Poly (lactic-co-glycolic acid) nanoparticles were synthesized through an established emulsion method and were used to encapsulate SSZ (Almería et al., 2011; Park et al., 2011; Steenblock et al., 2011). To elaborate, 100 mg of Poly (lactic-co-glycolic acid) with an inherent viscosity of 0.55–0.75 dL/g (ester terminal) and 10 mg of SSZ were dissolved in chloroform and slowly added drop-wise to a 5% solution of poly (vinyl alcohol) (Sigma-Aldrich). The mixture was sonicated three times and subsequently added to a 0.2% poly (vinyl alcohol) solution. Under stirring, the solvent was evaporated for 2 h, and the poly (lactic-co-glycolic acid) particles were centrifuged prior to lyophilization. The concentration of SSZ was determined by spectrophotometry (SpectraMax M5, Molecular Devices) at 565 nm. Pluronic F-127 (Sigma-Aldrich) was dissolved in PBS over a duration of 3 days at 4 °C, and the nanoparticles were added (50 mg particles/mL, 50 μg SSZ/mg NP [50% loading efficiency], mean NP diameter 380.5 nm; mean zeta-potential −38.8 ± 2.5 mV).

Sulfasalazine -containing nanoparticles (NP-SSZ) in pluronic gel were placed in a microdialysis system and the release of SSZ was monitored by periodic withdrawal of PBS samples. This was also performed with NP-SSZ without pluronic gel. The release was studied by placing a dispersion of NP-SSZ into a dialysis bag (cut-off 20,000 g/mol, Millipore) and immersing the bag into a vial containing PBS (1% Tween, 10 mL). At appropriate times, 1 ml of the release medium was collected from the vials and the medium was replaced by fresh PBS.

All animal laboratory studies were conducted in compliance with the guidelines on animal care of the Second Xiangya Hospital of Central South University, and were approved by the Institutional Animal Care and Use Committee. Male mice (C57BL/6J) aged 8–10 weeks and weighing between 23 and 30 g were used for this experiment. The mice were housed in a room maintained at 22 °C with a 12-h light/dark cycle, and provided with ad libitum access to food and water. The carotid artery procedure was performed as described in previous studies (Williams et al., 2022). Briefly, the mice were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine-HCl and 10 mg/kg xylazine-HCl. Both the left and right carotid arteries were exposed, and the left common carotid artery was ligated proximal to the bifurcation, while the right carotid artery was subjected to a sham procedure. For the NP-SSZ treatment group, NP-SSZ were dissolved in pluronic gel and applied to the ligated carotid artery adventitia prior to closing the skin. For the control group, only equivalent amount of NPs were dissolved in pluronic gel and applied to the ligated carotid artery adventitia prior to closing the skin.

For histological analysis, the carotid arteries were excised and embedded in paraffin and 5 μm-thick transverse sections on the glass slides were stained with hematoxylin and eosin (H&E), Elastica van Gieson (EVG), immunofluorescence was performed, according to the standard protocols; six sections taken from the middle portion of each artery, 28 days after the carotid artery injury, were examined; The histological sections of the neointimal area, medial area, and neointima/media (NI/M) ratio were analyzed using ImageJ software. Anti-Ki67, α-SMA, SM22α, MMP2, MMP9, Collage III, IBA-1 antibodies (all from Abcam), CD31/PECAM-1 (R&D), and other fluorescent secondary antibodies were obtained from Invitrogen.

Protein was extracted from carotid arteries and SMCs using RIPA lysis buffer containing a protease inhibitor cocktail (Thermo Scientific) with PhosSTOP (Roche) and boiled in SDS sample buffer for 6 min. Equal amounts of protein per sample were separated by SDS-PAGE, transferred electrophoretically to a nitrocellulose membrane (Bio-Rad Laboratories), and blotted with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling). Primary antibodies: anti‐p‐mTOR, anti‐mTOR, anti‐LC3B, anti‐P62 all from Abcam, HSP90 (Santa Cruz), Phospho-NF-κB p65 (Ser536), NF-κB p65, GAPDH, secondary antibody: anti-mouse HRP, anti-rabbit HRP all from Cell Signaling.

