivery, and sensor properties. Here, we present novel hyaluronic acid (HA) microneedle patches with these features by encapsulating aminobenzeneboronic acid‐modified gold nanoclusters (A‐GNCs) for infected wound management. The A‐GNCs loaded microneedle patches were derived from negative‐mold replication and showed high mechanical strength to penetrate the skin. The release of the A‐GNCs was realized by the degradation of HA, and the self‐monitor of the released actives was based on the dynamic bright orange fluorescence emitted from A‐GNCs under ultraviolet radiation. As the A‐GNCs could destroy bacteria membranes, the microneedle patches were with excellent in vitro antibiosis ability. Based on these features, we have demonstrated the bacteria inhibition, residual drug self‐monitoring, and wound healing promotion abilities of the microneedle patches in Escherichia coli‐ or Staphylococcus aureus‐infected wound management. These results indicated the great potential of such A‐GNCs loaded microneedle patches for clinical applications.


INTRODUCTION
[3] The management of these wounds is usually time-consuming and needs a careful nursing operation involving the utilization of effective and appropriate antimicrobial agents.10] Although much progress has been achieved, there are still some non-negligible problems that limit their practical wound healing performance.During the process, it is easy to form a scab on the wound surface for selfprotection, which would greatly impede further drug delivery.In addition, the poor stability and short lifetime of biogenic antibacterial agents encapsulated in the existing patches remains an obstacle to efficient wound healing. [5,11]Furthermore, the lack of dynamic monitoring of drug release in the infected wound management always leads to incorrect drug administration.Thus, a new intelligent patch for the effective management of infected wounds is still highly expected.
Herein, we present novel gold nanoclusters (GNCs) encapsulated hyaluronic acid (HA) microneedle patches with features of transdermal antimicrobial agents delivery and residual dose self-monitoring for infected wound management, as schemed in Figure 1.Microneedles are needle-like F I G U R E 1 Schematic illustration of the aminobenzeneboronic acid-modified gold nanoclusters (A-GNCs) encapsulated fluorescent microneedles array applied to the bacteria-infected skin wounds.The A-GNCs in microneedles are released into the infected wound with the hyaluronic acid (HA) degradation to exert its antibacterial effects, while displaying orange fluorescence under the ultraviolet (UV) light excitation.The wound healing and the residual drug self-monitoring were thus achieved.
[28] Although many reports have demonstrated the infected wound healing performances of microneedles or GNC-based nanomedicine, [13,[29][30][31][32] the integration of them remained unexplored.Thus, it is conceived that the encapsulation of antimicrobial GNCs into the microneedles would realize effective drug release and residual dose monitoring in the infected wound healing.
In this paper, taking advantage of the functionalized-GNCs and the microneedle device, we fabricated the desired intelligent microneedle patch for the smart management of infected wounds.Specifically, the GNCs were synthesized via a onestep reaction based on HAuCl 4 , and further modified by glutathione and aminobenzeneboronic acid (ABA) to form ABA-modified GNCs (A-GNCs).After dispersing the A-GNCs to HA solutions, the microneedle patch was generated by negative-mold replication.Owing to the penetration ability of the microneedle together with the degradation of HA, the A-GNCs could be efficiently delivered through the scab and released to perform their bacteria inhibition ability by destroying the bacteria wall.Moreover, benefitting from the intrinsic fluorescence of A-GNCs, the microneedle patch emitted decreased fluorescence under UV irradiation during the administration period, thus realizing the online resid-ual dose monitoring of A-GNCs.Based on these features, we have demonstrated the promising values of the resultant microneedle patch in Escherichia coli-or Staphylococcus aureus-infected wound management, including their bacteria inhibition, residual drug self-monitoring, and wound healing promotion abilities.Therefore, we believe that such smart microneedles would broaden the scope of wound healing patches and find wider applications in biomedical and clinical fields.

