{'fraction_non_alphanumeric': 0.045299589761368234, 'fraction_numerical': 0.022418674524758767, 'mean_word_length': 5.0327640292784945, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Incorporation of ROS-Responsive Substance P-Loaded Zeolite Imidazolate Framework-8 Nanoparticles into a Ca2+-Cross-Linked Alginate/Pectin Hydrogel for Wound Dressing Applications

Purpose Wound healing, especially of extensive full-thickness wounds, is one of the most difficult problems in clinical studies. In this study, we prepared a novel substance P (SP)-delivery system using zeolite imidazolate framework-8 (ZIF-8) nanoparticles. Methods We synthesized ZIF-8 nanoparticles using a modified biomimetic mineralization method. We then coated SP-loaded ZIF-8 nanoparticles (SP@ZIF-8) with polyethylene glycol-thioketal (PEG-TK) to fabricate SP@ZIF-8-PEG-TK nanoparticles, and encapsulated them in injectable hydrogel composed of sodium alginate and pectin and cross-linked using calcium chloride. The final hydrogel wound dressing containing SP@ZIF-8-PEG-TK nanoparticles was called SP@ZIF-8-PEG-TK@CA. Results The fabricated ZIF-8 nanoparticles had high SP-loading efficiency. SP-release assay showed that the SP@ZIF-8-PEG-TK nanoparticles maintained drug activity and showed responsive release under stimulation by reactive oxygen species. The SP@ZIF-8-PEG-TK nanoparticles promoted proliferation of human dermal fibroblasts, up-regulated expression levels of inflammation-related genes in macrophages, and exhibited favorable cytocompatibility in vitro. Full-thickness excision wound models in vivo confirmed that SP@ZIF-8-PEG-TK@CA dressings had excellent wound-healing efficacy by promoting an early inflammatory response and subsequent M2 macrophage polarization in the wound-healing process. Conclusion In conclusion, these findings indicated that SP@ZIF-8-PEG-TK@CA dressings might be useful for wound dressing applications in the clinic.


