Integrating Inflammation-Responsive Prodrug with Electrospun Nanofibers for Anti-Inflammation Application

Chronic inflammation plays a side effect on tissue regeneration, greatly inhibiting the repair or regeneration of tissues. Conventional local delivery of anti-inflammation drugs through physical encapsulation into carriers face the challenges of uncontrolled release. The construction of an inflammation-responsive prodrug to release anti-inflammation drugs depending on the occurrence of inflammation to regulate chronic inflammation is of high need. Here, we construct nanofiber-based scaffolds to regulate the inflammation response of chronic inflammation during tissue regeneration. An inflammation-sensitive prodrug is synthesized by free radical polymerization of the indomethacin-containing precursor, which is prepared by the esterification of N-(2-hydroxyethyl) acrylamide with the anti-inflammation drug indomethacin. Then, anti-inflammation scaffolds are constructed by loading the prodrug in poly(ε-caprolactone)/gelatin electrospun nanofibers. Cholesterol esterase, mimicking the inflammation environment, is adopted to catalyze the hydrolysis of the ester bonds, both in the prodrug and the nanofibers matrix, leading to the generation of indomethacin and the subsequent release to the surrounding. In contrast, only a minor amount of the drug is released from the scaffold, just based on the mechanism of hydrolysis in the absence of cholesterol esterase. Furthermore, the inflammation-responsive nanofiber scaffold can effectively inhibit the cytokines secreted from RAW264.7 macrophage cells induced by lipopolysaccharide in vitro studies, highlighting the great potential of these electrospun nanofiber scaffolds to be applied for regulating the chronic inflammation in tissue regeneration.


Introduction
Inflammation is a protective response in human-beings that prevents higher organisms from infection and injury [1]. The initial acute inflammatory response plays a vital role, acting as an indispensable phase in tissue healing and regeneration [2][3][4]. However, if not regulated tightly, acute inflammation may develop into chronic inflammation, which will cause an excessive release of noxious by-products and eventually lead to chronic diseases such as diabetic wounds [5,6], osteoarthritis [7,8], and others. Hence, it is important to ensure tissue regeneration by regulating chronic inflammation at the correct timing [2].
Clinically, oral administration of nonsteroidal anti-inflammatory drugs (NSAIDs) is often used to regulate inflammation, among which ibuprofen, indomethacin (IDCM), and diclofenac are commonly used [9,10]. However, with the existence of the carboxyl moieties in NSAIDs, long-term intake of NSAIDs can incur severe side effects [11,12], especially catalyze the hydrolysis of the ester bonds both in the prodrug and the PCL/gelatin matrix, leading to the generation of IDCM and the subsequent release to the surrounding. The CE enzyme-triggered release profile of the anti-inflammatory drug IDCM from the scaffolds and the efficacy of the anti-inflammation scaffolds on relieving the inflammation in lipopolysaccharide (LPS)-induced RAW264.7 cells model at the cellular level were studied. The results demonstrate a significant difference in drug release, and much more IDCM could be triggered to release from the scaffolds with the help of the CE enzyme. Furthermore, upon incubation of the scaffolds with LPS-induced RAW264.7 cells, the scaffolds could effectively inhibit the cytokines secreted from the RAW264.7 cells and regulate the inflammatory responses. This work offers a facile and widely-applicable strategy for the fabrication of smart biomaterials with the stimuli-responsive capability to promote tissue regeneration by regulating chronic inflammation. the prodrug PIDCM into the PCL/gelatin electrospun nanofibers. CE, mimicking the inflammation environment, was adopted to catalyze the hydrolysis of the ester bonds both in the prodrug and the PCL/gelatin matrix, leading to the generation of IDCM and the subsequent release to the surrounding. The CE enzyme-triggered release profile of the anti-inflammatory drug IDCM from the scaffolds and the efficacy of the antiinflammation scaffolds on relieving the inflammation in lipopolysaccharide (LPS)induced RAW264.7 cells model at the cellular level were studied. The results demonstrate a significant difference in drug release, and much more IDCM could be triggered to release from the scaffolds with the help of the CE enzyme. Furthermore, upon incubation of the scaffolds with LPS-induced RAW264.7 cells, the scaffolds could effectively inhibit the cytokines secreted from the RAW264.7 cells and regulate the inflammatory responses. This work offers a facile and widely-applicable strategy for the fabrication of smart biomaterials with the stimuli-responsive capability to promote tissue regeneration by regulating chronic inflammation. Figure 1. Schematic illustration of integrating inflammation-responsive prodrug with electrospun nanofibers for anti-inflammation application. The synthesis procedure of inflammation-responsive prodrug PIDCM, and schematic illustration of the enzyme-triggered release of IDCM from electrospun nanofibers loaded with inflammation-responsive prodrug.