Characterization of A-GNCs and GNCs
In a typical experiment, the A-GNCs were synthesized by a one-step reaction method.Glutathione was introduced as the surface ligand for the synthesis of A-GNCs and GNCs, ABA was applied as the modification agent for A-GNCs synthesizing, and the GNCs were prepared without ABA as a control.Both the A-GNC and GNC solutions were pale yellow under the sunlight and could be excited by the UV light (365 nm) to emit the intense orange fluorescence (Figure 2A).The zeta potential of synthesized A-GNCs and GNCs was further tested (Figure S1).Compared to the GNCs with negative zeta potentials, the positive value of A-GNCs suggested the successful modification of ABA on the surface of GNCs.The fluorescence intensity (FI) decreased gradually with the decrease of the nanomedicine concentration (Figure S2).The UV-vis absorption spectra of A-GNCs and GNCs, as well as the bi-exponential fitting curves of the fluorescence decay curves of A-GNCs and GNCs were obtained (Figure S3).On the basis of these, the average luminescence lifetimes of A-GNCs and GNCs were determined to be 1809.3711and 1809.2480ns, respectively.The clear lattice structures of A-GNCs and GNCs were characterized by the transmission electron microscopy (TEM) (Figure 2B,C).Besides, as shown in Figure 2D, their fluorescence emission peak wavelength was proved to be 622 nm (A-GNCs) and 640 nm (GNCs), respectively.There was a blue-shift of the emission peak of A-GNCs compared with that of GNCs, which was probably attributed to the changes in the intramolecular charge distribution caused by the ABA modification.
According to the dynamic light scattering (DLS) analysis (Figure 2E,F), the size distribution curve displayed that a majority of GNCs was at the size ranging from 1.12 to 4.19 nm, a very small amount of oversized or undersized particles may result from the excess of reactants or the aggregation of several GNCs.Moreover, the successful synthesis could also be demonstrated by X-ray photoelectron spectroscopy (XPS) analysis, where O (1s), N (1s), C (1s), S (2p), and Au (4f) electrons simultaneously presented (Figure S4).

Fabrication and characterization of the A-GNCs microneedles array
The synthesized A-GNCs were then encapsulated in the microneedle array via a two-step molding method (Figure 3A).In this process, A-GNCs were dispersed in deionized water, and mixed with HA as the pouring material.Then, the needle-like polydimethylsiloxane (PDMS)-negative mold was filled by the pouring material through vacuuming.The A-GNCs microneedles array was then obtained after solidifying the pouring material based on the evaporation of water under a high temperature and being demoulded from the PDMS mold.As shown in Figure 3B, the microneedles array possessed a 10 × 10 array on a 1.15 × 1.15 cm 2 support base.Owing to the ample A-GNCs contained in microneedles, the microneedles array displayed pale yel-low under the sunlight and could be excited by the UV light to emit the dazzling orange fluorescence, and the FI gradually weakened as the A-GNCs content decreased (Figure 3C).Then, the microstructure of microneedles was observed by applying the scanning electron microscopy (SEM).As displayed in Figure 3D, the microneedles exhibited a cone shape with the sizes of 430 and 950 µm in base diameter and height, respectively.As exhibited in Figure S5, although the A-GNCs microneedles patch appeared light yellow due to the additive A-GNCs, the letters on the paper could be clearly seen through the HA microneedle patch or the A-GNCs microneedle patch on it, which could confirm their high transparency.
The mechanical strength of the A-GNCs microneedles array containing different concentrations of HA was first estimated.In the compression test, the force-displacement curves indicated the displacement increased as the force mounted until the force reached the preset maximum value of 0.9 N/needle (Figure 3E), which greatly exceeded previously reported the magnitude of force to penetrate human skin (0.08 N/needle). [33]Basically, microneedles with higher HA content could withstand greater pressure at the same displacement.Young's modulus (YM) measurement also showed that the ability of microneedles to resist deformation enhanced with the increase of HA content (Figure 3F).Taken together, A-GNCs microneedles array could supply superior mechanical property with sufficient strength to pierce the target skin tissue, and the increased HA in the microneedle would enhance the mechanical strength of the produced microneedle.Then, the effect of the loaded A-GNCs on the mechanical property of microneedles was explored.As force-displacement curves and the YM measurement shown in Figure S6, the microneedles patches with different A-GNCs content exhibited no significant differences in mechanical integrity, which was likely due to the small volume of A-GNCs.The A-GNCs with higher concentration were utilized in many subsequent experiments for more satisfactory antibacterial effects and higher FI.
Before evaluating the release profile of A-GNCs from microneedle array, the fluorescence stability of A-GNCs was estimated by monitoring its FI in the simulated in vivo environment.As Figure S7 displayed, the FI of A-GNCs presented superior stability during the 7 consecutive days of testing.Based on this, the A-GNCs release property of the microneedles array was evaluated.As displayed in Figure 3G, there was a burst release of A-GNCs in the initial 24 h (over 35%) due to the hydrolysis of hyaluronidase and the simulated skin interstitial fluid.Then, the release of A-GNCs reached a plateau 5 days later, indicating a complete hydrolysis of HA.Differences in drug release could be seen among the microneedles made from different concentrations of HA.In detail, compared to microneedles made from 4% and 3% HA, the percentage of A-GNCs released from microneedles made from 5% HA was slightly lower in the early release stage.This may result from the fact that the denser structure coming from a higher HA concentration would decelerate the degradation and drug release processes.Despite of the attempts in the application of GNCs in microneedles for the visual detection of adenosine triphosphate (ATP), or in glass nanoparticles (BGN) for infected wound treatments, [34,35] the combination of the intrinsic fluorescence and the antibacterial activity of A-GNCs, along with the special structural and mechanical strength of microneedles has not been studied.In comparison with the functional nanomaterial-integrated films, the microneedle allowed the administration of payloads to the deep wound due to the penetration through the scab.
Moreover, the intrinsic fluorescence of A-GNCs enabled the microneedle to simultaneously achieve bacteria inhibition and the monitoring of the released actives.

