Engrailed-1 (En1) activation in fibroblasts. Our experiments demonstrate that PF-MNs promote scarless wound repair in mouse models of both acute and chronic wounds, and inhibit the formation of hypertrophic scar in rabbit ear models.Effective reprogramming of chronic wound healing remains challenging due to the limited drug delivery efficacy hindered by physiological barriers, as well as the inappropriate dosing timing in distinct healing stages. Herein, a core-shell structured microneedle array patch with programmed functions (PF-MNs) is designed to dynamically modulate the wound immune microenvironment according to the varied healing phases. Specifically, PF-MNs combat multidrug-resistant bacterial biofilm at the early stage via generating reactive oxygen species (ROS) under laser irradiation. Subsequently, the ROS-sensitive MN shell gradually degrades to expose the MN core component, which neutralizes various inflammatory factors and promotes the phase transition from inflammation to proliferation. In addition, the released verteporfin inhibits scar formation by blocking  Effective reprogramming of chronic wound healing remains challenging due to the limited drug delivery efficacy hindered by physiological barriers, as well as the inappropriate dosing timing in distinct healing stages. Here, the authors report a core-shell structured microneedle array patch with programmed functions which dynamically modulates the wound immune microenvironment according to the varied healing phases However, impaired wound healing with dysfunctions of immunoregulation could lead to chronic wounds, which are usually stagnated in the phase of inflammation3. The delayed wound healing could be ascribed to extensive inflammatory responses primarily driven by bacterial infection, which subsequently cause the failure of macrophage polarization and excessive secretion of pro-inflammatory cytokines including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α)6. Consequently, the imbalance of immune microenvironment impedes the healing process by restraining the formation of granulation tissue, neovascularization, and re-epithelialization8. Noteworthily, persistent inflammatory response in early stages is directly associated with deficiencies in dermal appendage, leading to insufficient skin functions and subsequent scar formation1. Therefore, the specific modulation of wound microenvironment in varied repair phases is tightly demanded in clinics, especially for chronic wounds.The classical wound healing process mainly consists of four stages, namely hemostasis, inflammation, proliferation, and remodeling, wherein the regulation of immune microenvironment serves as a key factor to promote the progress in a sequential manner9, the implantation of naturally derived or synthetic scaffolds12, as well as cell therapy14. However, the skin barrier system which includes physical, chemical, immunological, and microbiome barriers significantly hinders the transport of active ingredients, thereby limiting the therapeutic efficacy15. The presence of bacterial biofilms in wounds can further exacerbate inflammation and restrict the efficacy of antibiotic treatments due to their intrinsic resistance16. Although topical antibiotics can help mitigate surface-level infections, they may not be effective against deeper infections because of the limited permeability across the skin barrier. Polymeric microneedles (MNs)17, emerging as effective tools for transdermal drug delivery via perforating the stratum corneum, have been utilized for diagnosis and treatment of various diseases including cancer18, diabetes mellitus19, obesity20, and infection21. MNs encapsulated with antibiotics or metals have demonstrated superior efficacy in eliminating biofilms over other strategies23. However, there are still several challenges remaining in MN-mediated wound healing fields24. For example, most MNs were designed to inhibit bacterial infection only through the controlled release of encapsulated therapeutics, yet dynamical program of immune microenvironment corresponding to distinct healing stages has not been explored.In the last decade, several strategies have been explored for the treatment of chronic wounds by targeting the inflammatory microenvironment around the wound region, such as the delivery of cytokinesEngrailed-1 (En1) activation in fibroblasts to facilitate scarless skin regeneration26. Meanwhile, the exposed cHP core initiated modulation of immune microenvironment by binding and neutralizing various inflammatory factors to induce macrophage polarization from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype, forwarding the phase transition from inflammation to proliferation. In addition, the modulation of immune microenvironment promoted angiogenesis and extracellular matrix deposition, and finally attributed to scarless regeneration. Upon this design, scarless wound healing process was accelerated on both acute wound and diabetic mouse models, as well as on rabbit ear scar models.In this study, we describe a core-shell structured MN patch for the regulation of the inflammation, proliferation, and remodeling phases in a programmed manner Fig. . The MN N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diaminium  at a volume ratio of 2:1  PVA, 2%) and a ROS-sensitive linker % and a R MN Fig. . The hol MN Fig.  and conf MN Fig. . The mecdle Fig. .Fig. 2Th29. Sustained ROS generation by VP under laser irradiation was demonstrated as the increased fluorescence signal of 2′,7′-dichlorodihydrofluorescein diacetate  of macular degeneration clinically30. However, constant presence of pro-inflammatory macrophage could lead to chronic inflammation. In order to promote phase transition from inflammation to proliferation, the cHP was designed to be sheathed by the degradable MN shell during the earlier period of the inflammation stage (first early 24 h), but latterly exposed to neutralize pro-inflammatory cytokines32. To investigate the degradation behavior of the VP@PVA shell, 0.1 mM H2O2 was selected to simulate the inflammatory microenvironment of chronic wounds33. The release profiles of VP from shells with different ratios of PVA and TSPBA were investigated  activation by inhibiting Yes-associated protein (YAP), yielding skin regeneration with less alpha-smooth muscle actin (α-SMA), a marker of activated myofibroblasts that contributed to scar formation26. Western blot analysis confirmed these down-regulations of YAP and α-SMA protein expression in fibroblasts treated with VP  and IFN-γ were served as positive control groups to promote the M1 phenotype transition of RAW 264.7 cells. In contrast, the polarization of macrophage was significantly restricted by neutralizing IFN-γ after the treatment of PF-MNs  was determined by incubating differently charged MNs with IFN-γ. Heparin-formulated MNs (negatively charged) could bind with IFN-γ to reach over 90% neutralization. In contrast, only less than 30% of IFN-γ was neutralized by polyethylene glycol (PEG)-formulated MNs . The crosslinked PVA shell was also validated to isolate the inner heparin core from the outer environment with limited binding of IFN-γ Fig. . SimilarMNs Fig. .34. PF-MNs with laser irradiation (PF-MN + L group) showed nearly 100% inhibition towards Gram-positive Staphylococcus aureus (SA), Gram-negative Escherichia coli (E. coli), Pseudomonas aeruginosa (PA), and Methicillin-resistant Staphylococcus aureus (MRSA), indicating its broad and efficient antibacterial property  often leads to chronic or unhealable wounds1. Thus, we explored the polarization of macrophages around the wounds on day 3 post-treatments through flow cytometry. We found that the administration of PF-MNs significantly reduced the proportion of M1 phenotype (F4/80+CD86+) macrophages. Moreover, the percentage of M2 phenotype (F4/80+CD206+) macrophages showed an elevation in the blank PF-MN group (16.12%) and PF-MN group (23.52%) compared to the Control group (8.46%) and VP group (13.37%), indicating that PF-MNs could promote the polarization of macrophage towards anti-inflammatory phenotype in the presence of cHP core. Besides macrophages, regulatory T cells (Tregs) also make important contributions to the regulation of immune responses  in the PF-MNs involved groups were enhanced, which was favorable for wound recovery. Moreover, haematoxylin and eosin (H&E) staining also showed that tightly connected epidermis was formed in the blank PF-MN group and PF-MN group on day 7 . A variety of pro-inflammatory cytokines including IFN-γ, IL-6, and MCP-1 were decreased after both blank PF-MN and VP-loaded PF-MN treatments , one of the major complications in diabetic people, is hard to heal spontaneously due to its vulnerability to bacterial infection, prolonged inflammation, impaired angiogenesis, and limited cell proliferationMRSA biofilms were generated two days post the introduction of MRSA into the wounds created on the back of streptozotocin (STZ)-induced diabetic mice  Control group (PBS), (2) Anti group (ampicillin), (3) VP + L group (VP solution with laser irradiation), (4) NMN + L group (VP-loaded non-degradable MNs with laser irradiation), (5) PF-MN group (VP-loaded PF-MNs), and (6) PF-MN + L group (VP-loaded PF-MNs with laser irradiation). The wound areas in different groups were measured and photographed on day 0, 3, 7, and 14. As shown in Fig. In the chronic wound model, ice Fig. 34,39. ThWe also found the levels of various pro-inflammatory cytokines mainly released by neutrophils and M1 macrophages, such as IFN-γ, MCP-1, TNF-α, IL-1β, and IL-6, were down-regulated in the PF-MN group and PF-MN + L group Fig. , furtherThe immunofluorescence staining showed the total amounts of macrophage in the wounds with different treatments almost remained the same , while the inflammation was relieved after the treatment of PF-MNs with laser irradiation , with 218 up-regulated and 509 down-regulated genes shown in the volcano plots  Control group (PBS), (2) VP group (VP solution), (3) blank PF-MN group (blank PF-MNs without VP), and (4) PF-MN group (VP-loaded PF-MNs). As shown in Fig. 45. Masson Trichrome staining and Sirius Red staining results also revealed that the collagen fibers (blue) in skin treated with VP solution and PF-MNs were significantly reduced compared to that in other two groups  to M2 (anti-inflammatory) and maintaining appropriate levels of inflammatory factors are essential for forwarding the wound healing stages from inflammation