Introduction
The skin is the body's largest organ and encloses all the other organs in the human body. It functions as a barrier between the outside and internal environments to protect the body from external injury and invasion, and to prevent the loss of nutrients, electrolytes, and body fluids. [1][2][3] Breaking the barrier may, thus, have serious implication for human health and life. 3 Numerous strategies have been developed and applied for wound-healing repair, including electrospun nanofibers of cellulose acetate, 4 self-assembled tripeptide glycyl-L-lysine nanoparticles, 5 curcumin loaded sodium hyaluronate immobilized vesicles, 6 allantoin-incorporated organ oil enriched liposomes 7 and tocopherol-loaded vesicle transfersomes. 8 Notably, wound healing, especially of extensive full-thickness wounds usually takes a long time. 3 There is, thus, an urgent need for an efficient wound dressing that can promote wound closure, accelerate wound healing, and reduce scar formation. 9,10 In addition, wound dehydration can destroy the conditions needed for injury repair, and the ideal wound dressing should thus also maintain a moist environment to promote wound healing. To date, researchers have developed various wound dressings, such as hydrogels, 11,12 glass microfibers, 10 polycaprolactone, 13 nanofibers 14 and silk fibroin. 15 Hydrogel dressings are widely applied because of their advantages of maintaining oxygen permeation, absorbing tissue exudates, cooling the injury surface to relieve patient discomforts, and thus maintaining a favorable environment for wound-healing progression. [16][17][18][19] Injectable hydrogel dressings in particular have unique properties, including in situ drug encapsulation, and attachment and padding to the shape of the wound, thus protecting the injured skin from the outside environment. 20 In addition, stimuli-responsive bio-based polymeric systems have attracted significant attention for diverse biomedical applications. 21,22 However, reactive oxygen species (ROS)responsive wound dressings have not yet been developed.
Sodium alginate is a natural linear polysaccharide mainly extracted from brown algae, 23 while pectin is also a linear polysaccharide polymer, which can be extracted from diverse plant materials. 24,25 Their characteristics of biocompatibility, biodegradability, and film-forming ability mean that the combination of sodium alginate and pectin (SA/PC hydrogel) has been widely used for wound dressings. 26 However, sodium alginate and pectin lose their shape and integrity when exposed to the physiologic environment due to their hydrophilic nature. 27 Fortunately, ionic crosslinking, especially by Ca 2+ , can improve the polymers' properties by enhancing their stability, tensile strength, and light transmission and decreasing water-vapor permeation. 26,28 SA/PC hydrogels were therefore selected and subjected to cross-linking by Ca 2+ for the fabrication of wound dressings.
Substance P (SP) is a 10-amino acid neuropeptide generated by both immune and neuronal cells. It is mainly found in the junction between the adventitia and media in the muscle layer, in connective tissues around blood vessels and in the epidermis and dermis. SP has been shown to regulate wound healing and inflammation. 29,30 It is also an endogenous damage-induced factor that mobilizes CD29 (+) stromal-like cells into the circulation and accelerates wound healing. [31][32][33] Metal-organic frameworks (MOFs) are highly crystalline materials combining metal ions or clusters with rigid organic ligands and represent promising materials for biomedical applications. 34 Due to their excellent characteristics of high surface area and porosity, tunable pore shape and size, adjustable structure, biodegradability, and variable internal traits depending on the guest molecules, MOFs have been selected for various applications, including catalysis, gas storage and delivery, selective gas absorption and separation, and drug delivery. 34,35 Notably, the emerging MOF zeolite imidazolate framework-8 (ZIF-8) has attracted wide attention. ZIF-8 has a high loading efficiency and is thus widely used for drug delivery, [36][37][38][39][40] and has also been used as an enhanced delivery system for nucleic acids, 41 oligodeoxynucleotides 42 and proteins. 43,44 However, to the best of our knowledge, ZIF- 8 has not yet been developed as a carrier for the delivery of SP for wound healing. We, therefore, loaded ZIF-8 with SP to enhance its delivery to wound sites.
In this study, SP-loaded ZIF-8 (SP@ZIF-8) nanoparticles were fabricated and coated with polyethylene glycolthioketal (PEG-TK) to obtain a ROS-responsive trait. The obtained SP@ZIF-8-PEG-TK nanoparticles were loaded into a hydrogel consisting of sodium alginate and pectin, and then cross-linked using calcium chloride. ZIF-8 and SP@ZIF-8-PEG-TK nanoparticles were characterized by scanning and transmission electron microscopy, and by Brunauer-Emmett-Teller (BET) and thermogravimetric (TGA) analyses. The ROS-responsiveness of SP@ZIF-8-PEG-TK nanoparticles was assessed by drug-release assay. The effects of the SP@ZIF-8-PEG-TK nanoparticles on human dermal fibroblasts (HDFs) and macrophages were evaluated in vitro. The above resulting SP@ZIF-8-PEG-TK@CA dressing was characterized by scanning and transmission electron microscopy. The dressing with improved properties was used as wound dressing and its function was confirmed in vivo using a fullthickness excision wound model.

Materials and Methods
Fabrication of SP@ZIF-8 Nanoparticles and Dressings SP@ZIF-8 nanoparticles were synthesized as described previously. 45 Firstly, 0.75 mg SP was added into 0.9 mL aqueous solution of 2-methylimidazolate (MeIM, 3.15 mmol) with vigorous stirring, and the mixture was stirred constantly for 10 min at 30°C, followed by the addition of 0.1 mL aqueous solution of Zn(NO 3 ) 2 ▪6H 2 O (0.045 mmol) and agitation for 10 min at 30°C. SP@ZIF-8 nanoparticles were finally obtained after centrifugation at 3500 rpm for 20 min. Free SP on the surface of the SP@ZIF-8 nanoparticles was removed by rinsing several times with ethanol followed by 5% sodium dodecyl sulfate (w/w) at 50°C. The pure SP@ZIF-8 nanoparticles were lyophilized, weighed, and stored at 4°C.
Characterization ZIF-8 nanoparticle (100 µg/mL) and SP@ZIF-8-PEG-TK nanoparticle solutions (100 µg/mL) were prepared separately by diluting in double distilled water and characterized. The ultrastructures of the ZIF-8 and SP@ZIF-8-PEG-TK nanoparticles were examined by scanning electron microscopy (SEM, Hitachi FE-SEM S-4800) and transmission electron microscopy (TEM, JEM-2100F). The particle sizes of the ZIF-8 and SP@ ZIF-8 nanoparticles were measured by dynamic light scattering (DLS) at 25°C using a Malvern Zetasizer NanoZS instrument. The BET surface areas of the ZIF-8 and SP@ ZIF-8 nanoparticles were examined by N 2 adsorption-desorption. TGA of the ZIF-8 and SP@ZIF-8-PEG-TK nanoparticles was performed using a TAQ500 thermogravimetric analyzer, and the data were obtained from 25~800°C increasing at 10°C/min of scan rate.