Biocompatibility verification
Before they are applied to in vivo wound management, the biocompatibility, including blood compatibility and cell cytocompatibility, of HA, ABA, GNCs, A-GNCs, and A-GNCs microneedles array was confirmed.The hemolysis test was conducted to evaluate the blood biocompatibility, where the hemolysis rate (HR) of rat erythrocytes co-incubated with different materials was determined, and optical images were taken.The blood compatibility of GNCs and ABA was studied firstly.As Figure S8 exhibited, both ABA with the concentration from 50 to 200 mM, and GNCs with the concentration from 20 to 160 µg/mL caused no significant hemolysis.Similar results could be found in the evaluation of HA, A-GNCs, and microneedles.It demonstrated that the blood compatibility of HA was excellent when its content (w/v) was between 2% and 8% (Figure 4A).As for the A-GNCs, when its concentration ranged from 20 to 160 µg/mL, no significant hemolysis of co-cultured rat erythrocytes could be found (Figure 4B).Therefore, the microneedle patch developed by combining these two possessed superior blood compatibility (Figure 4C).These all indicated that the A-GNCs microneedles had a wide safe content range for blood-contacting applications.Subsequently, the cell cytocompatibility of HA, ABA, GNCs, A-GNCs, and A-GNCs microneedles was studied by co-culturing them with NIH/3T3 cells.Considering the materials will be utilized to manage the infected skin wounds, the NIH/3T3 cells were chosen because they are the main functional cell during the process of wound healing.As manifested in Figures 4D-F and S8c,d, the morphology of NIH/3T3 cells co-cultured with different concentrations of HA, ABA, GNCs, A-GNCs, and A-GNCs microneedles were similar to these of control groups, demonstrating great cell viabilities in these testing groups.Furthermore, the cell viability of NIH/3T3 cells co-cultured with different concentrations of these materials was also measured by the cell counting kit (CCK-8) assay.As shown in Figures 4G-I and S8e,f, the cell viability of all experimental group samples was above 80%.All of these results demonstrated that neither the base material of the microneedle array, HA, nor the additive functional nanomedicines, A-GNCs, had obvious toxicity on NIH/3T3 cells.Thus, this A-GNCs loading microneedle array could be used in in vivo applications with high biological safety.

Antibacterial activity estimation
The loading of A-GNCs also imparts the microneedle array with antibacterial capability, which is significant in the infected wound healing treatment.The antibacterial mechanism was first estimated.Some nanomaterials can simulate the catalytic process mediated by natural enzymes such as oxidase and peroxidase, and increase the intracellular reactive oxygen species (ROS) level, thus killing bacteria by generating the damaging ROS. [36,37]40] Due to the intrinsic enzyme-mimicking catalytic property, these GNCs are capable of inhibiting bacteria growth by producing toxic ROS to cause oxidative stress damages and destroy the cell structure. [35,41]Besides, the cationic surface of GNCs could generate electrostatic interactions between positively-charged nanoparticles and negatively-charged bacteria, promoting efficient binding and further destroying the integrity of cell membrane, thus leading to the extraordinary antimicrobial effect. [42,43]erein, two bacteria (E. coli and S. aureus) were treated with HA, ABA, GNCs, A-GNCs, ABA microneedle, GNCs microneedle, and A-GNCs microneedles, respectively.After cultivation, the morphology of bacteria in different groups was characterized by SEM.As exhibited in Figure 5A,B, there was no obvious difference in bacterial growth status between the HA-treated group and the control group, indicating that HA did not play a major role in antibiosis.More notably, SEM images displayed bacteria cell wall collapse caused by A-GNCs in corresponding experimental groups, and some of them even merged.All of these were corresponding to the previous report that individual GNCs or ABA exhibited no bacteriostasis, while modifying GNCs with ABA could effectively inhibit bacteria by destroying its cell wall. [44,45]The minimum inhibition concentration (MIC) of the A-GNCs or A-GNCs microneedles against E. coli and S. aureus was also investigated by the Luria-Bertani (LB) dilution method.The photos of E. coli or S. aureus suspensions treated by different concentrations of A-GNCs or the leaching solution of A-GNCs microneedles were taken, and the corresponding bacterial concentration was measured as well.The decreased turbidity of bacteria suspension suggested an increased inhibition effect of the increased A-GNCs content (Figure S9a-d).The lowest turbidities visible to naked eye corresponded to the 8 µg/mL A-GNCs and the 10 µg/mL A-GNCs microneedles groups, respectively, suggesting the MIC of A-GNCs to E. coli or S. aureus was 8 µg/mL, and the MIC of A-GNCs microneedles to E. coli or S. aureus was 10 µg/mL.In order to achieve effective antibacterial effects, the A-GNCs concentration applied in the followup studies was higher than the MIC within the biosafety range.
To further verify the in vitro antibacterial activity, HA, A-GNCs, and A-GNCs microneedles were co-cultured with E. coli or S. aureus, respectively.The bacterial live/dead staining results showed that after 24 h of co-culture, both the mortality of E. coli and S. aureus increased significantly with the increasing concentration of the nanomedicine (Figure 5C,D).Besides, the bacteriostatic rate (BR) of each group was also calculated.Similar to the results of bacterial live/dead staining, compared with the control group, the BR of both the A-GNCs-treated group and the A-GNCs microneedle-treated group increased substantially after the co-culture (Figure 5E-G).In contrast, the live/dead stained bacteria images of GNCs, ABA, ABA microneedle, or GNCs microneedle-treated groups, and the corresponding low BR implied the GNCs, ABA, ABA microneedle, or GNCs microneedle had no bacteria inhibition effects (Figure S10).These features demonstrated the potent antimicrobial effect of A-GNCs microneedles array.