to regeneration. Glycosaminoglycan has been reported to neutralize various inflammatory cytokines31. In this work, heparin was chosen to formulate the core of MN for binding and neutralizing inflammatory cytokines, such as IFN-γ, MCP-1, TNF-α, IL-1β, and IL-6. Furthermore, the inhibition of bacteria and depletion of pro-inflammatory factors also contributed to the polarization of macrophages from M1 phenotype to M2 phenotype, thus remarkably accelerating wound regeneration.Besides bacterial infection, macrophages play an important role in regulating inflammatory response1. In addition, persistent inflammation and dysfunction of immunoregulation are also associated with scar formation26. Recent studies revealed Engrailed-1 lineage-positive fibroblasts (EPFs) were crucial in aggravating scar formation, and suppression of En1 activation by inhibiting YAP using VP could facilitate scarless skin regeneration25. Effective delivery of VP to the deeper tissues by transdermal MNs in our study was able to remold the ultrastructure of the scar tissue. The remarkable decrease in the thickness of scar, the proportion of collagen, and the ratio of type I/III collagen were observed in the wounds treated by this approach.During the remodeling stage, excessive scar formation represents another major obstacle in skin regeneration, resulting from excessive collagen synthesis, deficient matrix degradation, and failed coordination of various cells, especially under pathological conditionsMRSA-infected diabetic mice, and also attenuate HS in rabbit ear scar models. For potential human use, the PF-MNs could be readily tailed to fit the size of wound and applied onto the wound area. After a short administration of laser irradiation, the PF-MNs allow the wound to experience a self-regulated healing process without any change of the dressing for patients. In addition, the degradation rate of the shell and the release profile of VP could be further dynamically tuned according to the inflammatory condition in the wounds, potentiating personalized treatment in the clinic. Overall, our work presents a strategy to deal with non-healing wounds via core-shell structured MNs, and highlights the significance of programmed modulation of the repair process.In summary, we developed a core-shell structured PF-MN patch to advance the wound healing process in a programmed manner. In the early stage of inflammation, PF-MNs generated ROS under laser irradiation for bactericidal effects. Afterwards, the VP@PVA shell responsively degraded upon the highly oxidative wound environment and released VP to promote scarless skin regeneration. Meanwhile, the exposed cHP core modulated immune microenvironment via neutralizing excessive inflammatory cytokines and inducing macrophage polarization. Our findings suggested that this core-shell structured PF-MN patch could promote chronic wound healing in the N-hydroxysuccinimide , 1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride , 2-hydroxy-4′-(2-hydroxyethoxy)−2-methylproplophenone (≥98.0%), ethylene glycol dimethacrylate (98%), and tween 20 were purchased from Aladdin. N,N,N′,N′-tetramethyl-1,3-propanediamine (≥99%), 2-aminoethyl methacrylate hydrochloride , and poly (ethylene glycol) diacrylate (PEG) were purchased from Sigma-Aldrich. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Med Chem Express USA. Live & dead bacterial staining kit and Luria-Bertani (LB) medium were purchased from Yeasen Biotech. LB Agar (CM159) was purchased from Beijing land bridge technology. Cell Counting Kit-8 (CCK-8) was purchased from Beijing Lablead Biotech.Heparin sodium (Mw 15 k-19 kDa), verteporfin , 4-(bromomethyl) phenylboronic acid (98%), MES (≥99.5%), ampicillin, streptozotocin , and crystal violet (98%) were purchased from Macklin. Poly  , poly  , N,N,N′,N′-tetramethyl-1,3-propanediamine  was added into the mixture and stirred for 24 h. Then, the light yellow solution was precipitated in tetrahydrofuran (THF) (150 mL) and filtrated quickly. The resulting white solid was further washed with THF for three times to remove the excess DMF. Pure TSPBA was obtained after volatilizing THF under vacuum overnight47. The structure was characterized by 1H NMR (BRUKER AVIII500M).4-(Bromomethyl) phenylboronic acid  was dissolved in dimethyl formamide (DMF) (60 mL) at 60 °C. 1H NMR : δ(ppm) = 7.77 , 7.47 , 4.49 , 3.30 , 3.02 , 2.34 .1H NMR48.Heparin (1 g) was dissolved in MES buffer  under stirring. Then, NHS (203 mg) and EDC (676 mg) were added into the solution to activate carboxyl groups of heparin. After reacting for 1 h, AEMA (438 mg) was further added to the above solution. The mixture was stirred at room temperature for 24 h. Then, the solution was precipitated with acetone, and the supernatant was discarded. The white solid was dried in a fume hood, and then hydrated to 1% with deionized (DI) water. After dialysis for three days, m-heparin was obtained with the method of lyophilization and characterized by 1H NMR : δ(ppm) = 6.13 , 6.09 , 1.98 .To fabricate the PF-MN patch with a core-shell structure, PVA  and TSPBA (2%) were first dissolved in DI water, and then mixed at the volume ratio of 2:1. 2 mL of the mixture was casted into the PDMS mold. The PDMS mold was placed under vacuum to allow the mixture to fill down to the MN tip. Then, the crosslinked PVA, the shell of PF-MN patch, was obtained after air-dried overnight at room temperature. m-heparin (0.1 g), ethylene glycol dimethacrylate  and photoinitiator  dissolved in DI water (0.5 mL) were added to the above of hollow MN patch and then filled up the cavity under vacuum. After drying at room temperature in the dark overnight, the blank PF-MN patch was obtained under UV exposure for 20 min. The excessive heparin served as the backing of MN patch and then 30 μg VP was further loaded to the above MNs to obtain PF-MNs. The non-degradable MN patch was obtained with PVA (99% hydrolysis) using the same method. The morphology of PF-MNs was obtained by the scanning electron microscope  and confocal laser scanning microscopy .2O2 solutions (5 mL) at room temperature on an orbital shaker (200 rpm), respectively. At predetermined time points, 200 μL of the supernatant was taken out to 96-well plates for analysis. Absorbance at 689 nm was read out with the Hybrid Multi-Mode Reader  to measure the release profiles of VP. The release profiles of VP from MN shells of the ratio of 2:1 in H2O2 solutions with different concentrations  were measured using the same method.To evaluate the ROS-responsive behavior of PF-MNs, the PF-MN shells loaded with 150 μg VP with different ratios of PVA:TSPBA  were incubated in 0.1 mM H12. The PF-MN patch was irradiated under 690 nm laser (25 mW/cm2). The fluorescence intensity (488 nm/525 nm) of DCFH was continuously monitored with Hybrid Multi-Mode Reader at predetermined time points. Furthermore, 104 L929 cells were cultured in the complete cell media of dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin liquid  in a 24-well plate and incubated at 37 °C for 24 h. VP (5 μg/mL) was added to incubate with cells for 4 h. DCFH-DA (5 μM) was added and incubated for 20 min. Afterwards, VP in the cells was irradiated with 690 nm laser (25 mW/cm2) for 10 min to generate ROS and further observed with a fluorescent microscope (Nikon).DCFH-DA was used as the ROS indicator. First, sodium hydroxide was used to convert DCFH-DA to DCFH, and then PF-MNs loaded with VP (3 μg) were added into DCFH solution  in a 48-well plateL929 fibroblasts were cultured in a 12-well plate for 24 h. Then, different concentrations of VP were added to the wells for another 24 h. 200 μL of RIPA lysis buffer (Beyotime) was added to each well for 20 min at 4 °C after washing one time with PBS. The concentration of protein was determined with a BCA protein assay (Beyotime). The amount of protein was equally added for later gel electrophoresis and blotted onto nitrocellulose membranes (Beyotime). After being blocked by 10% nonfat milk for 2 h, the membranes were incubated with the primary antibodies including YAP, α-SMA and β-tubulin (Proteintech) overnight at 4 °C. Then, the membranes were washed three times (each time for 10 min) with Tris Buffered Saline with Tween (TBST) and further incubated with the secondary antibody (Proteintech) for 1.5 h at RT. After washing three times with TBST, the bands were imaged. All antibodies were diluted 2000 times before used.Mouse IFN-γ (Biolegend) was incubated with HP MNs, PF-MNs, and PEG MNs at 37 °C at a concentration of 1000 pg/mL in PBS. 100 μL of the supernatant was taken out at intervals of 0, 1, 3, 6, 12, and 24 h, and stored at −20 °C until quantification with the ELISA kit (Biolegend). Moreover, the capability of MNs to scavenge MCP-1, TNF-α, IL-1β, and IL-6 was also investigated by multiplex immunoassay after 24 h.4 RAW 264.7 cells were cultured in a 24-well plate and incubated at 37 °C for 24 h. Then, LPS (100 ng/mL), IFN-γ (100 ng/mL), and IFN-γ (100 ng/mL) after being incubated with PF-MNs for an hour were added to induce M1 polarization. 