Crushed carotid arteries or scraped cells were immersed in lysis buffer and total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. RevertAid First-Strand cDNA Synthesis Kit (Thermo, Waltham, MA, United States of America) was performed for reverse transcription according to the manufacturer’s specifications. Subsequently, SYBR Green-based real-time PCR was performed in triplicate using SYBR Green master mix (Thermo Fisher Scientific) on an Applied Biosystems StepOnePlus real-time PCR machine (Thermo Fisher Scientific). For analysis, the threshold cycle (Ct) values for each gene were normalized to expression levels of GAPDH. Analysis was performed using the Bio-Rad CFX Manager software. Primer sequences were mouse GAPDH: forward, 5′-TGA​AGC​AGG​CAT​CTG​AGG​G-3′, reverse 5′-CGA​AGG​TGG​AAG​AGT​GGG​AG; TNF-α: forward, 5′-CCT​CCC​TCT​CAT​CAG​TTC​TA-3′, reverse,5′-ACTTGGTGGTTTGCTACGAC-3′ MCP-1: forward, 5′-CTT​CTG​GGC​CTG​CTG​TTC​A-3′, reverse, 5′-CCA​GCC​TAC​TCA​TTG​GGA​TCA-3′, ICAM-1: 5′-CAA​TTT​CTC​ATG​CCG​CAC​AG-3′, reverse, 5′-AGC​TGG​AAG​ATC​GAA​AGT​CCG-3′; VCAM-1: forward, 5′-CTT​CAT​CCC​CAC​CAT​TGA​AG-3′, reverse, 5′-TGA​GCA​GGT​CAG​GTT​CAC​AG-3′.

HA-VSMC cells were seeded in six-well plates at 2.5 × 10⁵ cells/well and cultured until cell monolayers formed. Monolayers were wounded in the center of the confluent cell monolayer by manual scraping with a 200-μL micropipette tip and washed with fresh media. The cells were then incubated with fresh medium supplemented with or without SSZ (200uM SSZ) for 24 h. Wound repair was analyzed by measuring the injured area covered by cells counted from the edge of the scratched lesion with the ImageJ software.

CCK‐8 Assay Kit (MedChem- Express) was used for assessing the viability of the VSMCs. The cells were seeded in the 96‐well plate, followed by different treatments. Then the cells were incubated with CCK‐8 reagent at 37 °C in the dark according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader.

Data are represented as mean value ±SEM. All data were analyzed using Prism 8 software (GraphPad Software, Inc., La Jolla, CA). Data were assumed to be from a normal distribution with equal variance, as suggested by normality (D’Agostino and Pearson test or Shapiro-Wilk test) and F tests. Statistical significance for these analyses was determined using Student t test or ANOVA (with repeated measures for time series data) with the Sidak post hoc correction. p values < 0.05 were considered statistically significant.

The pharmacokinetics of SSZ release from NP-SSZ was assessed in vitro; cumulative release of SSZ was sustained for at least 21 days (Figure 1A; green). In addition, NP-SSZ that were dissolved in pluronic gel showed less cumulative release of SSZ, with approximately 55% by day 21 (Figure 1A; red). These data show that NP-SSZ that were dissolved in pluronic gel have a longer release time could be better with a single dose delivered to the adventitia at the time of surgery.

The injured artery was followed by vascular remodeling which presented prominent luminal narrowing with NH. SMCs play a critical role in NH, including proliferation, phenotypic switching, and ECM deposition. In this study, the NH was measured as the ratio of intima to media (I/M), the ratio of I/M was increased after ligation injury at day 28, while the ratio was significantly reduced in the NP-SSZ treatment group (Figures 1B,C), indicating that NP-SSZ inhibited NH which induced by ligation injury. SMCs proliferation makes a big contribution to the NH, Ki-67, a maker of proliferation, was detected and compared in this study to see if there are any differences between the NP-SSZ treatment group and the control group. The dual positive nuclei of Ki-67 and α-SMA were 47.83 ± 9.15%, whereas only 29.83 ± 5.98% in the SSZ-treated group (p < 0.05) (Figure 2 A, C). The immunostaining shows that Ki-67 in SMCs was decreased in the NP-SSZ treatment group, indicating that NP-SSZ inhibited SMCs proliferation.