2.5
Evaluation of E. coli-or S. aureus-infected skin wound models on rats Because both Gram-positive and Gram-negative bacterial infections may occur at the wound site, E. coli and S. aureus were utilized to build infection skin wound models as two representative pathogens.Sprague-Dawley (SD) rats were infected by E. coli or S. aureus after the fullthickness cutaneous wound on their back was created.Each infection model was divided into the treatment group and the control group.Then, A-GNCs microneedle patches were applied to the wounds of the treatment group rats, while the gauze was utilized to cover wounds of the control group rats.During the wound healing process, the wound site was recorded under both sunlight and UV lights (Figure 6A,B).It could be seen that the microneedles exhibited obvious orange fluorescence under UV lights due to the encapsulation of fluorescent A-GNCs and transparent HA.Basically, the FI gradually reduced showing the release of A-GNCs in microneedles during the wound healing, and it almost became zero on the seventh day indicating the full-release.Compared to the in vitro release study, the total release time of A-GNCs from microneedle was prolonged.This may be attributed to the complicated microenvironment of wound sites.Notably, the release period was fully corresponding to the change of healed wound areas.As shown in Figure 6C,D, after 10 days of treatment, the healing ratio of the wound area was 84.69% (E.coli-infected wound) and  87.44% (S. aureus-infected wound) in A-GNCs microneedletreated groups.These results illustrated that the A-GNCs encapsulated microneedle patches effectively expedited the healing of the bacteria-infected wounds and at the same time showed self-monitoring property.
During the healing process, the wound constriction, the proliferation of fibroblasts as well as the progress of the epithelization were observed and recorded by hematoxylineosin (HE) staining.It could be seen that the wound range of A-GNCs microneedle group was significantly reduced compared to those of the control group, which was in consistency with the statistical results of wound area (Figure S11a,b).The successful construction of the infected wound model was demonstrated by the exhibition of inflammatory responses.As shown in Figure 7A,B, the infiltration of neutrophils or lymphocytes (indicated by yellow arrows) can be observed at the wound site, indicating the severe infection there.After 7 days of microneedle patch treatment, compared to the control group, elongated fibroblasts (indicated by light blue arrows), newborn capillaries (indicated by green arrows), proliferated epidermal cells (indicated by orange arrows), and reduced inflammatory cells could be observed in the A-GNCs microneedle-treated group.These implied that the A-GNCs microneedle patches had constructive effects on inflammation regression and re-epithelization in infectious wounds.In addition, the thinned and compacted epidermal layer, the generation of hair follicles (indicated by dark blue arrows), as well as sebaceous glands (indicated by black arrows) demonstrated in the A-GNCs microneedle patches-treated group also proved this.
Besides, to further investigate the therapeutic effect of microneedles patch, the HA microneedles, the A-GNCsloading film and the A-GNCs microneedles without UV irradiation, were also used as comparison groups.The wound tissues were studied by HE staining (Figure S12).Although neovascularization and elongated fibroblasts existed in the HA microneedles group, infiltration of neutrophils or lymphocytes can still be observed due to the lack of antibacterial activity.In addition, the tissue remodeling was inferior compared to the A-GNCs involving groups.While in the groups treated by both HA and A-GNCs, the increase in angiogenesis, fibroblasts as well as the thickness of the epidermal layer, and the reduction of inflammatory cells, showcased the effective antibiosis of A-GNCs and healing promotion abilities of the composites.Notably, as shown in Figure S13, the live/dead stained bacteria images indicated that the mortality of bacteria significantly increased after the co-culture with A-GNCs-loading film.Besides, the BR of the A-GNCs-loading film-treated group was preferable (over 80%), indicating its antibacterial effect.However, compared to the A-GNCsloading film, the increased number of newly-formed hair follicles and sebaceous glands together with the denser epidermal layer in the A-GNCs microneedles group suggested better reconstruction of the wound tissue.This may be attributed to the antibacterial effects of A-GNCs in deep tissues delivered via microneedle penetration.The effect of UV on wound healing could be eliminated based on the similar wound healing effect of A-GNCs microneedles without UVirradiation group.It could be seen that no apparent difference in histological staining observation between microneedles with or without UV-irradiation groups, which was probably owing to the very short period of UV irradiation only for residual dose monitoring of A-GNCs purpose.
The wound healing conditions including collagen deposition and inflammation were further investigated by Masson's trichrome staining.Compared with the control groups, the collagen fibers in the A-GNCs microneedle patches-treated groups aligned regularly (Figure S14).Besides, the A-GNCs microneedle patches-treated wounds had more uniform epithelial cell layers with hair follicles, sebaceous glands and blood capillary compared to the control ones.These all demonstrated that the A-GNCs microneedle patch possessed excellent abilities to promote the healing of infected wound and the remodeling of tissue.The inflammation was evaluated by studying the expression of interleukin-6 and tumor necrosis factor-α at the infected wound site (Figure 8).Immunofluorescence staining revealed that the FI decreased in the A-GNCs microneedle patches-treated group during wound healing, in contrast to the strong intensity observed in the control group.The corresponding FI analysis was consistent with the results shown in the immunofluorescence staining images (Figure S15).These findings suggested that the A-GNCs microneedle patches have a significant antiinflammatory effect, as proved by the downregulation of two inflammatory factors.To verify the biosafety of the A-GNCs microneedle during in vivo wound healing application, blood samples of SD rats were collected at fixed time points and subjected to routine blood tests (Tables S1 and S2) and serum tests (Figure S16).The infection by E. coli or S. aureus could also be revealed in the higher level of lymphocytes and lower level of neutrophils compared with the normal 3 days post-surgery.However, the treatment of A-GNCs microneedle patches downregulated the level of lymphocytes and upregulated the level of neutrophils to the normal, implying that the A-GNCs microneedles had successfully cured the infection of the wounds.Apart from these, the serum test results showed that levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) as well as uric acid (UREA) of the A-GNCs microneedletreated groups showed no significant difference from those of control groups, indicating that the A-GNCs microneedle patches would not cause adverse effects on liver and kidney functions during the infected wound healing.Taken together, the A-GNCs encapsulated HA microneedle patches posed good biosafety and exhibited excellent antibacterial as well as healing promoting effects, which were greatly beneficial in bacterial-infected wound management.