24 h later, the solutions were centrifuged at 350×g for 5 min at 4 °C. The supernatant was discarded and resuspended in 1 mL of flow cytometry buffer (2% FBS in PBS). Then the cells were analyzed with the method of flow cytometry (Beckman CytoFlex S).10MRSA, SA, E. coli, and PA solutions (106 CFU/mL) were added into 48-well plates respectively, and further treated with PBS, laser irradiation, antibiotics , PF-MNs, VP with laser irradiation (30 μg/mL), and PF-MNs with laser irradiation, respectively. The laser irradiation was applied for 10 min (25 mW/cm2). After different treatments, the plate was incubated at 37 °C for 2 h. 10 μL of diluted solution was plated on the LB agar and incubated at 37 °C overnight before calculating the CFU49.To evaluate the antibacterial ability of PF-MNs, 1 mL of MRSA solution (107 CFU/mL) was added into a 24-well plate per well with circle microscope cover glass. After incubation at 37 °C for 6 h, the medium was replaced with different agents: (1) Control group (fresh LB), (2) Laser group (fresh LB with laser irradiation), (3) Anti group , (4) VP + L group , (5) PF-MN group (PF-MNs in fresh LB), and (6) PF-MN + L group (PF-MNs in fresh LB with laser irradiation)39. The laser involved groups were exposed under 690 nm laser irradiation for 10 min (25 mW/cm2). After different treatments, the plate was incubated at 37 °C for another 36 h. The plate was washed three times with PBS, and stained with 500 μL crystal violet (1 g/L) for 10 min after incubating with anhydrous methanol for 10 min. The stained biofilms were washed three times with PBS, and photographed after air-dried. The absorbance at 570 nm was obtained using the Hybrid Multi-Mode Reader when the stained biofilms were dissolved in 500 μL of 95% ethanol. Meanwhile, biofilms with different treatments were stained with DMAO in live & dead bacterial staining kit for 20 min, and then imaged by CLSM (Olympus FV1000) to obtain 3D structures of biofilms using ImageJ. Besides, biofilms treated with the above method, were fixed with 4% paraformaldehyde  for 10 min, and then dehydrated with ethanol of 50%, 60%, 70%, 80%, 90%, and 100% in sequence over 10 min50. Finally, the samples were observed by SEM.To evaluate the ability of PF-MNs to prevent biofilm formation, 1 mL of MRSA solution (107 CFU/mL) was added into a 24-well plate per well with circle microscope cover glass and incubated at 37 °C for 48 h to obtain mature biofilms. Fresh LB was replaced every 24 h51. Then, the medium was replaced with the above agents. After different treatments, the plate was incubated at 37 °C for another 24 h. The biofilms were then assessed using crystal violet staining, live & dead staining and SEM in the same way above. The anti-biofilm activity of PF-MNs on PA-formed biofilms was also investigated with the above methods.To evaluate the ability of destructing mature biofilms by PF-MNs, 1 mL of Male BALB/c mice (6-8 weeks) were obtained from Shanghai Slac Laboratory Animal Co., Ltd. All animal procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang University (ZJU20220079). The mice were randomly divided into four groups (15 mice per group) and anesthetized with isoflurane (RWD). Then two round wounds with a diameter of 8 mm were made on the back of each mouse after depilation and then treated with 10 μL of PBS (Control group), 10 μL of 3 mg/mL VP (VP group), PF-MNs without VP (blank PF-MN group), and PF-MNs loaded with 30 μg VP (PF-MN group), respectively (day 0). The mice were individually housed in cages to prevent the interference among mice. A medical tape was applied on the back of MN patches to further immobilize them on the mice. And the mice of other groups were also stuck with the medical tape as control. The MNs were removed on day 2. On days 3, 7, and 14, the wounds were photographed and wound areas were measured. Meanwhile, the wounds after different treatments were collected and stored in 4% PFA for H&E staining. All the histological slices were recorded using a virtual slide microscope (Olympus VS200) and the epidermal thickness of wounds was analyzed using ImageJ. Besides, the wounds after different treatments on day 3 were collected in flow cytometry buffer for flow cytometry analysis.52. STZ was dissolved in citric acid sodium citrate buffer (pH 4.4), which was placed on ice till injection. The injection was finished within 30 min. A week later, the mice were determined as diabetic ones if the blood glucose >16.7 mmol/L. The diabetic mice were anesthetized with isoflurane, and two round wounds with the diameter of 8 mm were made on the back of each mouse after depilation. Then, the wounds were infected with 107 CFU of MRSA (109 CFU/mL). Two days later, the diabetic mouse infection model was established.The diabetic mouse model was established by intraperitoneal injection of 50 mg/kg STZ (10 mg/mL) to male BALB/c mice (6–8 weeks) for five consecutive daysMRSA were randomly divided into six groups (12 mice per group). The mice were treated with 10 μL of PBS (Control group), 10 μL of ampicillin (200 μg/mL) (Anti group), 10 μL of VP (3 mg/mL) with laser irradiation (VP + L group), non-degradable MNs with laser irradiation (NMN + L group), PF-MNs (PF-MN group), and PF-MNs with laser irradiation (PF-MN + L group) (day 0). The laser irradiation was applied for 10 min . All the MNs were loaded with VP (30 μg) and removed on day 2. The mice were individually housed in cages and the MNs were immobilized with a medical tape as the method above. On days 3, 7, and 14, the wounds were photographed and the wound areas were measured. Meanwhile, the wounds after different treatments were collected and stored in 4% PFA for H&E staining, Masson Trichrome staining, Sirius Red staining and immunofluorescence staining 40. All the histological slices were recorded using a virtual slide microscope (Olympus VS200) and statistical analysis was analyzed using ImageJ. On day 3, the wounds were collected in PBS (500 μL) and then homogenized with a homogenizer (KZ-III-FP). The homogenates were divided into two parts: one part was for bacterial plate counting (10 μL), and the other one was for inflammatory factors quantitative analysis. Serially diluted homogenates were plated on LB agar to quantify the bacterial colonies. Then the homogenates were centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatants were taken out to detect the inflammatory factors by multiplex immunoassay (Biolegend), including IFN-γ, TNF-α, MCP-1, IL-1β, and IL-6.The diabetic mice infected with g for 5 min at 4 °C before being plunged through a 40 μm filter to obtain a single cell suspension. Polyclonal antibodies for staining including: Anti-CD16/32, Anti-F4/80, Anti-CD86, Anti-CD206, Anti-CD45, Anti-CD4, and Anti-FoxP3 (Biolegend). All the amounts of antibodies for staining were followed the manufacturer’s instructions. All data were acquired on a CytoFLEX flow cytometer and analyzed with FlowJo10.For healthy mice and diabetic mice, the wound tissues were collected on day 3 and chopped. Then, the tissues were digested with hyaluronidase/collagenase II (5 mL) at 37 °C for 30 min and terminated by flow cytometry buffer. The solutions were centrifuged at 350×MRSA infection was collected for further analysis after different treatments on day 3. Besides, the major organs on day 14  of mice were also collected for H&E staining and all the histological slices were recorded using a virtual slide microscope (Olympus VS200).To estimate the biosafety of PF-MNs, the blood of diabetic mice with The wound tissues of diabetic mice on day 7 were collected and washed with saline. Then, the tissues were stored at –80 °C before sequencing . Differential expression analysis was conducted using R Bioconductor package “edgeR”. The R package “ClusterProfiler” (version 4.0.5) was applied for GO enrichment and KEGG pathway enrichment analysis.4 L929 cells were seeded in a 24-well plate at 37 °C for 24 h. And then they were exposed to different concentrations of VP and PF-MNs for 24 h, in which cells with PF-MNs were exposed to 690 nm laser irradiation . The cells were then washed with PBS and incubated with complete media containing 10% CCK-8 for 2 h at 37 °C, before reading the absorbance at 450 nm using the Hybrid Multi-Mode Reader.Cytotoxicity of different concentrations of VP was evaluated using the CCK-8 assay. Briefly, 10New Zealand white male rabbits (3–4 months) were anaesthetized by intravenous injection of pentobarbital (30 mg/kg). Then each ear received three wounds using an 8 mm biopsy punch and the perichondrium was completely removed using a scalpel. Then, the wounds were treated with PBS (10 μL), VP , blank PF-MNs, and PF-MNs, respectively. The MNs were stabilized by a sterilized bandage and removed after two days. The wounds were photographed during the treatments and the scars were collected on day 30 for H&E staining, Masson Trichrome staining and Sirius Red staining. All the histological slices were recorded using a virtual slide microscope (Olympus VS200) and the SEI, percentage of collagen, and type I/III collagen ratio were analyzed by ImageJ.43. In detail, after anaesthetization with pentobarbital (30 mg/kg), three wounds were established using an 8 mm biopsy punch on each ear. The perichondrium was completely removed using a scalpel. 30 days later, the wounds were completely healed and hypertrophic scars were formed. Then the rabbits were randomly divided into four groups: Control group (no treatment), Laser group (laser irradiation), PF-MN group (PF-MNs), and PF-MN + L group (PF-MNs with laser irradiation). The laser irradiation was applied for 10 min (25 mW/cm2) and the MNs were immobilized by a sterilized bandage for two days. The scars were photographed during the treatments and the wounds were collected on day 15, 25, and 35 for H&E staining, Masson Trichrome staining, and Sirius Red staining. All the histological slices were recorded using a virtual slide microscope (Olympus VS200) and the SEI, percentage of collagen, and type I/III collagen ratio were analyzed by ImageJ.The rabbit ear HS model was established according to the previously methodP < 0.05 was considered statistically significant .The GraphPad Prism software (version 8.2.1) and ImageJ software (version 2.1.0) were used for the statistical analysis. All data were obtained from at least three independent experiments with at least three parallel samples per condition in each experiment and are expressed as mean ± standard deviations (SD). Multiple comparisons were performed using one-way two-sided analysis of variance (ANOVA) with Tukey’s multiple comparison test. A probability value of Further information on research design is available in the Supplementary informationReporting Summary