Preparation of IDCM Prodrug
MIDCM was polymerized by radical polymerization to prepare poly (MIDCM) (further labeled as PIDCM) [39]. The molecular weight of PIDCM was controlled by varying the reaction time. Briefly, 0.5 g of MIDCM (1.1 mmol) and 5 mg of AIBN (0.03 mmol) were firstly dissolved in 5 mL of DMF under a nitrogen atmosphere and then sealed. After stirring for 8 h and 36 h in an oil bath at 70 • C, respectively, the reactions were stopped, and the solutions were precipitated in DI water. After drying under vacuum for 24 h, PIDCM with two different molecular weights were obtained, and the M n were measured by gel permeation chromatography (GPC, Waters 1525-2414 system, Wasters Corporation, Milford, MA, USA). Polystyrene and tetrahydrofuran were the standard reference and the eluent for calibrating the GPC, respectively. The columns were Waters Styragel HT3 THF, Waters Styragel HT4 THF, and Waters Styragel HT5 THF. The detector was the 2414 differential refractive index detector. The prodrugs with a molecular weight of about 3500 g/mol and 1400 g/mol were prepared and labeled as PIDCM 35 and PIDCM 14 , respectively.

Fabrication of Electrospun Nanofibers
PCL and gelatin were used as the matrix to fabricate the PIDCM prodrug-loaded electrospun nanofibers. Electrospinning was carried out according to the previous report [45,46], and the type of spinning machine was purchased from Beijing Xinrui Baina Technology Co., Ltd. (TEADFS-103, Beijing, China). In brief, PCL and gelatin (50/50 w/w) were dissolved in TFE to prepare the homogeneous electrospinning solution, and then the prodrugs PIDCM 35 and PIDCM 14 in the range of 0-60 wt.% were separately added to the electrospinning solutions. The solution was added to a 10 mL syringe and fed by a syringe pump at a rate of 1 mL/h. At the same time, a roller wrapped with aluminum foil was applied as the collector, and the rotating rate was set to 270 rpm. Between the needle and the grounded collector, a high voltage (12 KV) was applied. The distance between the needle and the ground collector was set as 15 cm. As shown in Table 1, the prepared electrospun nanofibers with PIDCM 35 of 20 and 60 wt.% were labeled as PGPI 35 20 and PGPI 35 60, respectively. The electrospun nanofibers with PIDCM 14 of 20, 40, and 60 wt.% were labeled as PGPI 14 20, PGPI 14 40, and PGPI 14 60, respectively. As a control, PCL/gelatin electrospun nanofiber was also prepared, which was labeled as PG0.

Morphology Characterization of Electrospun Nanofibers
The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM, S4800, Hitachi, Japan). The diameter of electrospun nanofibers on the SEM micrographs was measured by Image J software according to the previous report [47].

Chemical Characterization of Electrospun Nanofibers
The surface characterization of electrospun nanofibers was investigated by energy dispersive spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS), which could provide the information of different elements on the surface. The Cl content of the electrospun nanofibers' surfaces was investigated by EDS, which was carried out on a HORIBA X-Max20 detector (HORIBA Corporation, Kyoto, Japan) attached to the SEM. At the same time, the surface composition and functional groups of the electrospun nanofibers were investigated by XPS, which was performed on an ESCALAB 250 (Thermo Electron Corporation, Waltham, MA, USA) according to the previous report [48,49].