CONCLUSIONS
In summary, we developed a brand-new nanomedicineencapsulated microneedles array for the smart care of bacteria-infected wounds.This microneedles array was fabricated by loading the A-GNCs synthesized by the one-step reaction method to the HA microneedle substrate duplicated from PDMS-negative molds.The HA matrix imparted the microneedle with high transparency and biosafety, whereas A-GNCs inside the microneedle tips showed potent antibacterial activity and bright orange fluorescence under UV light.The penetration of A-GNCs microneedle patch into the wound tissue together with the degradation of HA enabled the release of A-GNCs to inhibit bacteria.Meanwhile, with the release of A-GNCs, the observed FI decreased, achieving an online residual dose monitoring during wound healing.The desirable practical performance of these resultant microneedle patches was then explored by applying them to E. colior S. aureus-infected full-thickness cutaneous wounds.It was demonstrated that bacteria inhibition and residual drug self-monitoring of our microneedle patches facilitated the treating and evaluation of wound healing process.These results indicated that such smart microneedles were promising candidates in bacteria-infected wound management and related biomedical fields.

Materials and animals
ABA (Mw = 136.94),glutathione (Mw = 307.33),tetrachloroauric acid (HAuCl 4 ⋅3H 2 O), HA, and hyaluronidase were purchased from Sigma.The PDMS-negative mold was purchased from Taizhou Research Institute of Zhejiang University.The deionized water was purified using a Milli-Q Plus 185 water purification system (Millipore) with a resistivity of 18.25 MΩ × cm.All reagents were of analytical grade and used as received.The male SD rats of 4-week-old were supplied by Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health).Animals were treated in strict accordance with the recommendations in the "Guidelines for the Care and Use of Laboratory Animals in China".All the animal care and experimental protocols were reviewed and approved by Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health).