The Wnt/β-catenin pathway is a critical regulator of development and stem cell maintenance. Mounting evidence suggests that the outcome of Wnt signaling is determined by the collaborative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. However, the contribution of FOX transcription factors to Wnt signaling has not been investigated in a systematic manner. Here, we performed complementary screens of all 44 human FOX proteins to identify new Wnt pathway regulators. By combining β-catenin reporter assays with Wnt pathway–focused qPCR arrays and proximity proteomics of selected candidates, we determine that most FOX proteins are involved in the regulation of Wnt pathway activity. As proof-of-principle, we additionally characterize class D and I FOX transcription factors as physiologically relevant regulators of Wnt/β-catenin signaling. We conclude that FOX proteins are common regulators of Wnt/β-catenin–dependent gene transcription that may control Wnt pathway activity in a tissue-specific manner. The Wnt/β-catenin signaling pathway, also referred to as the canonical Wnt pathway, is a central homeostatic signaling cascade, whose defining feature is the stabilization of β-catenin induced by WNT family proteins, which results in the differential expression of Transcription factor 7/Lymphoid enhancer-binding factor (TCF/LEF) target genes . Wnt/β-cAmong these additional Wnt pathway regulators are forkhead box (FOX) transcription factors. FOX proteins comprise one of the largest transcription factor families, with 44 main family members in humans that are subdivided into 19 classes (A through S) based on sequence similarity , 8, 9. SBecause both the Wnt/β-catenin pathway and FOX transcription factors are critical for mammalian physiology and pathobiology, it is of considerable interest to better understand their reciprocal regulation. We therefore assessed the impact of the entire FOX transcription factor family on Wnt/β-catenin signaling using complementary, uniform gain-of-function screens in model cell lines. Overexpression of FOXs resulted in pronounced changes in Wnt pathway activator and inhibitor expression, which was associated with differential β-catenin/TCF reporter activity and TCF/LEF target gene transcription. Based on these findings, we characterize class D and I FOXs as Wnt signaling activators and inhibitors, respectively. We conclude that the induction of various FOX transcription factors causes a comprehensive transcriptional rewiring of the canonical Wnt pathway that is partially independent of β-catenin and TCF/LEF. Our study identifies FOX proteins as common regulators of Wnt/β-catenin–dependent gene transcription and provides mechanistic clues for the apparent functional redundancy of some FOXs in the Wnt signaling pathway.A). In both assays, we induced FOX proteins by transient overexpression of a functionally validated, uniform FOX plasmid library , as well as in HCT116 colorectal cancer cells with constitutively active Wnt signaling. qPCR assays were done in 293T cells pretreated with R-spondin 3 to sensitize cells to subsequent pathway activation.To identify potential new Wnt pathway regulators within the FOX family, we performed gain-of-function screens using two complementary standard assays for Wnt/β-catenin pathway activity: the β-catenin/TCF transcriptional reporter TOPflash  and a quFig. 1 library . TOPflasB, and S1A). We performed unsupervised k-means clustering of the aggregated results to group FOXs into three discrete clusters (B). The first cluster contained several previously identified positive regulators of Wnt/β-catenin signaling, including FOXB2, FOXP1, FOXQ1, and FOXK2 (B). Additionally, we performed TOPflash assays in 293T cells with genetic deletion of LRP5/6, β-catenin, or TCF/LEF/β-catenin (penta-KO) (C and D). In these cells, most FOX proteins had essentially no effect and did not synergize with Wnt3a/R-spondin 3 in TOPflash activation. Lastly, we repeated TOPflash experiments in HeLa  and PC3 (prostate cancer) cell lines (E). Most putative Wnt pathway regulators differentially regulated TOPflash activity in these cells as well, especially in HeLa cells.We observed that most FOX transcription factors significantly altered TOPflash activity across multiple independent experiments  , 21, whienta-KO) , C and Dll lines E. Most pbona fide TCF/LEF target genes in 293T cells    showed a significant interassay correlation  that was primarily driven by the differential regulation of AXIN2, LEF1, and MYC (C). We thus clustered the gene expression data as before and observed that most FOX proteins were grouped consistently with the TOPflash data (C and D). Collectively, this approach shortlisted ten likely activators and 11 candidate inhibitors of Wnt/β-catenin signaling in the FOX family (D), including FOXs that have not been studied in this context previously.We then performed qPCR arrays of five D1, SP5) , as wellR5, MYC) C and S2.C) C and , 21, andproteins . Accordiproteins , B and C and MYC C. We thuash data , C and DX family D, includA and WNT4, WNT7A/B, and RSPO1, among others. Consistently, inhibition of WNT protein secretion using the porcupine inhibitor LGK974 (B and C). However, we observed the same pattern of Wnt pathway agonist induction by candidate activators as well as inhibitors, and across all FOX transcription factors, Wnt pathway agonist induction was not generally indicative of TOPflash activation or TCF/LEF target gene expression (D and E). Similarly, while several FOXs differentially regulated various secreted Wnt pathway antagonists (WNT5A and WNT11 (A), which may prime cells further towards activation of canonical Wnt signaling . Of these, 144 (68%) were associated with four or more FOX proteins, suggesting that they are common FOX interactors. Notably, this number is considerably higher than shared interactors identified in previous coimmunoprecipitation/mass spectrometry assays of 36 FOX transcription factors (A), likely due to the better performance of proximity proteomics in screens of chromatin-associated proteins .FOX proteins occupy the promoter regions of various TCF/LEF target genes , and we Fig. 3 factors , 26 . These included chromatin remodeling as well as histone acetylation and methylation complexes known to regulate TCF/LEF target gene expression , possibly indicating cell type–specific functions. In contrast, FOXIs inhibited Wnt reporter activity across all tested cell types. Moreover, overexpression of FOXD1 and FOXI1 in HCT116 and HeLa cells differentially regulated TCF/LEF target gene expression (B and S6A). Curiously, contrary to results from 293T, FOXI1 strongly induced LEF1 in these cells, possibly due to tissue-specific regulation of this gene . FOXD1 depletion also reduced the expression of multiple TCF/LEF target genes (D), although FOXI1 knockdown had no effect in these assays presumably due to low siRNA efficiency (B and C). Finally, we determined the effect of FOXD1 and FOXI1 gain-of-function on colony formation in HCT116, which depend on β-catenin/TCF signaling for efficient proliferation (E and F). Taken together, these findings support a physiologically relevant role of class D and I FOXs in the regulation of Wnt/β-catenin signaling.Conversely, RNA interference of FOXD1 reduced TOPflash activity in 293T, while FOXI1 knockdown increased reporter activity C. FOXD1 et genes D, althouficiency , B and Cferation . We obseferation , E and FA). Moreover, FOXD1-3 were unable to activate TOPflash in 293T ΔLRP5/6 cells expressing constitutively active LRP6 lacking its ligand-binding extracellular domain . Interestingly, FOXD4 still activated TOPflash in this assay, suggesting that its mode of action is distinct. Because WNT7A/B were among the most highly induced WNT genes, we depleted WNT7B or the WNT7 coreceptor RECK by RNA interference using previously validated siRNAs . Additionally, FOXD1 and FOXD3 in particular were able to modestly activate WNT7B and WNT1 promoter reporters, consistent with direct transcriptional regulation of these genes . Taken together, these results suggest that FOXD1-3 activate Wnt signaling by inducing agonistic WNT proteins.Given that FOXD1-3 strongly induced the expression of multiple WNT genes, we asked if this mechanism may sufficiently explain their activity in the Wnt pathway, as we have previously shown for FOXB2 . ConsistFig. 5E). Several FOX family members such as FOXOs inhibit Wnt signaling by competitive binding of β-catenin (D and E). However, we did not detect physical interaction of FOXIs with β-catenin (A), arguing against β-catenin sequestration. Instead, we noted a destabilization of β-catenin protein by FOXIs and reduced nuclear β-catenin levels at least in the presence of FOXI2/3 (F and S7B). Consistently, proteasomal inhibition released the effect of FOXI2/3 on TOPflash activity in HCT116 (G). We conclude that FOXIs may inhibit Wnt signaling at least in part by promoting the degradation of β-catenin.FOXIs, on the other hand, had a limited impact on WNT gene expression, and their inhibitory activity was not affected by treatment with the porcupine inhibitor LGK974 E. Severa-catenin . We obse-catenin , D and E-catenin A, arguin FOXI2/3 F and S7Bn HCT116 G. We conA). It is thus likely that their effects are mediated by few specific cofactors. Pairwise analysis of the TurboID data confirmed that most identified interactors were shared between FOXD1 and FOXI1 (A). Accordingly, pathway-focused analyses did not reveal any significant results due to the low number of unique hits. However, among the FOXD1-exclusive hits, we detected the ubiquitin/SUMO ligases PIAS4 and TRIM33 (B), which have been implicated in the regulation of Wnt signaling (B), which has been shown to control the expression of multiple WNT genes including WNT7A/B, and which was also identified as a candidate FOXB2 interactor (C), which may conceivably mediate its specific effects.FOXD1 and FOXI1 have opposing functions in the Wnt pathway despite a largely overlapping interactome and virtually identical DNA recognition motifs A. It is nd FOXI1 A. Accordd TRIM33 B, which ignaling , 39, 40.ignaling B, which teractor , 41. In teractor C, which A). Accordingly, gene set enrichment analyses against a curated list of TCF/LEF target genes (B). We previously reported that regulation of Wnt/β-catenin signaling is just one of many functions of FOXQ1 . Wnt3a and control conditioned media were collected from stably transfected or parental L cells, following the supplier’s guidelines. R-spondin 3 conditioned media were generated by transient transfection of Rspo3ΔC  and the American Type Culture Collection (ATCC). 