Hydrophilicity Characterization of Electrospun Nanofibers
The water contact angles of the electrospun nanofiber scaffolds were determined by a SL200A type Contact Angle Analyzer (SOLON TECH., Shanghai, China) at room temperature according to the previous report [45].

Cytotoxicity and Proliferation of Cells on the Surfaces
MC3T3-E1 and L929 cells are typical cells for testing cytotoxicity [50,51]. The viability of the cells proliferated on the electrospun nanofiber scaffolds loaded with PIDCM 35 and PIDCM 14 were evaluated using MC3T3-E1 osteoblasts and L929 fibroblasts by using CCK-8 assay, respectively. Briefly, the electrospun nanofiber scaffolds were cut into 13 mm diameter sheets with a 13 mm diameter cutter, sterilized by an Ultraviolet lamp, then fixed in a 24-well plate, and 4.0 × 10 3 cells were plated onto the surfaces of samples carefully, followed by moving to an incubator at 37 • C. The number of cells that proliferated on the scaffolds at a determined time interval (day 1, 3, 5, and 7) was quantified using the CCK-8 assay referring to previous reports [39,52]. As for the electrospun nanofiber scaffolds loaded with PIDCM 35 , the samples co-cultured with cells were firstly washed with PBS three times, followed by fixing cell morphology via 3% glutaraldehyde. After dehydration and lyophilization, the morphology of MC3T3-E1 cells on the scaffolds loaded with PIDCM 35 was observed by SEM. As for the electrospun nanofiber scaffolds loaded with PIDCM 14 , the samples co-cultured with cells were washed with PBS three times, and then L929 cells were stained by DAPI and Alexa Fluor 568-phalloidin and observed under a fluorescent inverted microscope (Axio Observer 3).

Drug Release Profiles
The drug release profiles of the PGPI 35 20 and PGPI 35 60 were determined as follows. Each sample was cut into 13 mm round discs, and all the samples were accurately weighed. Then, the samples were incubated at 37 • C in 1 mL PBS (pH 7.4) with or without CE (10 U/mL) to investigate the release kinetics of IDCM. High-performance liquid chromatography (HPLC) was used to detect the amount of released IDCM [39]. The percentage of IDCM release from triplicate samples was then determined based on the amount of IDCM in the prodrug incorporated into electrospun nanofiber scaffolds.

In Vitro Anti-Inflammatory Activity
RAW264.7 cells were used to evaluate the anti-inflammatory effect of the electrospun nanofiber scaffolds. Briefly, RAW264.7 cell suspension (5 × 10 5 cells/mL, 1 mL/well) was added to a 24-well plate. After incubation for 24 h, LPS was added to the DMEM solution with 10% FBS at a concentration of 5 µg/mL, followed by replacing the old culture medium. After incubation for another 24 h, the LPS-treated RAW264.7 cells were incubated with PG0,  35 20, and PGPI 35 60 for 24 h and 48 h, respectively. Finally, the levels of IL-6 and NO in the supernatants were determined by the ELISA kit and Griess reaction, respectively [39].

Statistical Analysis
All quantitative data were expressed as mean ± standard deviation. OriginPro 8 software (Hampton, OriginLab) was used to perform the statistical analyses. The statistical differences between independent samples were performed by using the student's t-test. * p < 0.05, ** p < 0.01, and *** p < 0.005 were regarded as statistically significant among independent groups.