Synthesis and characterization of A-GNCs
Glutathione (100 mM), HAuCl 4 (10 mM), and ABA (100 mM) were prepared into the aqueous solution, separately.Then, glutathione solution (0.6 mL), HAuCl 4 solution (5 mL), and the ABA solution (0.3 mL) were mixed.The final volume of the reaction system was kept at 20 mL, stirring with the rpm of 500 at 70 • C for 24 h.The GNCs were prepared using the same formula without adding ABA.Subsequently, to get rid of the unreacted chemicals, the solution was dialyzed in the deionized water in the dialysis bag (3000 Da Mw) for 72 h.Next, the dispersion was sterilized by the filter (0.22 µm) and then kept in cold storage at 4 • C for further use.
TEM (FEI Talos) was applied to characterize the morphologies of A-GNCs.The elemental gold was measured by the inductively coupled plasma mass spectrometry (Agilent 7850) to determine the A-GNCs concentration.The size distribution and the zeta potential of A-GNCs and GNCs were assayed by a DLS device (Malvern, Zetasizer Nano ZS ZEN3600).XPS (Thermo Fisher Scientific) was utilized to characterize the chemical element composition of the A-GNCs.The fluorescence spectra and the UV-vis absorption spectra of A-GNCs and GNCs were acquired by the microwell plate reader (Thermo Fisher Varioskan LUX).The fluorescence decay curves of A-GNCs and GNCs were obtained using a steady state/transient fluorescence spectrometer (FLS 980).The average luminescence lifetime (T) of A-GNCs and GNCs was determined according to the following formula: where A 1 and A 2 represent the amplitude of the decay of the first lifetime component and the second lifetime component, respectively, and t 1 and t 2 represent the lifetime of the first component and the second component, respectively.
To verify the stability of the fluorescence of A-GNCs in the stimulated in vivo environment, A-GNCs were added to phosphate-buffered saline (PBS) buffer containing fetal bovine serum (10%) and placed at 37 • C, then its FI (excitation wavelength: 365 nm, emission wavelength: 622 nm) was measured for 7 consecutive days.

Fabrication and characterization of the A-GNCs microneedles array
A-GNC solution (160 µg/mL) was applied as the solvent to prepare the A-GNCs/HA (5%, w/v) solution, and the loading percentage of A-GNCs in the microneedle was 0.32% (w/w).The PDMS-negative mold was treated by the plasma surface treatment instrument for 3 min.Then, 300 µL of the above A-GNCs-HA solution was added into the PDMS-negative mold and then injected into needle-shaped pores of the mold by vacuuming.Next, the negative mold containing A-GNCs-HA solution was placed at 37 • C until the water was completely evaporated.Then, the above steps were repeated twice to increase the A-GNCs content.Then, the demolding process was carried out, and the A-GNCs microneedles array was prepared.
The optical images of the microneedles array were obtained by applying the stereomicroscope (Olympus SZX16).The microneedles' morphology was observed by the scanning electron microscope (HITACHI S-3400N).Laser scanning confocal microscopy (Nikon A1) was utilized to obtain the fluorescence images.Then, the mechanical strength test was conducted.The microneedles array was put on the sample stage with their needles vertically upward.Next, a vertical downward force was exerted and the displacement-force curve and YM of the microneedles array were obtained by recording the displacement when the sensing element of the testing machine (Instron, 5944) touched the needles.

The A-GNCs release curve of A-GNCs microneedles array
The A-GNCs release performance of the microneedles array was studied based on the fluorescence spectrophotometry method.To monitor the A-GNCs released from the microneedles, the microneedles were soaked in PBS buffer (pH 7.35) containing 50 ng/mL hyaluronidases at 37 • C. At certain time points, the leaching liquor of samples was collected and the corresponding FI at 622 nm was measured by the microwell plate reader, then the concentration was obtained based on the standard curve, and the release percentage was further determined.