293T ΔCTNNB1, Penta-KO, and ΔLRP5/6 cells have been described elsewhere , 21. MCF Rspo3ΔC  into 293et al. . Cell transfection was performed using Lipofectamine 2000, Lipofectamine 3000, or Lipofectamine stem transfection reagent (Thermo Fisher Scientific), or jetOPTIMUS (Polyplus Transfection), according to the supplier’s recommendations. Other plasmids used in this study include the following: M50 Super 8x TOPflash and M51 Super 8x FOPflash  or R-spondin 3 conditioned media (standard dilution 1:1000), or a combination of both (indicated as W/R). The dual luciferase assay was conducted as described previously  with fewqPCR array experiments were performed using custom 384-well TaqMan Gene Expression Array Cards (Thermo Fisher Scientific) containing inventoried probes for 48 gene targets, including three controls . Data were normalized to the geometric mean of the controls. Experiments were done in 293T cells pretreated with 5 ng/ml recombinant human R-spondin 3 (R&D Systems). Experiments were performed with three biological replicates and two technical replicates. For control of R-spondin effects, we included two untreated, empty vector transfected samples. Data were acquired on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher Scientific). All gene expression data averaged from the technical replicates, excluding the untreated controls, can be found in the supplemental materials.qPCR was performed using standard protocols. Briefly, RNA was extracted using a Qiagen RNeasy mini kit and reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Complementary DNA was amplified using validated custom primers, with SYBR green dye. Data were acquired on Bio-Rad CFX96 touch thermocycler and normalized to HPRT1 control.Cells were harvested in PBS and lysed in lysis buffer (1% NP-40 in PBS with 1× protease inhibitor cocktail). Lysates were boiled in Laemmli sample buffer with 50 mM DTT, separated on 10% polyacrylamide gels (Bio-Rad), transferred onto nitrocellulose membranes, and incubated in blocking buffer (LI-COR). Primary antibodies were detected using near-IR fluorophore-labeled secondary antibodies (LI-COR). Blots were scanned on a LI-COR CLx imager. Antibodies used in this study are as follows: mouse anti-Flag M2 (F3165) and anti-Flag affinity gel (A2220) from Sigma Aldrich; rabbit anti-HSP70 (AF1663) from R&D Systems; rabbit anti-non phospho (Active) β-catenin (8814) from Cell Signaling Technology; mouse anti-FOXI (TA800145) from Thermo Fisher Scientific.2. Cells were surface washed with ice-cold PBS for three times to remove excess biotin and then harvested centrifuging at 1500 rpm for 15 min. Cells were washed thrice with ice-cold PBS buffer by centrifugation to remove any remaining biotin. Cells were lysed in RIPA buffer containing 1x protease inhibitor cocktail for 15 min on ice. Prewashed streptavidin beads  were added to the cell lysate and incubated overnight at 4 °C with end-over-end rotation. The beads were washed once with 1 ml of RIPA buffer, once with 1 ml of 1 M KCl, once with 1 ml of 0.1 M Na2CO3, once with 1 ml of 2 M urea in 10 mM Tris–HCl (pH 8.0), and twice with 1 ml RIPA lysis buffer. The beads then transferred to new Eppendorf tube and washed twice with 50 mM Tris–HCl buffer (pH 7.5) and 2 M urea/50 mM Tris (pH 7.5) buffer. Beads were incubated with 0.4 μg of trypsin (Thermo Fisher) in 2 M urea/50 mM Tris containing 1 mM DTT for 1 h at 25 °C with end-over-end rotation. After incubation, the supernatant was collected and the beads were washed twice with 60 μl of 2 M urea/50 mM Tris buffer (pH 7.5) and the washes were combined with the collected supernatant. The supernatant was reduced with 4 mM DTT for 30 min at 25 °C with end-over-end rotation. The samples were alkylated with 10 mM iodoacetamide for 45 min in the dark at 25 °C with end-over-end rotation. For the complete digestion of the sample, an additional 0.5 μg of trypsin was added and incubated at 25 °C overnight with end-over-end rotation. After overnight digestion, the samples were desalted with C18 Thermo Fisher Scientific pipette tips and then dried with vacuum centrifuge.The labeling and sample preparation of TurboID experiments was performed as described previously , 24. BriHomo sapiens UniProt database (release 2019-12-16) with Sequest HT search engine. The search parameters were as follows: taxonomy: H. sapiens; enzymes; trypsin with two missed cleavages, no variable modifications; fixed modification: Carbamidomethyl; Peptide Mass Tolerance, 10 ppm; MS/MS Fragment Tolerance, 0.02 Da. Quantification of the analyzed data were performed with Scaffold 5.1.0  using total spectral count. Protein and peptide identifications were accepted if they could be established at greater than 95 and 90% probability, respectively and if protein identification contained at least two identified peptides.TurboID samples were analyzed by mass spectrometry, using an Easy nano LC 1200 system interfaced with a nano Easy-Spray ion source (Thermo Fisher Scientific) connected Q-Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The peptides were loaded on a precolumn  and the chromatographic separation was performed using an EASY-Spray C18 reversed-phase nano LC column . The nano LC was operating at 300 nl/min flow rate with a gradient (6–40% in 95 min and 5 min hold at 100%) solvent B (0.1% (v/v) formic acid in 100% acetonitrile) in solvent A (0.1% (v/v) formic acid in water) for 100 min. Separated peptides were electrosprayed and analyzed using a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific), operated in positive polarity in a data-dependent mode. Full scans were performed at 120,000 resolutions at a range of 380 to 1400 m/z. The top 15 most intense multiple charged ions were isolated (1.2 m/z isolation window) and fragmented at a resolution of 30,000 with a dynamic exclusion of 30.0 s. Raw data were processed by Proteome Discover 2.0 (Thermo Fisher Scientific) searching against the HCT116 were seeded on 6-well plates at a density of 1000 cells after 24 h of transfection. Cells were grown for 14 days. At the end of the experiment, cells were washed with PBS and stained with 2% methylene blue in 50% methanol. Stained plates were photographed and colonies were counted manually. Colonies with less than 50 cells were excluded.in silico prediction of transcription factor binding to Wnt pathway–related gene promoters, we retrieved positional weight matrices for 27 human FOX family proteins and all four human TCF/LEF proteins from the JASPAR 2022 database  for the target genes included in our qPCR array, as well as their mouse orthologs, from the Eukaryotic Promoter Database. Co-occupancy was predicted using MCAST v5.5.1 . Proteomics data were processed using SAINTexpress, CRAPome, and ProHits-viz. (et al. (et al. (Fig. S5A are from Li et al. (Most statistical analyses were performed in R v4.2.0  and wereits-viz. , 68, 69,its-viz. , includi (et al.  followin (et al. : GSE1085 (et al. . Gene se (et al.  were per (et al. . Proteomi et al. . Only hivia the PRIDE (TOPflash and Taqman qPCR array data can be found in the supplemental materials. TurboID proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Bletilla striata polysaccharide (BSP) are prepared as the transmucosal delivery system. The prepared TA@MPDA-HA/BSP MNs exhibit well-arranged microarrays, high mechanical strength and fast solubility (<3 min) properties. In addition, the hybrid structure improves the biocompatibility of TA@MPDA and expedites oral ulcer healing in the SD rat model through the synergistic anti-inflammatory and pro-healing effects of microneedle ingredients , with 90% less amount of TA compared with Ning Zhi Zhu®. TA@MPDA-HA/BSP MNs are shown to be their great potential as novel ulcer dressings for OM management.Oral mucositis (OM) is the most common disease of the oral mucosa, which affects people’s daily production and life. Triamcinolone ointment is the common clinical drug for OM treatment. However, the hydrophobic properties of triamcinolone acetonide (TA) and the complex microenvironment of the oral cavity led to its low bioavailability and unstable therapeutic effects on ulcer wounds. Herein, dissolving microneedle patches (MNs) composed of mesoporous polydopamine nanoparticles (MPDA) loaded with TA (TA@MPDA), sodium hyaluronic acid (HA), and  Oral mucositis (OM) is an inflammatory disease caused by necrosis and shedding of oral mucosal tissue, which can involve the epithelial layer and connective tissue . At the Bletilla striata polysaccharide (BSP) is a kind of natural glucomannan with high biosafety and low price. BSP has anti-inflammatory, pro-coagulant, antibacterial and antioxidant activities, thus promoting wound healing , as a highly effective transdermal drug delivery form that can break through the mucosal barrier, have attracted extensive attention in the treatment of oral diseases . MNs can healing . Hu et a healing . The res healing . HA in t healing . We have healing . In thisSo far, many materials have been used as carriers to synthesize composite materials, such as montmorillonite , polydopvia a one-casting fabrication process for oral mucositis treatment. The schematic illustration of the TA@MPDA-HA/BSP MN patch is shown in Here, we may reasonably hypothesize that i) MPDA NPs may be the ideal carrier for TA, both to improve the bioavailability of TA and to exert the anti-inflammatory effects of MPDA NPs; ii) the combination of TA@MPDA NPs with dissolving HA/BSP microneedle may produce synergistic effects in the treatment of OM, reducing the dosage of the hormone administered. To test our hypotheses, TA@MPDA NPs were prepared through π-π stacking and encapsulated in the HA/BSP dissolving microneedle patch Escherichia coli O55:B5 (LPS) and phenol were purchased from Aladdin . Pluronic® F-127 (F127) was purchased from Sigma . Bletilla striata polysaccharide  was acquired from Xi’an Zelang Biotechnology . Sodium hyaluronic acid  was obtained from Freda Biology Co., Ltd. . The polydimethylsiloxane mold  had 225 (15 × 15) MNs, and the height and width of each needle were 700 and 300 μm, respectively. The distance among needles was 600 μm. TRITC-Phalloidin, 4′,6-diamidino-2-phenyllindole (DAPI) was purchased from Solarbio. Cell Counting Kit-8 (CCK-8) was purchased from Absin Bioscience Inc . Human Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit was purchased from Elabscience Biotechnology Co., Ltd. . Methanol was the chromatographic grade for chromatographic analysis when High-performance liquid phase (HPLC) was used, and other chemical reagents were analytical grade, both provided by Sinopharm Chemical Reagent Co., Ltd. , and used directly.1,3,5-Trimethylbenzene , Tris (hydroxymethyl) aminomethane , dopamine hydrochloride , Triamcinolone acetonide (TA), Lipopolysaccharides from 2, Human Oral Keratinocytes (HOK) and Human gingival fibroblasts (hGFs) were cultured using the complete medium. The complete medium includes 90% Dulbecco’s Modified Eagle Medium , 10% fetal bovine serum  and 1% penicillin/streptomycin .In a humidified incubator  at 37°C with 5% COWe purchased adult Sprague Dawley rats  from Jinan Pengyue laboratory animal breeding center  and maintained them in Qingdao University’s Laboratory Animal Center in a specific pathogen-free (SPF) environment. All procedures performed in the study were in accordance with the Ethics Committee of Qingdao Stomatological Hospital Affiliated of Qingdao University Certificate (contract grant 2021KQYX031) and with the National Research Council’s Guide for the Care and Use of Laboratory Animals ethical standards.The preparation of MPDA NPs was carried out according to the methods reported in the previous literature . BrieflyDifferent concentrations of MPDA NPs suspension were prepared by dispersing MPDA NPs  uniformly in 10 mL of methanol under the action of sonication. TA solution  was added dropwise to the MPDA NPs dispersion and stirred at 45°C for 3 h. Finally, TA@MPDA NPs were collected by centrifugation, dried by vacuum and stored at 4°C for further use. The amount of TA loaded into MPDA was calculated by subtracting the mass of TA in the supernatant from the total mass of the TA in the initial solution.