Characterization of Prodrug
To investigate the influence of the different molecular weights of prodrugs on the anti-inflammatory properties of the electrospun nanofiber scaffolds: firstly, two PIDCM prodrugs with the molecular weight of 1400 g/mol and 3500 g/mol were readily obtained via free radical polymerization, respectively. The molecular weights of the two PIDCM prodrugs were measured using GPC, as shown in Figure 2, indicating the low polydispersity index. Then, the as-synthesized prodrug was incorporated with PCL/gelatin matrix to prepare the PIDCM-loaded electrospun nanofibers through electrospinning. chromatography (HPLC) was used to detect the amount of released IDCM [39]. The percentage of IDCM release from triplicate samples was then determined based on the amount of IDCM in the prodrug incorporated into electrospun nanofiber scaffolds.

In Vitro Anti-Inflammatory Activity
RAW264.7 cells were used to evaluate the anti-inflammatory effect of the electrospun nanofiber scaffolds. Briefly, RAW264.7 cell suspension (5 × 10 5 cells/mL, 1 mL/well) was added to a 24-well plate. After incubation for 24 h, LPS was added to the DMEM solution with 10% FBS at a concentration of 5 μg/mL, followed by replacing the old culture medium. After incubation for another 24 h, the LPS-treated RAW264.7 cells were incubated with PG0, PGPI3520, and PGPI3560 for 24 h and 48 h, respectively. Finally, the levels of IL-6 and NO in the supernatants were determined by the ELISA kit and Griess reaction, respectively [39].

Statistical Analysis
All quantitative data were expressed as mean ± standard deviation. OriginPro 8 software (Hampton, OriginLab) was used to perform the statistical analyses. The statistical differences between independent samples were performed by using the student's t-test. * p < 0.05, ** p < 0.01, and *** p < 0.005 were regarded as statistically significant among independent groups.

Characterization of Prodrug
To investigate the influence of the different molecular weights of prodrugs on the anti-inflammatory properties of the electrospun nanofiber scaffolds: firstly, two PIDCM prodrugs with the molecular weight of 1400 g/mol and 3500 g/mol were readily obtained via free radical polymerization, respectively. The molecular weights of the two PIDCM prodrugs were measured using GPC, as shown in Figure 2, indicating the low polydispersity index. Then, the as-synthesized prodrug was incorporated with PCL/gelatin matrix to prepare the PIDCM-loaded electrospun nanofibers through electrospinning.

Morphology of the Electrospun Nanofibers
As shown in Figure 3a, SEM micrographs show that the PG0 nanofibers were randomly arranged with smooth surfaces, whereas the surfaces of PGPI 35 20 and PGPI 35 60 nanofibers contained needle-like bumps, and the number of the bumps increased with the increase in the content of PIDCM 35 . The average fiber diameters of PG0, PGPI 35 20, and PGPI 35 60 were 0.58 ± 0.10, 0.63 ± 0.11, and 0.78 ± 0.26 µm, respectively, as shown in Figure 3b. We also tested the fiber diameter distribution of PGPI 35 20 and PGPI 35 60 including the needle-like bumps in Figure S1, the average fiber diameters of PGPI 35 20 and PGPI 35 60 including the needle-like bumps were 0.72 ± 0.11 and 1.06 ± 0.27 µm, respectively. On the contrary, SEM micrographs (Figure 3c) of the PGPI 14 20, PGPI 14 40, and PGPI 14 60 nanofibers showed smooth topography and no apparent drug crystals. As shown in Figure 3d, the average diameters of PGPI 14 20, PGPI 14 40, and PGPI 14 60 were 0.46 ± 0.17, 0.50 ± 0.09, and 0.76 ± 0.18 µm, respectively, and the diameters of the nanofibers were increased with the increase in PIDCM 14 content. Due to the higher molecular weight of PIDCM 35 than PIDCM 14 , it was easier for PIDCM 35 to precipitate from the nanofibers during electrospinning. average diameters of PGPI1420, PGPI1440, and PGPI1460 were 0.46 ± 0.17, 0.50 ± 0.09, and 0.76 ± 0.18 μm, respectively, and the diameters of the nanofibers were increased with the increase in PIDCM14 content. Due to the higher molecular weight of PIDCM35 than PIDCM14, it was easier for PIDCM35 to precipitate from the nanofibers during electrospinning.
significantly changed. For the other five samples, one new C 1s peak with th energy of about 285.4, 285.4, 286.55, 286.55, and 286.65 eV appeared, which attributed to the new carbon region of C-Cl for PGPI3520, PGPI3560, PGPI1420 and PGPI1460, respectively. These results suggested that PIDCM35 and PIDC loaded in the PCL-gelatin nanofibers. To further confirm the successful preparation of the PIDCM loaded into electrospun nanofibers, the surface composition and functional groups on electrospun nanofibers were investigated by XPS. Figure 4b,d show the C 1s spectra of the electrospun nanofibers. In the C 1s spectrum, the surface of pure PG0 nanofibrous mats presented four expected peaks with binding energy at 283.7, 284.6, 286.8, and 287.6, indicating the existence of four carbon regions of C-C, C-N, N-C=O, and O-C=O, respectively. After loading with PIDCM 35 and PIDCM 14 , the chemical compositions of the nanofiber surfaces were significantly changed. For the other five samples, one new C 1s peak with the binding energy of about 285.4, 285.4, 286.55, 286.55, and 286.65 eV appeared, which could be attributed to the new carbon region of C-Cl for PGPI 35 20, PGPI 35 60, PGPI 14 20, PGPI 14 40, and PGPI 14 60, respectively. These results suggested that PIDCM 35 and PIDCM 14 were loaded in the PCL-gelatin nanofibers.

