A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Abstract

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI (QK) are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases. However, conventional topical drug delivery often results in a burst release of the drug, leading to transient retention (inefficacy) and undesirable diffusion (toxicity) in vivo. Therefore, a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke. Matrix metalloproteinase-2 (MMP-2) is gradually upregulated after cerebral ischemia. Herein, vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG (TIMP) and customizable peptide amphiphilic (PA) molecules to construct nanofiber hydrogel PA-TIMP-QK. PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro. The results indicated that PA-TIMP-QK promoted neuronal survival, restored local blood circulation, reduced blood-brain barrier permeability, and restored motor function. These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Introduction

Cerebral ischemia is characterized by a considerable reduction in cerebral blood flow, destruction of the blood–brain barrier (BBB), and substantial neuronal death. Increasing blood supply and neurogenesis in the hypoxic-ischemic brain would be beneficial for functional recovery. Growth factors have been extensively used for treating cerebral ischemia through the induction of angiogenesis and neurogenesis (Hatakeyama et al., 2020; Dordoe et al., 2021; Zhu et al., 2021). Vascular endothelial growth factor (VEGF), as the most potent angiogenic factor, is commonly used to treat ischemic diseases (Zhang et al., 2000; Geiseler and Morland, 2018; Liu et al., 2023; Niu et al., 2023; Wu et al., 2024). In cerebral ischemia, VEGF promotes blood vessel regeneration by stimulating endothelial cell proliferation and migration, and it also acts as an essential neuroprotective factor by inducing neurogenesis and neural progenitor cell proliferation. However, VEGF has a relatively short half-life in vivo, and excessive administration of VEGF in the early stage after cerebral ischemia can increase BBB leakage in ischemic tissue, leading to edema and a subsequent increase in intracranial pressure that can further obstruct blood flow (Zhang et al., 2000). Therefore, modified administration routes of VEGF have been proposed to promote functional recovery of cerebral ischemia. For example, VEGF binding with heparan sulfate was shown to control VEGF release, which improved the repair of cerebral ischemia (Chan et al., 2020). VEGF embedded in different nanofiber membranes or microspheres was shown to protect the brain from ischemia/reperfusion (I/R) injury (Wang et al., 2023; Wu et al., 2023). In another study, specific peptides derived from various VEGF receptors were shown to specifically bind and release VEGF from a scaffold, which enhanced angiogenesis and neurogenesis in ischemic brain (Yin et al., 2022).

Alternatively, the VEGF-derived angiogenic peptide QK, with the sequence KLTWQELYQLKYKGI, has been shown to activate the VEGF receptor and mimic VEGF’s biological activity (Feng et al., 2020). In a permanent middle cerebral artery occlusion model, delivery of QK via the intracerebroventricular, intravenous, or intranasal routes markedly reduced cerebral ischemic tissue damage by up to 40% compared with control. Notably, QK was found to decrease vessel permeability compared with VEGF (Pignataro et al., 2015). Additionally, it has been reported that the use of excessive VEGF or QK peptides between 1–3 hours and 24 hours may have detrimental effects as described above, but that administration later than 24 hours after cerebral ischemia may promote neuroprotection and increase blood vessel density (Geiseler and Morland, 2018). Therefore, controlling the release of VEGF or QK is crucial for its clinical application in cerebral ischemia.

Smart stimulus-responsive hydrogels have been increasingly used in drug delivery and tissue engineering, and offer superior control over the spatiotemporal release of drugs for tissue regeneration than conventional inert hydrogels (Chen et al., 2023a). In the context of cerebral ischemia, these hydrogels can be designed to respond to specific biological cues from disease-associated enzymes. One such enzyme that has garnered attention is matrix metalloproteinase-2 (MMP-2), a member of the zinc and calcium-dependent extracellular endopeptidases family, which is progressively upregulated in various ischemic diseases. In cerebral ischemia, MMP-2 degraded tight junction proteins and disrupted the BBB, which was closely related to hemorrhage and vasogenic edema (Jickling et al., 2014). It was reported that MMP-2 levels increase 1 day after hypoxia and persist for over 2 weeks in the ischemic brain (Magnoni et al., 2004; Jin et al., 2012; Younis and Mohamed, 2023), which was also confirmed by our results. Because of this release profile, MMP-2 represents a potential candidate for initiating the release of VEGF or QK peptides 1 day after cerebral ischemia.