−1 were characterized using a Nicolet iN10 Fourier transform infrared (FTIR) spectrometer  performed 32 scans with a scan resolution of 2 cm−1. TA was determined by a Shimadzu LC-20A instrument  equipped with a SHIMADZU Shim-pack GIST C18  analytical column (P/N: 277-30017-08) using methanol-deionized water  as the mobile phases with a flow rate of 1.0 mL/min at 25°C. The regression equation with TA concentration as the independent variable and peak area as the dependent variable is Y = ×27681 − 27556 (r2 = 0.9979)  at 25°C. All the tests were conducted in triplicate, and all the data were expressed as the mean ± standard (SD). The morphology of nanoparticles was observed by a JEM-2100 transmission electron microscopy . The functional groups of MPDA and TA@MPDA in the range of 500–4,000 cm 0.9979) . The pea0.1 mg MPDA or TA@MPDA was evenly dispersed in 1 mL deionized water by ultrasonic vibration. Then, HA and BSP were mixed in the ratio of 1:2, 1:1, 2:1, respectively, and dissolved in the above dispersion solution. The final concentration of HA was 30%. The microneedle molds injected with deionized water were placed in a vacuum at 0.08 mPa for 20 min so that each well was filled with deionized water, and the excess deionized water was removed. After that, the MPDA-HA/BSP or TA@MPDA-HA/BSP matrix solution was poured into the microneedle molds and then maintained 30 min under ultrasound until the cavity of the tips was filled with solution. The sample was then left at room temperature for approximately 48 h to dry completely. The corresponding MPDA-HA/BSP MNs or TA@MPDA-HA/BSP MNs were gently separated from the mold and stored in a dryer. In addition, HA/BSP MNs were prepared by the same method with HA/BSP as matrix solution.The morphology of TA@MPDA-HA/BSP MNs was visualized by a dermoscope . In order to observe the microscopic structure of MNs, they were gilded by sputtering to increase conductivity after the edges of MNs were trimmed properly and then imaged using a scanning electron microscope  at an accelerating voltage of 10 kV. The size of MNs was measured by ImageJ. The mechanical properties of MNs were tested by compression method at a temperature of 22°C ± 5°C and relative humidity of 40% ± 10%. TA@MPDA-HA/BSP MNs (array: 15 × 15) were pressed into the pig skin using thumb force. The insertion marks left were observed. In addition, TA@MPDA-HA/BSP MNs were inserted into the mucous membrane of rat tongue with the same force for 3 min, and hematoxylin and eosin (H&E) staining was performed to observe the depth of insertion.The dissolution rates of TA@MPDA-HA/BSP MNs were studied on the tongue abdomen of SD rats. At the predetermined time points, MNs were removed from the tongue and observed by a microscope . The size variations of MNs were analyzed by ImageJ.Citrated whole blood from healthy donors was diluted with normal saline (NS) to 2% (v/v). Then 50 μL of the diluted blood was added into the TA@MPDA-HA/BSP MNs dispersed NS solution with different concentrations , and kept at 37°C for 1 h. Afterward, the mixtures were centrifuged at 1,500 rpm/min for 5 min. The absorbance of the supernatant was detected at 540 nm by a UV-Vis spectrophotometer . In addition, deionized water and NS solution were mixed with an equal volume of diluted blood to form the positive and negative control group, respectively. The hemolysis ratio was calculated as the following formula:3 cells per well and incubated in at 37°C with 5% CO2 using the complete medium for 24 h. The cell culture medium was replaced by the test preparation diluted with culture medium for 24 h. After that, the medium was discarded, and the cells were cultured with 100 μL of CCK-8 reagent (10%) solution per well for 2 h. Subsequently, the optical density (OD) values were read with a microplate reader  at a wavelength of 450 nm. Pristine culture medium was used as a control group. Cell viability was calculated according to the formula:Preparation, ODControl, and ODBlank refer to the OD values of the test preparation-treated group, pristine medium-treated group, and the culture medium without cells, respectively. Each group was carried out in three replicates.The cytotoxicity of MPDA, TA@MPDA, MPDA-HA/BSP MNs and TA@MPDA-HA/BSP MNs were evaluated using a standard CCK-8 assay. Human Oral Keratinocytes (HOK) were seeded into 96-well plates at a density of 3 × 104 cells per well. After incubation for 24 h, the original medium of experimental group was replaced by a MN-contained medium  and the control group was replaced by the complete medium. Cells were fixed with 4% (v/v) paraformaldehyde to maintain morphology, and treated with 0.5% (w/v) Triton X-100 for 3 min and 5% bovine serum albumin for 30 min, with phosphate buffered saline (PBS) used for cleaning. Then, cells were stained with TRITC-Phalloidin and DAPI under dark conditions. The morphology of the cells was photographed using a fluorescence microscope .TRITC-Phalloidin and DAPI staining were used to detect the effect of the MN patches on cell morphology. Human gingival fibroblasts (hGFs) were inoculated into 24-well plates at a density of 5.0 × 105 cells each well and cultured for 24 h. To verify that cell migration was caused by drug action rather than cell proliferation, cells were maintained in a serum-free starvation state for 24 h to minimize proliferation. Then, three straight lines were drawn evenly using a 10 μL tip for the formation of microdamage and the fallen cells were washed and removed with PBS, after which the microneedle-contained medium (2 mg/mL) was then introduced into each well. Wound closure was monitored and imaged using an optical microscope  in 12 h intervals. ImageJ was used to quantify the wound area. The formula is as follows:0 refers to the initial wound area (0 h), and SA refers to wound area of 12 and 24 h. Each group was carried out in three replicates.The cell scratching assay was performed using hGFs. Briefly, hGFs were seeded on 6 well culture-treated plates with a density of 5 × 103 per well for 24 h. After removing the original medium, the cells were co-cultured with LPS and MN patches for 24 h. Only adding medium was used as blank control group. LPS and culture medium were used as the positive control group. The final concentrations of LPS and MN patches were 0.1 and 2 mg/mL, respectively. The level of TNF-α in the supernatant was determined by enzyme-linked immunosorbent assay (n = 3).To verify the anti-inflammatory ability of the prepared microneedle patches, LPS-induced hGFs were used to construct a cell inflammation model. hGFs were inoculated into 96-well plates at a density of 3 × 10To evaluate the safety of TA@MPDA-HA/BSP MNs insertion, the MNs were inserted into the tongue mucosa of rats using thumb force to observe the recovery of the mucosa. After 3 min, the MNs were completely dissolved, and then were removed. Before the insertion of the MNs, immediately and 5, 10, and 30 min after the insertion, photos of the tongue mucosa were recorded. Twenty-4 hours later, the rats were euthanized, and the tongue tissue was removed for pathological section preparation and H&E staining.®) group, HA/BSP MNs group, MPDA-HA/BSP MNs group, TA@MPDA-HA/BSP MNs group. Three rats were assigned for each group, and the healing of the ulcer was recorded using a digital camera for analysis. On the fifth day after administration, the rats were euthanized, and tongue tissue was excised. H&E staining and Masson’s Trichrome staining were used to detect oral ulcer healing degree. The levels of TNF-α and CD31 in each group were observed by immunohistochemistry (IHC) to evaluate the ulcer repair.The model of oral ulcer was constructed by chemical burning as our previous studies . SD ratsp < 0.05 was considered to be statistically significant.All quantified data were presented as mean ± standard deviation (SD). All statistical analysis graphs were drawn using GraphPad Prism 9 or Origin 2021. One-way analysis of variance (ANOVA) with Tukey`s test was used to determine the differences between groups. A value of Max) demonstrated that the successful loading of TA into MPDA NPs  in cell viability between 100 μg/mL and 200 μg/mL of the two kinds of particles. The statistical results showed that there was a statistical difference between 0.5 and 2 mg/mL MPDA-HA/BSP MNs groups (p < 0.05) and there were no statistical differences between TA@MPDA-HA/BSP MNs groups (p > 0.05)  . Two kinThe cytoskeleton was stained with TRITC-Phalloidin and DAPI to observe the effect of microtargeting on cell morphology. After staining, the cell membrane of hGFs showed red spindle shapes, and the cell nucleus was filled, and the nucleus showed blue round shapes, which corresponded to the cell membrane one by one . The resp < 0.05, The process of wound-scratch testing was shown in p < 0.05), and remarkably, the TA@MPDA-HA/BSP MNs group exhibited the lowest TNF-α concentration. From these data we conclude that the superimposition of multiple components of the microneedles enhance their anti-inflammatory effect, which may achieve the goal of low-dose hormones for suppression of inflammation.To our knowledge, MPDA and Chinese herbs extract BSP have been widely explored in the treatment of inflammatory diseases . To veriThe safety of TA@MPDA-HA/BSP MNs was evaluated by inserting MNs into the tongue mucosa of rats and observing the inflammatory response of the mucosa. Normal SD rats tongue mucosa were selected for the experiment. As was shown in ® and microneedle samples were shown in ® and nontreated groups (p < 0.05). Moreover, the MPDA-HA/BSP MNs group showed better therapeutic effects than the HA/BSP MNs (p < 0.05), suggesting that MPDA NPs are effective in treating oral ulcers  loaded with Triamcinolone acetonide (TA) for the treatment of oral mucositis (OM). TA can be successfully loaded into MPDA, thus improving bioavailability of TA. TA@MPDA-HA/BSP MNs had well-arranged microneedles array, high penetration efficiency, rapid dissolution in 3 min, excellent biocompatibility and anti-inflammatory properties. When TA@MPDA-HA/BSP MNs were used to treat oral ulcers, the area of oral ulcers and inflammatory factor level such as TNF-α and CD31 were significantly reduced. TA@MPDA-HA/BSP MNs can not only improve the bioavailability of TA, but also play the role of MPDA, BSP and HA in the combination treatment of OM, becoming a new clinical comfortable and effective alternative for the treatment of OM.