Effects of PIDCM Encapsulation on Hydrophilicity of Scaffolds
Hydrophobicity of materials plays a vital role in tissue regeneration [45]. The hydrophilicity of the scaffolds was investigated by the water contact angles test, as shown in Figure 5. Among them, the water contact angles of the electrospun nanofiber scaffolds loaded with PIDCM 35 or PIDCM 14 were increased with the increase in PIDCM content. Specifically, PGPI 35 20 and PGPI 14 20 had good hydrophilicity, and their water contact angles were 26.0 • and 28.5 • , respectively, smaller than the contact angle of PG0 without drug loading. The hydrophilicity property of the scaffolds could provide them with a stronger protein adsorption capability, which plays an important role in providing cues to interact with cells and surrounding tissues.  respectively. (c) The EDS spectra and (d) normalized C 1s XPS spectra of PGPI1420, PGPI1440, an PGPI1460, respectively.

Effects of PIDCM Encapsulation on Hydrophilicity of Scaffolds
Hydrophobicity of materials plays a vital role in tissue regeneration [45]. Th hydrophilicity of the scaffolds was investigated by the water contact angles test, as show in Figure 5. Among them, the water contact angles of the electrospun nanofiber scaffold loaded with PIDCM35 or PIDCM14 were increased with the increase in PIDCM conten Specifically, PGPI3520 and PGPI1420 had good hydrophilicity, and their water contac angles were 26.0° and 28.5°, respectively, smaller than the contact angle of PG0 withou drug loading. The hydrophilicity property of the scaffolds could provide them with stronger protein adsorption capability, which plays an important role in providing cue to interact with cells and surrounding tissues.