Biocompatibility of HA, A-GNCs, and the A-GNCs microneedles array
The hemolysis assay was exerted to test the blood compatibility.First, the fresh rat blood was centrifuged with the rpm of 1500 for 15 min and then resuspended with saline.Next, different concentrations of HA (2%, 4%, 6%, and 8%, respectively), A-GNCs (20, 40, 80, and 160 µg/mL, respectively), ABA (50, 100, 150, and 200 mM, respectively), GNCs (20,  40, 80, and 160 µg/mL, respectively), as well as microneedle arrays encapsulated with different concentrations of A-GNCs (20, 40, 80, and 160 µg/mL, respectively) were added into the erythrocyte solution and incubated at 37 • C for 3 h.The deionized water was the positive control group and the saline was the negative control group.After the samples were centrifuged at 1500 rpm for 5 min, their optical densities were measured at 567 nm (OD 567nm ) by a microwell plate reader.The optical photographs of the samples were also taken to observe the hemolysis.Then, the OD 567nm value was processed according to the following formula to determine the HR: where OD sample was the OD 567nm value of experimental group samples, OD negative was the OD 567nm value of the negative control group, OD positive was the OD 567nm value of the positive control group.The cytotoxicity of HA, A-GNCs, and the A-GNCsencapsulated microneedles array was evaluated by the cell viability test.First, the cells (3T3, 10 5 cells/mL) were cultured in DMEM with the addition of 1% penicillinstreptomycin (Gibco) and 10% fetal bovine serum (Gibco), at 37 • C with 5% CO 2 .The cells were co-cultured with different concentrations of HA (0%, 3%, and 5%, respectively), A-GNCs (0, 10, and 20 µg/mL, respectively), ABA (0, 5, and 10 mM, respectively), GNCs (0, 10, and 20 µg/mL, respectively), and 5% HA encapsulated different concentrations of A-GNCs (0, 10, and 20 µg/mL, respectively) for 24, 48, and 72 h.The cell viability at different time points was measured by applying CCK-8.Subsequently, the cells were stained by Calcein/PI and the fluorescent images of their morphology were observed and then obtained by the fluorescence microscope (ZEISS, Axio Vert.A1).

Antibacterial activity of A-GNCs and A-GNCs microneedles array
The bacteria (E. coli or S. aureus) was co-cultured with different concentrations of HA (0%, 3%, 4%, and 5%, respectively), A-GNCs (0, 40, 80, and 160 µg/mL, respectively), A-GNCs microneedles array (prepared using 5% HA and the A-GNC solution with concentrations of 0, 40, 80, and 160 µg/mL, respectively), ABA (0, 50, 100, 150, and 200 mM, respectively), GNCs (0, 20, 40, 80, and 160 µg/mL, respectively), GNCs microneedles array (prepared using 5% HA and the GNC solution with the concentration of 160 µg/mL), ABA microneedles array (prepared using 5% HA and the ABA solution with the concentration of 200 mM), and the A-GNCs-loading film (prepared using 5% HA and the A-GNC solution with the concentration of 160 µg/mL) at 37 • C for 24 h, respectively.After the cultivation, the absorbance at 600 nm (OD 600nm ) of the bacteria suspension was measured.Then, the OD 600nm value was processed according to the following formula to determine the BR: where OD control was the OD 600nm value of the control group, OD experiment was the OD 600nm value of the experimental group, OD blank was the OD 600nm value of the blank medium control group.The bacteria were stained by Calcein/PI and the fluorescent images of the bacterial live/dead staining were obtained by applying the fluorescence microscope.The bacteria were fixed using 2.5% glutaraldehyde and then dehydrated sequentially with ethanol of different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% [v/v]).Next, the images of the morphology of the two bacteria were acquired by the SEM.
The MIC of the A-GNCs and the A-GNCs microneedles patch was investigated by the LB dilution method.The bacteria (E. coli or S. aureus) were cultured in the LB medium (pH 7) for 24 h, then the original concentration was diluted to 1 × 10 4 CFU/mL.Next, the bacteria (E. coli or S. aureus) was co-cultured with A-GNCs of different concentrations (0, 2, 4, 6, 8, 10, 12, and 14 µg/mL, respectively), or the leaching solution of A-GNCs microneedles patch (prepared using 5% HA and the A-GNC solution with concentrations of 0, 2, 4, 6, 8, 10, 12, and 14 µg/mL, respectively) in LB medium for 24 h.The blank LB medium and the bacteria in LB medium without any other addition were set as control groups.Then, photos of the samples were obtained to observe the bacteria growth.

Animal experiment and histological analysis
Five-week-old male SD rats were anesthetized, after the anesthesia took effect, their back hair was shaved.Then, the 1 cm diameter full-thickness cutaneous wound was created on rat's back.Subsequently, 100 µL bacteria (E. coli or S. aureus) suspension with a concentration of 1 × 10 8 CFU/mL was dripped onto the wound and stayed for 1 h then wiped with gauze.Twenty-four hours after the operation, the rat model was established when the obvious infection foci formed.Then, the infected wounds of the rats in the experimental groups were treated with the A-GNCs-encapsulated microneedles patch (prepared using the A-GNCs solution with the concentration of 160 µg/mL), the HA microneedle patch (prepared using 5% HA), or the A-GNCs-loading film (prepared using the A-GNCs solution with the concentration of 160 µg/mL), respectively.The infected wounds of the control group rats were covered with gauze.The photos of wounds under sunlight or UV light were taken to record and monitor the wound healing condition and the residual A-GNCs in the microneedles patch.The rats were euthanized on days 3, 7, and 10 after the treatment and the tissue of the wound site was excised.Then the excised tissues were immersed in 4% paraformaldehyde (PFA) for 24 h to fix, and then embedded in paraffin for the histological analysis.Meanwhile, the blood samples of rats were collected for the serum tests and the routine blood tests.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F I G U R E 2
Characterization of aminobenzeneboronic acid-modified gold nanoclusters (A-GNCs) and GNCs.(A) Optical photos of GNC and A-GNC solutions under sunlight and ultraviolet (UV) light.(B and C) Transmission electron microscopy (TEM) images of (B) A-GNCs and (C) GNCs.(D) Fluorescence emission spectra of A-GNCs and GNCs.(E and F) Particle size distribution curve of (E) A-GNCs and (F) GNCs.The inserted images showed the crystal structure of the nanomedicines.The scale bars are 5 nm in (B) and (C), and 1 nm in the insert.