Computational models of infectious diseases have become valuable tools for research and the public health response against epidemic threats. The reproducibility of computational models has been limited, undermining the scientific process and possibly trust in modeling results and related response strategies, such as vaccination. We translated published reproducibility guidelines from a wide range of scientific disciplines into an implementation framework for improving reproducibility of infectious disease computational models. The framework comprises 22 elements that should be described, grouped into 6 categories: computational environment, analytical software, model description, model implementation, data, and experimental protocol. The framework can be used by scientific communities to develop actionable tools for sharing computational models in a reproducible way. Computational models have become valuable tools for the global response against infectious disease outbreaks and pandemics, including the Coronavirus Disease 2019 (COVID-19) pandemic . As in aReproducibility can be especially limited for computational studies such as computational modeling and artificial intelligence, due to their methodological complexity and heterogeneity of, or restricted access to, data sources ,8. The rScientific and government agencies, including the US Government Accountability Office and the US National Academies of Sciences, Engineering, and Medicine (NASEM), have published recommendations to enhance the reproducibility of computational research ,13. MostVarious initiatives have emerged in the scientific community to improve scientific inference based on computational models and to improve public health decision-making during outbreaks. Several initiatives have developed methods for comparing results across multiple models. For example, multi-model comparison studies have been conducted for rotavirus and dengue to better understand the effects of vaccination ,17. ReceThe success and impact of model comparison and combination projects depend on methods and technologies that enable researchers to share their modeling experiments in a transparent and reproducible way that characterizes model similarities, discrepancies, and uncertainties. Thus far, the various model combination projects and modeling hubs have been dependent on ad hoc methods to describe models and share results. No methodological framework currently exists that can directly be translated into tools for sharing computational models of infectious diseases in a transparent and reproducible manner.We developed an implementation framework for representing computational models of infectious diseases in a reproducible format, grounded in previous research on reproducibility from a broad range of scientific disciplines . The fraWe identified 22 elements in 6 categories that together provide a complete representation of the reproducibility for a computational model , based oThe computational environment comprises the combined software and hardware used to conduct the experiment. The analytical software includes the software, e.g., R or Python, used for the computational analysis. The computational model is usually described as a combination of narrative text, diagrams, and mathematical equations (model description). The model is then represented in its computational format as a set of commands, functions, and operations encoded in the model source code (model implementation). The data are ingested by the model implementation and operated on to compute the model results. The entire computational experiment is documented in the experimental protocol. A complete description of all the elements will represent a reproducible computational model.The implementation framework provides a foundation that can be further developed by scientific communities into tools for sharing computational models of infectious diseases in a reproducible way, based on community-specific preferences. For example, the framework can be formalized into a structured metadata schema with prescribed structured vocabularies and ontologies that could render a machine-actionable metadata object compliant with the Findable, Accessible, Interoperable, and Reusable (FAIR) guiding principles –24. MachThe implementation framework can also be developed into a checklist to represent the degree to which a computational modeling experiment is reproducible, as we did as part of the iterative testing process during the development of the framework . SimilarAnother highly promising method for sharing computational models in a transparent and reproducible manner is through containerization in executable workflow objects, such as Galaxy , Open CuThe lack of reproducibility for computational models of infectious diseases can undermine the scientific credibility of modeling results among researchers, policymakers, and even among the public, where skepticism regarding scientific research is already on the rise . It is eTo identify the 6 categories and 22 elements of the implementation framework, we reviewed guidelines published by the NASEM, the FAIR guiding principles, and peer-reviewed literature in a variety of scientific domains including general computational science, computer science, statistics, epidemiology, genetics, computational biology, psychology, and ecology .We identified peer-reviewed literature by querying PubMed articles published between January 1, 2000 and January 1, 2020, using keywords “reproducible,” “reproducibility,” “computational,” “research,” “data,” and “code.” We limited our search to studies published in English and excluded papers about animal-models or clinical research. We extracted quotes that referenced information that researchers believed was relevant to represent reproducible and transparent computational modeling studies.We grouped the 22 elements into 6 categories and mapped the relationships between each of the categories to the conceptual model of an infectious disease modeling workflow . The worTo validate the implementation framework categories and elements, we structured the framework into a checklist . The chen = 3,664). Based on title and abstract review, if the paper was not related to infectious disease modeling, we randomly selected another paper until we identified 10 infectious disease modeling studies. For the second trial, we randomly selected 20 COVID-19 modeling papers without replacement from a list of 229 papers authored by MIDAS members between January 1, 2020 and March 31, 2020.For the first trial, 10 publications were randomly selected without replacement from all publications authored by Models of Infectious Disease Agent Study (MIDAS) members before September 20, 2019 Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe checklist consists of questions related to the 6 categories: (1) computational environment; (2) analytical software; (3) model description; (4) model implementation; (5) data; and (6) experimental protocol. The center column provides examples for each category and element.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The building construction materials are responsible for a large amount of energy and natural resource consumption. In light of the current challenges of resource scarcity and global climate change, the circular economy (CE) is a promising strategy to mitigate pressure on the environment, improve supplying of raw materials, and increase new market and employment opportunities. Developing eco-friendly thermal insulation materials based on agro-waste is a new waste management trend to achieve the sustainability of the resource and energy consumption in the construction sectors. In this work, banana-polystyrene composites were prepared by mixing the banana peels powder (BP) with polystyrene (PS) in different weight ratios . The physical and thermal properties such as thermal conductivity, electrical conductivity, Fourier Transform Infrared (FTIR), crystallographic structures of the fibers, X-Ray Diffraction (XRD), Thermogravimetric Analysis (TGA), and Differential Scanning Calorimetry (DSC) were carried out on BP and BP-PS1 that were prepared with ten wt.% and 20 wt.% of polystyrene powder (BP-PS2). The bio-composites results showed low thermal conductivity ranging from 0.028 to 0.030 W/m.K. The BP-PS2 exhibited a lower thermal conductivity of 0.027 W/m.K, while the pure peel powder demonstrated notable thermal stability, indicated by a total weight loss of 66.4% and a high crystallinity value of 56.1%. Furthermore, the thermal analysis (TGA) and X-Ray Diffraction (XRD) demonstrated that the pure banana peel has the highest thermal stability and crystallinity. These findings indicate that using banana peel-polystyrene composites represents an innovative solution for thermal insulation in buildings as an alternative to conventional materials to reduce energy and resource consumption. In order to reduce the ecological effects, economic losses, and resource consumption, thermal insulators play a crucial role in lowering the heating and cooling load by reducing the heat transmission through the building envelope2. Several insulating materials are commercially available such as fiberglass, urethane, cork, and Extruded Polystyrene3. However, the environmental concerns related to the manufacturing process of conventional thermal insulators justify the need to develop materials with eco-friendly features4.The construction industry is responsible for several negative effects5. Nonetheless, new trends have shown that using agricultural waste in construction materials manufacturing is a feasible solution to tackle the identified challenges6.Conversely, agricultural waste disposal is also a significant problem in developing countries. For instance, in Egypt, over 39 thousand metric tons of vegetal waste was reported  for