In Vitro Biocompatibility of Electrospun Nanofibers Loaded with Prodrug
For cell-material interactions, attachment and proliferation are the first stages, thu the biocompatibility of material has a significant effect on the proliferation an morphology of cells [45]. Among them, MC3T3-E1 cells and L929 cells are widely used t test the biocompatibility of materials [45,46]. Thus, the cell proliferation assay of PG0 PGPI3520, and PGPI3560 was tested by MC3T3-E1 cells. Meanwhile, the cell proliferatio assay of PGPI1420, PGPI1440, and PGPI1460 was tested by L929 cells. As shown in Figur 6a, the numbers of MC3T3-E1 cells progressively increased during the 7 days, indicatin that MC3T3-E1 cells were adhered and in a proliferative state on PG0, PGPI3520, an PGPI3560. Similarly, PG0, PGPI3520, and PGPI3560 had good effects on promoting ce proliferation and showed good biocompatibility. At the same time, the morphologies o the MC3T3-E1 cells proliferating on PGPI3520 for 1, 3, 5, and 7 days were observed by SEM (Figure 6b), respectively. The expanding area of MC3T3-E1 cells on the surface of th electrospun nanofibers increased with the extension of the co-culture time, and the cel on the surface of the PGPI3520 reached 70-90% confluence on day 7.
As shown in Figure 6c, the numbers of L929 cells on PG0, PGPI1420, PGPI1440, an PGPI1460 were continuously increased during 7 days of incubation, indicating th scaffolds loaded with prodrugs were nontoxic and supported cell proliferation. As show in the fluorescence microscopy images (Figure 6d), the cells reached 70-90% confluenc after incubation for 7 days. Furthermore, L929 cells exhibited healthy morphologies afte

In Vitro Biocompatibility of Electrospun Nanofibers Loaded with Prodrug
For cell-material interactions, attachment and proliferation are the first stages, thus the biocompatibility of material has a significant effect on the proliferation and morphology of cells [45]. Among them, MC3T3-E1 cells and L929 cells are widely used to test the biocompatibility of materials [45,46]. Thus, the cell proliferation assay of PG0, PGPI 35 20, and PGPI 35 60 was tested by MC3T3-E1 cells. Meanwhile, the cell proliferation assay of PGPI 14 20, PGPI 14 40, and PGPI 14 60 was tested by L929 cells. As shown in Figure 6a, the numbers of MC3T3-E1 cells progressively increased during the 7 days, indicating that MC3T3-E1 cells were adhered and in a proliferative state on PG0, PGPI 35 20, and PGPI 35 60. Similarly, PG0, PGPI 35 20, and PGPI 35 60 had good effects on promoting cell proliferation and showed good biocompatibility. At the same time, the morphologies of the MC3T3-E1 cells proliferating on PGPI 35 20 for 1, 3, 5, and 7 days were observed by SEM (Figure 6b), respectively. The expanding area of MC3T3-E1 cells on the surface of the electrospun nanofibers increased with the extension of the co-culture time, and the cells on the surface of the PGPI 35 20 reached 70-90% confluence on day 7.
As shown in Figure 6c, the numbers of L929 cells on PG0, PGPI 14 20, PGPI 14 40, and PGPI 14 60 were continuously increased during 7 days of incubation, indicating the scaffolds loaded with prodrugs were nontoxic and supported cell proliferation. As shown in the fluorescence microscopy images (Figure 6d), the cells reached 70-90% confluence after incubation for 7 days. Furthermore, L929 cells exhibited healthy morphologies after incubation with all groups. We can conclude that the PGPI 14 20, PGPI 14 40, and PGPI 14 60 could support the proliferation of L929 cells. incubation with all groups. We can conclude that the PGPI1420, PGPI1440, and PGPI14 could support the proliferation of L929 cells.

In Vitro Drug Release Profile of Electrospun Nanofibers Loaded with Prodrug
The prodrug encapsulation efficiency is significantly influenced by the interacti between the PCL/gelatin polymer matrix chains and PIDCM prodrug molecules [4 PIDCM is hydrophobic, contains carbonyl groups and amide bonds, and is capable interacting with the hydroxyl and carboxyl groups of gelatin chains via hydrog bonding. In the system, the cleavage of ester groups depended on the slow hydrolytic a fast enzymatic cleavage. Compared to PIDCM35, PIDCM14 was more likely to be releas from the nanofibers through diffusion due to its smaller molecular weight, which mea a less controllable release manner [40]. Our study aimed to design an inflammatio responsive scaffold, which could release anti-inflammatory drugs under the stimulati of the inflammation to regulate the excessive inflammation. Therefore, we chose PGPI35 and PGPI3560 to study the release profile of IDCM from the electrospun nanofib scaffolds upon enzyme stimulation.
Firstly, we tested the drugs after released from prodrugs by mass spectrum (Figu S2), in which the peak at 358.0845 was corresponding to IDCM. As shown in Figure 7, t enzyme-rich body fluid of living organisms during the occurrence of inflammation w  14 20, PGPI 14 40, and PGPI 14 60 for 1, 3, 5, and 7 days, respectively. (d) Fluorescence images of the L929 cells cultured on PG0, PGPI 14 20, PGPI 14 40, and PGPI 14 60 for 7 days, respectively. The F-actins of the cells were stained with Alexa Fluor 568-phalloidin (red), whereas cell nuclei were stained with DAPI (blue).