SP-Release Assay
We explored the effects of ROS on SP release from SP@ZIF-8-PEG-TK nanoparticles by drug-release assays in phosphate buffer (PBS, pH 7.4) containing different concentrations of H 2 O 2 (0, 5, and 20 mM) at 37°C. SP@ZIF-8-PEG-TK nanoparticles (50 mg) were added into the buffer (10 mL) and the mixture was shaken mildly (50 rpm/min) for 48 h. The fluorescence intensity of the solution was then read at intervals with a Lambda Bio40 UV/visspectrometer (Perkin-Elmer).

Cell Proliferation
The viability of HDFs treated with SP alone or SP@ZIF-8-PEG-TK nanoparticles was determined by the cell counting kit-8 (CCK-8) assay. HDFs (2.0 × 10 4 cells/cm 2 ) were plated on a cell culture dish and the absorbance was detected by spectrophotometry after 1, 4 and 7 days of incubation, using a microplate reader at a wavelength of 450 nm.

Cell Spreading
HDFs were incubated with SP, ZIF-8 and SP@ZIF-8-PEG-TK nanoparticles and their morphologies were observed by confocal laser scanning microscopy (CLSM). HDFs were collected after 24 h of incubation, fixed for 30 min with 4% paraformaldehyde, and then stained with rhodamine-labelled phalloidin (Abcam) for 45 min, followed by DAPI (Sigma) for 10 min. Finally, the cells were rinsed with PBS and observed by CLSM to visualize the nuclei and cytoskeleton.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
The expression levels of inflammation-related genes in LPSinduced macrophages with or without SP@ZIF-8-PEG-TK treatment were determined by RT-qPCR. The primer sequences are shown in Table 1.

In vivo Full-Thickness Excision Wound Healing Mouse Model
BALB/c mice (4 weeks old) were used in the animal study. All animal experiments were approved by the Animal Ethics Committee of Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University and carried out in accordance with its guidelines. The mice were anesthetized by intraperitoneal injection of pentobarbitone sodium (50 mg/kg). Their backs were then shaved, the exposed skin was sterilized with 75% ethanol, and a full-thickness wound (diameter 1 cm) was incised on the exposed dorsum. The wound was then treated with 100 μL of SP@CA or SP@ZIF-8-PEG-TK@CA dressings and covered with a bandage. Wound healing was observed 3, 7 and 15 days post-wounding. The reduction wound area was calculated according to the following equation: Where Ai is the initial wound area and At is the area of the wound at a specific time point.

Immunofluorescence Analysis
After different treatments, HDFs or wound tissues were collected and fixed with 4% paraformaldehyde. M1 and M2 phenotype macrophages were detected in HDFs or wound tissues sections after staining with CD32 and CD206 (Santa Cruz Biotecknology), respectively. DAPI (Sigma) was used to stain the nuclei.

Statistical Analysis
The data were recorded as mean ± standard deviation. Statistical analysis was carried out using IBM SPSS Statistics (version 20.0). Comparisons between two groups were made using Student's t-test and comparisons among multiple groups by one-way analysis of variance followed by Turkey's test. P<0.05 indicated a significant difference.
In this study, SP@ZIF-8 was coated by PEG-TK through ROS-responsive TK linkers. Stimulation with ROS would  (Figure 2). ROS are known to be generated at wound sites. 46 release SP in an incisional wound microenvironment containing high levels of ROS.

SP@ZIF-8-PEG-TK Nanoparticles Promoted HDFs Proliferation
To investigate the effects of SP@ZIF-8-PEG-TK nanoparticles on the proliferation of HDFs, we incubated HDFs with SP alone or SP@ZIF-8-PEG-TK nanoparticles and determined their viability by CCK-8 assay. There was no significant difference among the three groups after 1 and 4 days of culture ( Figure 3). However, the SP and SP@ZIF-8-PEG-TK groups showed significantly higher cell viabilities than the control group by 7 days, and the viability of the SP@ZIF-8-PEG-TK group was higher than that of the SP group. These finding indicated that SP@ZIF-8-PEG-TK nanoparticles promoted the proliferation of HDFs. We observed the spreading of HDFs cultured with SP, ZIF-8 and SP@ZIF-8-PEG-TK nanoparticles by CLSM (Figure 4). All the HDFs possessed a typical shape and had abundant actin filaments linking them to the surrounding cells after 24 and 72 h of incubation. However, the density and morphological characteristic of the HDFs treated with SP and SP@ZIF-8-PEG-TK were superior to those without treatment or treated with pure ZIF-8 nanoparticles after 72h of incubation.