Since MMP-2 and MMP-9 were abundantly expressed in proliferative neointima, we can detect them as another marker to evaluate the severity of NH in the NP-SSZ treatment group and control group. The immunostaining results show that both the MMP-2 and MMP-9 were decreased in the NP-SSZ treatment group (p < 0.05, p < 0.05, respectively) compare to the control group (Figure 3). Next, we studied the VSMC characteristics of the injured artery under the NP-SSZ treatment since the MMP family promotes synthetic VSMC characteristics (Newby, 2006). The immunostaining results show that the expression of SM-22α was significantly increased in the NP-SSZ treatment group, while the expression of collagen type III was decreased (Figure 2 B, D, E).

The quantitative PCR was performed to investigate whether NP-SSZ can affect the inflammatory response, the results showed that the levels of the targeted inflammatory genes (TNF-α, VCAM-1, ICAM-1, MCP-1) were lower in the NP-SSZ treatment group compared with the control group (Figure 4). Moreover, the IF results showed that the Ionized calcium-binding adaptor molecule 1(IBA-1) was decreased in the NP-SSZ treatment group (p < 0.05). Therefore, those data demonstrate that NP-SSZ suppressed the inflammatory response after artery injury.

The proliferation and migration of VSMCs make a great contribution to neointima hyperplasia. VSMCs were treated with TNF-α to induce cell proliferation and migration for investigating the effects of SSZ on VSMCs. The CCK‐8 assay results demonstrated that the cell viability of VSMCs was markedly increased in the TNF-α treatment group (Figure 5C), whereas SSZ treatment inhibited this effect. In addition, a cell scratch assay was performed in the groups of TNF-α+Vehicle and TNF-α+ SSZ group for evaluating the effect of SSZ on VSMCs migration. As reflected in the scratch assay, the migration rate of the TNF-α group was significantly higher than that of the control group, however, the migration rate was decreased in the SSZ treatment group, indicating that SSZ suppressed migration of VSMCs (Figure 5D). Western blot was performed to evaluate the proliferation of VSMCs. Compare to the vehicle group, the PCNA expression was significantly decreased in the SSZ treatment group, which was consistent with the results in vivo (Figure 6A). The above results suggested that the SSZ treatment can inhibit VSMC proliferation and migration which was induced by TNF-α.

It is reported that autophagy inhibits the proliferation and migration of VSMCs to reduce neointima hyperplasia. To investigate whether NP-SSZ induces autophagy in the injured carotid arteries, a Western blot was performed to detect the hallmarks of autophagy. The results showed that P62 protein were higher in the NP-SSZ group than the vehicle group, indicating that NP-SSZ promotes autophagy in carotid arteries after injury (Figure 6A). We use the TNF-α to induce the VSMC proliferation and migration, similarly, the Western blot results showed the LC3 II and P62 protein expression were higher in the group co-treatment with SSZ (Figures 6F,H,J). Taken together, those results demonstrated that SSZ promotes autophagy both in vivo and in vitro.

It is reported that SSZ can inhibit NF-kB activation in vivo (Wahl et al., 1998; Robe et al., 2004). In this study, WB was performed to detect the effects of SSZ on NF-kB activation. The WB results showed that P62 expression in the SSZ treatment group was lower than the control group with a significant difference (p < 0.05) (Figures 6F,H). Those results demonstrate SSZ inhibits NF-kB activation.

Since SSZ promotes autophagy and previous studies have shown that mTOR plays a pivotal role in autophagy, we hypothesize that SSZ affects the mTOR expression inducing autophagy to inhibit the VSMCs proliferation and migration. To prove this hypothesis, MHY1485, an agonist of mTOR was used to attest whether NF-kB/mTOR pathway is essential for SSZ -inducing autophagy. The Western blot results have shown that the phosphorylation of NF-kB (p‐NF-kB) and the phosphorylation of mTOR (p‐mTOR) were decreased in the TNF-α + SSZ group, whereas the P62 and LC3 II expression levels were increased. However, the expression level of p‐mTOR, P62, and LC3 II was reversed after co‐treatment with MHY1485, whereas the p-NF-kB expression level was without any changes (Figure 6F). Taken together, these data indicate that SSZ promotes autophagy through NF-kB/mTOR pathway.