In Vitro Drug Release Profile of Electrospun Nanofibers Loaded with Prodrug
The prodrug encapsulation efficiency is significantly influenced by the interaction between the PCL/gelatin polymer matrix chains and PIDCM prodrug molecules [45]. PIDCM is hydrophobic, contains carbonyl groups and amide bonds, and is capable of interacting with the hydroxyl and carboxyl groups of gelatin chains via hydrogen bonding. In the system, the cleavage of ester groups depended on the slow hydrolytic and fast enzymatic cleavage. Compared to PIDCM 35 , PIDCM 14 was more likely to be released from the nanofibers through diffusion due to its smaller molecular weight, which means a less controllable release manner [40]. Our study aimed to design an inflammationresponsive scaffold, which could release anti-inflammatory drugs under the stimulation of the inflammation to regulate the excessive inflammation. Therefore, we chose PGPI 35 20 and PGPI 35 60 to study the release profile of IDCM from the electrospun nanofiber scaffolds upon enzyme stimulation.
Firstly, we tested the drugs after released from prodrugs by mass spectrum (Figure S2), in which the peak at 358.0845 was corresponding to IDCM. As shown in Figure 7, the enzyme-rich body fluid of living organisms during the occurrence of inflammation was mimicked by the CE enzyme. The cumulatively released percentage of IDCM at different time points was determined and calculated according to the standard curve [39]. For PGPI 35 20 and PGPI 35 60, the loaded amounts were 0.371 ± 0.033 µg and 0.305 ± 0.014 µg in one sample, respectively. In the absence of CE, both PGPI 35 20 and PGPI 35 60 showed an extremely slow release of IDCM and the total amounts of released IDCM were less than 50% in 24 h. In contrast, both PGPI 35 20 and PGPI 35 60 showed a significantly rapid release of IDCM in the presence of CE. Specifically, 100% IDCM was released from PGPI 35  one sample, respectively. In the absence of CE, both PGPI3520 and PGPI3560 showed an extremely slow release of IDCM and the total amounts of released IDCM were less than 50% in 24 h. In contrast, both PGPI3520 and PGPI3560 showed a significantly rapid release of IDCM in the presence of CE. Specifically, 100% IDCM was released from PGPI3520 in the first 8 h, and 100% IDCM was released from PGPI3560 in 24 h, showing the enzymetriggered release behavior of the PGPI3520 and PGPI3560. Therefore, it can be concluded that the PGPI3520 and PGPI3560 had the capabilities of delivering the drug component IDCM under the stimulation of the CE enzyme, allowing for the smart and on-demand drug release to avoid the chronic inflammation that occurs during tissue regeneration.

In Vitro Evaluation of the Anti-Inflammatory Effect of Electrospun Nanofibers Loaded with Prodrug
RAW264.7 cells can secrete various kinds of cytokines under the stimulation of LPS [1,36], such as IL-6 and NO [53]. The anti-inflammatory effect of PGPI3520 and PGPI3560 were evaluated using the inflammation model according to the previous study [39]. As shown in Figure 8, compared with the blank group, the concentration of IL-6 and NO were remarkably decreased when the RAW264.7 cells were cultured with PGPI3520 and PGPI3560, indicating that they could successfully suppress the inflammation reaction. With the extension of the co-culture time of PGPI3520 and PGPI3560 with RAW264.7 cells, the levels of IL-6 ( Figure 8a) and NO (Figure 8b) secreted from the LPS-induced cells were