Egypt–Tilasto.) the most common strategies to manage agriculture waste are composting, dumping in landfills, and open-air incineration, which as a result cause severe environmental concern7. Exploiting natural fibers as green raw materials produces thermal insulators that are non-toxic, recyclable, locally available, and characterized by a low carbon footprint and lower energy consumption of the manufacturing process2. On the other.hand, using natural by-products as thermal insulators has drawbacks such as nonresistant fire, the Influence of weather, and Lower durability8. In order to assess the suitability of thermal insulator production, several researchers studied the thermal performance and chemical characteristics of different natural fibers. The thermal conductivity values were found to range between 0.034 W/m.K and 0.042 W/m.K for sugarcane bagasse waste fibers9, 0.050 and 0.132 W/m.K for oil palm wood10, 0.0393–0.0478 W/m.K for rice straw11.Utilizing thermal insulation materials from agricultural waste could reflect the circular economy concept as an optimum solution to reduce waste generation and energy consumption12. Banana peels are an abundant waste for every 10 tons of bananas, one ton of waste is produced13. Banana peel is discarded as a useless material, and it represents about 40% of the Banana total weight, and this causes waste management problems because of its phosphorus and nitrogen content14. Banana peel contains 20–30% fiber and is rich in carbohydrates, proteins, dietary fibers, and mono and disaccharides, making this waste a promising source for the industry15. The banana peel is a low-cost, readily available, lightweight, eco-friendly material and could be transformed enzymatically, physically, and chemically into value-added products16. To produce green building materials, several researchers investigated the thermal conductivity of banana composites. The composite's thermal conductivity ranged from 0.245 to 0.363 W/m.K for banana and jute fiber17, 0.149 W/m.K for banana-solid waste18, 0.0183 to 0.03168 W/m.K for Banana leaves and polystyrene19, 0.6 W/m.K for banana, bagasse and cement brick20 and 0.3880 to 0.04495 W/m.K for banana fiber21.The banana crop is one of the most significant crops in the world. In 2019, the banana production was more than 116 million tons globally, and around 1.3 million tons in Egypt22. Polystyrene (PS) is a synthetic thermoplastic polymer fabricated from styrene. Recently, polystyrene was used as an essential material in thermal insulation panels composition due to its low cost, low water absorption, low density, low acoustical absorption, and low thermal conductivity 0.038 W/m K Though the expanded polystyrene (EPS) products have 35% of the total insulation market in Europe, it has numerous drawbacks, including toxicity, flammability, and poor durability23.Polymers are categorized into thermosets and thermoplastics24.The circular economy principles could be achieved by closing the loop of agriculture materials consumption. This involves valorizing agricultural wastes, giving them another life, and utilizing them across different industries, rather than disposing of them in the landfill. The comprehensive literature review revealed that information is scarce regarding Egyptian agriculture waste, including banana peel characterizations and potential applications in the construction industry. Geographically, it cannot not rely on the thermal properties of banana waste in other regions of the world due to the impact of climatic conditions, soil nature, and water quality on the biological features of the banana tree. This includes the cellulose and lignin content which directly affect the physical features and the thermal conductivity properties. Consequently, this will affect its efficiency for insulating propertiesThe present study aims to manufacture thermal insulating material based on a banana peel fiber. Different composites will be prepared by adding a polystyrene filler to banana peel powder, to optimize the thermal characterization of the prepared composites. Therefore, the pure banana peel and the prepared composites will be subjected to thermal conductivity, Infrared analysis (FT-IR), X-ray Diffraction (XRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) to assess the possibility of the tested banana peel composite to produce an economical insulation material. A summary of the preparation process and the findings are presented in the graphical abstract Fig. .Figure 25.The banana fruit (peels) used were obtained from an available local fruit market, Beni-suef, Egypt. As seen in Fig. Polystyrene (PS), which is one of the largest volume vinyl polymers, was synthesized by free radical oxidation polymerization of a volume of 5 ml styrene free from inhibitor that was dissolved in a book of 20 ml dimethylformamide (DMF), using 5 gm of ammonium persulfate dissolved in 10 ml distilled water at 60 °C under nitrogen for 3 h. PS was filtered at room temperature after 12 h., washed with water/DMF mixture, and dried under vacuum at 60 °C.23.To produce the banana-polystyrene composites (BP-PS) with different filler contents, the polystyrene was added to banana peel powder with varying ratios of 10%, 20%, 30%, and 40% weight PS . The DC conductance of the samples was estimated from the measured DC current–voltage characteristics.From Fig. BP, BP-PS1, and BP-PS2 crystallographic nature were examined by the XPERT-PRO X-Ray Diffractometer, equipped with Cu radiation (λ = 1.540 Å) source operated at a tension of 40 kV and current of 40 mA. XRD data were recorded within the range of scattering angles (2θ) of 10 to 90° at room temperature. The BP, BP-PS1, and BP-PS3 Crystalline index (CI) and crystallinity (Cr) were evaluated by Segal methods using Eqs.   date and polystyrene (0.053 W/m.K)31 and camilina and expanded perlite (0.082 W/m.K)32, and rice straw and gypsum (0.2–011 W/m.K)33 . Meanwhile, the thermal conductivity coefficient of banana peel was higher than banana leaves and polystyrene (0.0183 W/m.K)19. Moreover, the BP-PS thermal conductivity coefficients were within the same range as those of conventional insulating materials such as rock wool (0.044 W/m.K), and sheep wool (0.038–0.054 W/m.K)34.The Fig. The DC current–voltage characteristics of the banana peel and the prepared composites  were evaluated using a Keithly measurement source unit (model:2400). As noticed in the present study, the pure banana peels showed non-ohmic behavior that was tuned to linear Ohmic after the PS was blended. Therefore, the experimental data were linearly fitted, and the electrical conductance of the composites were calculated and are listed in Table 35. The diffractograms obtained for BP, BP-PS1, and BP-PS2 are shown in Fig. 36. The narrow peaks observed in the graphs could be due to contamination present in the fibers, probably from an inorganic material37.In order to better understand the crystallization of banana peel composites, a high crystallinity index is expected to result in stiff, durable, and highly thermal stable fibers37, banana peel-HDPE composite by 65.8% . As depicted in Fig. 29.During the second degradation stage, which ranged from 180 to 350 °C, the BP, BP-PS1, and BP-PS2 showed a weight loss of 26.48%, 26.33%, and 16.92% at 268 °C, 270 °C and 272 °C, respectively. The fast degradation of the peel samples could be a result of hemicellulose degradation45.In the third stage (200–400 °C), the samples exhibited significant weight loss (37–50%). This can be attributed to the degradation of α-cellulose, cellulose I, and lignin. The weight loss of BP, BP-PS1and BP-PS2 was 47.26%, 47.57%, and 38.65% at 345 °C, 354, and 391 °C, respectively46. The total weight loss of the samples at the temperature of 750 °C was 66.4%, 80.75%, and 81.52% for BP, BP-PS1, and BP-PS229.The last stage was lignin decomposition, which was the most challenging component to decompose compared to other organic members. The degradation temperature started from 250 to 800 °C because of its complex structure of aromatic rings composed of heavy cross-linked molecules besides the polysaccharides47. The residual weights of the samples obtained at 750 °C are listed in Table48. The XRD results confirmed that the sample with the highest thermal stability had the highest crystallinity. The addition of polymers lowered the composite stability because of the weak intermolecular bonding of the composites49.The TGA and DTGA profiles of the banana composites showed thermal stability up to 190 °C, which confirms the possibility of using natural composites for insulating applications23. These results are in agreement with those of Banana leaves and polystyrene19, high-density polyethylene banana peel fibers  analysis was used to determine the thermal energy absorbed or released via chemical reactions of the constituents of the composite during heating. The DSC thermograms of BP, BP-PS1, and BP-PS2 are shown in Fig. 51. As a result, the crystallization and melting temperatures of the BP-PS1 and BP-PS2 composites were lower than their corresponding values for pure banana peel. A good explanation was suggested by52, who related these findings to the formation of tiny crystals due to the strong nucleation on the surfaces of fibers and the consequent shortened time of Ps crystallization. The lower melting peak recorded in the current study was probably caused by the tiny crystals formed by the strong nucleation on the fiber's surfaces and the consequent shortened time of PS crystallization52.The crystallization temperatures (Tc) of BP-PS1 and BP-PS2 composites were separated into double peaks Fig. . The reaIn this study, banana peels and polystyrene were investigated to produce insulating composites. The results demonstrate the potential for developing new recyclable, environmentally friendly materials for building insulation applications. All the bio-composites showed similar thermal conductivity values, ranging from 0.028 to 0.030 W/m.K. In contrast, the pure peel powder offers high thermal stability with 66.4% total weight loss and high crystallinity 56.1%. Based on these findings, replacing conventional thermal insulators with the developed bio-composites will lead to a lower environmental impact by increasing energy efficiency and resource management. However, further studies are required to evaluate the mechanical characteristics and investigate the characteristics required to transform the prepared composites for industrial applications.