F I G U R E 3
Fabrication and characterization of the aminobenzeneboronic acid-modified gold nanoclusters (A-GNCs) microneedles array.(A) Schematic of the A-GNCs microneedles array manufacturing process.(B) The digital graphs and partially enlarged images of the A-GNCs microneedles array under (i and ii) sunlight and (iii and iv) ultraviolet (UV) light.(C) Photographs of the microneedles array containing different concentrations of A-GNCs under (i) sunlight and (ii) UV light.(D) Scanning electron microscopy (SEM) image of the A-GNCs microneedles array.(E) The displacement-force curve of the compression tests on different microneedles arrays.(F) Young's modulus (YM) measurement of microneedles containing different concentrations of hyaluronic acid (HA).(G) The release curve of encapsulated A-GNCs in microneedles.The scale bars are 400 mm in (i) and (iii) of (B), 600 µm in (ii) and (iv) of (B), and 300 µm in (D), respectively.Error bars indicate the standard error of the mean.

F I G U R E 5
Antibacterial activity estimation.(A and B) Scanning electron microscopy (SEM) characterization of the morphology of (A) Escherichia coli and (B) Staphylococcus aureus treated by different concentrations of hyaluronic acid (HA), aminobenzeneboronic acid-modified gold nanoclusters (A-GNCs), and A-GNCs microneedles.(C and D) Live and dead bacteria staining of (C) E. coli and (D) S. aureus by co-culturing them with different concentrations of HA, A-GNCs, and A-GNCs microneedles.(E-G) Bacteriostatic rate (BR) of bacterial suspension treated by different concentrations of (E) HA, (F) A-GNCs, and (G) A-GNCs microneedles.The scale bars are 600 nm in (A) and (B), and 50 µm in (C) and (D).Error bars indicate the standard error of the mean.

F I G U R E 6
Evaluation of Escherichia coli-or Staphylococcus aureus-infected skin wound models on rats.(A and B) Photographs of wounds of different groups at different observation time points under sunlight and ultraviolet (UV) light and wound traces different groups.(C and D) Healing rate of (C) E. coli-infected or (D) S. aureus-infected wounds treated by different groups.The scale bars are 500 mm.Error bars indicate the standard error of the mean.**p < 0.01, ***p < 0.001, analyzed by the one-way analysis of variance (ANOVA) test.

F I G U R E 7
Histological analysis of Escherichia coli-or Staphylococcus aureus-infected skin wound models on rats.(A and B) Hematoxylin-eosin (HE) staining of the excised wound tissues of different groups at different time points.The scale bars are 200 µm.

F I G U R E 8
Immunofluorescence staining of the excised wound tissues.(A and B) The immunofluorescence staining image of (A) interleukin (IL)-6 and (B) tumor necrosis factor (TNF)-α of the Escherichia coli-infected group at (i) day 3, (ii) day 7, and (iii) day 10 after infection.(C and D) The immunofluorescence staining image of (C) IL-6 and (D) TNF-α of the Staphylococcus aureus-infected group at (i) day 3, (ii) day 7, and (iii) day 10 after infection.The scale bars are 200 µm.
Yi: Methodology; data curation; writing-original draft preparation.Yunru Yu and Yu Wang: Supervi-sion; writing-reviewing and editing.Lu Fan: Writingreviewing and editing.Li Wang: Writing-reviewing and editing.Yuanjin Zhao: Conceptualization; supervision; funding acquisition; writing-reviewing and editing.A C K N O W L E D G M E N T S This work was supported by the National Key Research and Development Program of China (2020YFA0908200), the National Natural Science Foundation of China (52073060 and 61927805), the Guangdong Basic and Applied Basic Research Foundation (2021B1515120054), and the Shenzhen Fundamental Research Program (JCYJ20210324133214038).