In Vitro Evaluation of the Anti-Inflammatory Effect of Electrospun Nanofibers Loaded with Prodrug
RAW264.7 cells can secrete various kinds of cytokines under the stimulation of LPS [1,36], such as IL-6 and NO [53]. The anti-inflammatory effect of PGPI 35 20 and PGPI 35 60 were evaluated using the inflammation model according to the previous study [39]. As shown in Figure 8, compared with the blank group, the concentration of IL-6 and NO were remarkably decreased when the RAW264.7 cells were cultured with PGPI 35 20 and PGPI 35 60, indicating that they could successfully suppress the inflammation reaction. With the extension of the co-culture time of PGPI 35 20 and PGPI 35 60 with RAW264.7 cells, the levels of IL-6 ( Figure 8a) and NO (Figure 8b) secreted from the LPS-induced cells were decreased, demonstrating that more content of IDCM was released from PGPI 35 20 and PGPI 35 60 through the inflammation-triggered release. There was no significant difference between PGPI 35 20 and PGPI 35 60, indicating the anti-inflammatory efficiency was similar between these two groups, due to the concentration of CE enzyme secreted from the RAW264.7 cells was in a certain level, which limits the amounts of ester linkages of PGPI 35 20 and PGPI 35 60 that could be cleaved by the CE. Taken together, the electrospun nanofiber scaffold loaded with the prodrug could inhibit the inflammation response, which will be beneficial for tissue regeneration. decreased, demonstrating that more content of IDCM was released from PGPI3520 and PGPI3560 through the inflammation-triggered release. There was no significant difference between PGPI3520 and PGPI3560, indicating the anti-inflammatory efficiency was similar between these two groups, due to the concentration of CE enzyme secreted from the RAW264.7 cells was in a certain level, which limits the amounts of ester linkages of PGPI3520 and PGPI3560 that could be cleaved by the CE. Taken together, the electrospun nanofiber scaffold loaded with the prodrug could inhibit the inflammation response, which will be beneficial for tissue regeneration.

Conclusions
In summary, enzyme-sensitive prodrugs were synthesized through the free radical polymerization of the IDCM-containing precursor and then loaded into PCL/gelatin electrospun nanofibers to develop an inflammation-responsive nanofiber scaffold. A large amount of IDCM was released from the anti-inflammation electrospun nanofiber scaffold under the stimulation of CE, whereas there was a minimal release of the drug in the absence of enzyme. Moreover, the inflammatory response could be significantly attenuated after incubating the scaffolds with LPS-treated RAW264.7 cells. CE was secreted by macrophages to a distinctly high concentration around inflammatory sites, further triggering the hydrolysis of ester bonds both in the prodrug and PCL/gelatin, resulting in the intensified degradation of the matrix and the rapid release of generated IDCM to relieve the chronic inflammatory response. This study offers a feasible and wide applicable strategy to deliver drugs in a smart, responsive, and effective manner and

Conclusions
In summary, enzyme-sensitive prodrugs were synthesized through the free radical polymerization of the IDCM-containing precursor and then loaded into PCL/gelatin electrospun nanofibers to develop an inflammation-responsive nanofiber scaffold. A large amount of IDCM was released from the anti-inflammation electrospun nanofiber scaffold under the stimulation of CE, whereas there was a minimal release of the drug in the absence of enzyme. Moreover, the inflammatory response could be significantly attenuated after incubating the scaffolds with LPS-treated RAW264.7 cells. CE was secreted by macrophages to a distinctly high concentration around inflammatory sites, further triggering the hydrolysis of ester bonds both in the prodrug and PCL/gelatin, resulting in the intensified degradation of the matrix and the rapid release of generated IDCM to relieve the chronic inflammatory response. This study offers a feasible and wide applicable strategy to deliver