SP@ZIF-8-PEG-TK Nanoparticles Promoted the Transfer to M2 Macrophages
CD32 is a marker of M1 phenotype macrophages. 50 In the current study, the immunofluorescence intensity of CD32 was increased in cells treated with SP@ZIF-8-PEG-TK nanoparticle compared with the control and SP treatments, suggesting  the generation of M1 phenotype macrophages ( Figure 6). An increase in M1 phenotype macrophages indicated the proinflammatory effect of SP@ZIF-8-PEG-TK nanoparticles and their consequent potential for wound healing.
Characterization of SP@ZIF-8-PEG-TK @CA Dressings SEM images of cross-sections of SP@CA and SP@ZIF-8-PEG-TK@CA dressings are shown in Figure 7. Both dressings had an interconnected porous architecture. The addition of SP@ZIF-8-PEG-TK had no significant effect on the pore size or interconnection between the pores of the hydrogel dressings. The SP@ZIF-8-PEG-TK nanoparticles were dispersed homogeneously as primary particles in the polymer matrix of the hydrogel films.

In vivo Wound Healing
We reduced the wound area by 12.6% after 3 days, compared with only 5.9% in the SP@CA group ( Figure 8). After 7 days of treatment, SP@ZIF-8-PEG-TK@CA had reduced the wound area by 62.5%, which was significantly more than the 42.5% in the SP@CA group. SP@ZIF-8-PEG-TK @CA had almost completely closed the wound (nearly 100%) after 15 days of treatment, which was also significantly higher than in the SP@CA group.
To investigate the mechanism underlying the effects of SP@CA and SP@ZIF-8-PEG-TK@CA dressings at the injured sites, we carried out immunofluorescence staining of CD32 at day 3 and CD206 staining at day 7. There were abundant CD32-positive cells at the injured sites in the SP@ZIF-8-PEG-TK@CA group ( Figure 9A). The observed effects of SP@ZIF-8-PEG-TK-CA dressings in vivo were consistent with those of the SP@ZIF-8-PEG-TK nanoparticles in vitro. SP@ZIF-8-PEG-TK@CA dressings promoted an inflammatory response and accelerated wound healing at an early stage of the wound-healing process.
M2 phenotype macrophage, characterized by high expression levels of CD206, are referred to as "wound healing"-macrophages and play an important role in tissue repair. [51][52][53] In the current study, the numbers of CD206positive cells at wound sites at the late stage were higher in mice treated with SP@ZIF-8-PEG-TK@CA compared with SP@CA dressings ( Figure 9B). All these findings suggested that SP@ZIF-8-PEG-TK@CA stimulated and promoted wound healing by M2 macrophage polarization. 54

Conclusion
In this study, we developed an injectable hydrogel for wound dressing application and confirmed that this dressing markedly promoted wound healing in a full-thickness excision mouse model. The SP@ZIF-8-PEG-TK@CA hydrogel dressing was prepared by incorporating SP-loaded ZIF-8-coated with PEG-TK (SP@ZIF-8-PEG-TK) nanoparticles into a hydrogel consisting of sodium alginate and pectin, and then cross-linked using calcium chloride. The SP@ZIF-8-PEG-TK nanoparticles exhibited high SP-loading efficiency and a novel ROS-stimulus -responsive release. The nanoparticles promoted the proliferation of human dermal fibroblasts, up-regulated the expression of inflammation-related genes in macrophages and exhibited favorable cytocompatibility in vitro. The SP@ZIF-8-PEG-TK @CA dressing also promoted an inflammatory response at the early stage and M2 macrophage polarization at the late stage of the wound-healing process, and thus, exhibited excellent wound-healing efficacy in vivo. Overall, these findings indicate that SP@ZIF-8-PEG-TK@CA dressing might be a promising candidate for clinical wound dressing applications.

Ethics Approval and Consent to Participate
All animal experiments were approved by the Animal Ethics Committee of Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University and carried out in accordance with its guidelines.