In the present study, we constructed an MMP-2-responsive self-assembly hydrogel, which consisted of VEGF mimic peptide QK, MMP-2 cleaved peptide from tissue inhibitors of MMP-2 (TIMP) with the sequence PLGLAG, and customizable peptide amphiphilic (PA) molecules. As MMP-2 was upregulated, the TIMP peptides were degraded by MMP-2 to release QK. In addition, TIMP peptides have been shown to exhaust endogenous MMP-2, thereby mitigating BBB disruption (Meng et al., 2021). Therefore, the PA-TIMP-QK nanofibers were hypothesized to promote angiogenesis and neuroprotection and attenuate BBB disruption after cerebral ischemia. We evaluated the physical and biological properties of the hydrogel in vitro, and investigated its therapeutic potential for repairing the ischemic brain in vivo.

Methods

Synthesis of self-assembling peptide and preparation of the hydrogel

The PA-TIMP-QK peptides (C16-VVAAPLGLAGKLTWQELYQLKYKGI-NH₂) and the PA-QK peptides (C16-VVAAKLTWQELYQLKYKGI-NH₂) were designed by Human Anatomy Laboratory of Qingdao University and produced by Sangon Biotech (Shanghai, China). The three-dimensional structure was predicted by ChemBioDraw Ultra 14.0 (CambridgeSoft, Cambridge, MA, USA). The biotin-modified PA-TIMP-QK and PA-QK peptides were also chemically synthesized by Sangon Biotech and were used for the collagen binding assay and release assay. All samples were purified using standard reverse-phase high-performance liquid chromatography with a purity of 98%. Then, the PA-TIMP-QK and PA-QK peptides were dissolved in double-distilled water at 1.5% (w/v) and mixed with 10 mM Na₂HPO₄ at 0.75% (w/v) final concentration (Webber et al., 2011) to induce hydrogel formation. Finally, the gel was observed and imaged using a Canon camera (EOSM50, Tokyo, Japan).

Scanning electron microscopy

The microstructure of the PA-TIMP-QK nanofiber hydrogel was visualized using a scanning electron microscope (SEM; Hitachi, Tokyo, Japan) operating at an accelerating voltage of 20 kV. PA-TIMP-QK peptides formed hydrogels in double-distilled water and were freeze-dried in a freeze dryer (Christ alpha1-2LDplus, Osterode, Germany) to prepare the imaging samples. The prepared samples were gold-spray coated at 5 nm in the sputter-coating chamber (EM ACE600, Leica, Hesse, Germany) and visualized by SEM.

Circular dichroism

The PA-TIMP-QK structure was tested on a circular dichroism spectrophotometer (J-815, JASCO Corporation, Tokyo, Japan). PA-TIMP-QK was dissolved at a working concentration of 0.075 mM and corrected for absolute peptide content. Measurement values within the 180–260 nm wavelength were collected.

Release assay of QK in response to MMP-2

Collagen hydrogel obtained from Sprague-Dawley male rats (n = 10, 6–8 weeks old, 250–280 g) purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (license No. SCXK (Lu) 2022-0006, Jinan, Shandong Province, China) was used as a scaffold material. It was then transferred into a 96-well plate, with 100 μL per well, and allowed to dry. The wells were washed with phosphate-buffered saline (PBS) five times at pH 8.0. The plates were then incubated with Traut’s reagent (2.5 mg/mL, 100 μL per well; I6256, Sigma, St. Louis, MO, USA) at 25°C for 2 hours, after which they were washed with PBS. Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC; 5 mg, Thermo Scientific, Waltham, MA, USA, A39268) was dissolved in 1 mL of distilled water. The biotin-labeled PA-TIMP-QK peptide and PA-QK peptide were then reacted with the Sulfo-SMCC solution (5 mg/mL) at 25°C for 1 hour, forming biotin-labeled peptide-sulfo-SMCC complexes at different concentration gradients (0, 25, 50, 100, 200, 300 μM). Next, the plates with Traut’s reagent were incubated with the biotin-labeled PA-TIMP-QK peptide-sulfo-SMCC complexes (100 μL/well) and biotin-labeled PA-QK peptide-sulfo-SMCC complexes (100 μL/well) in different concentrations (0, 25, 50, 100, 200, and 300 μM) at 25°C for 1 hour. After washing the plates with PBS three times for 5 minutes each, alkaline phosphatase-labeled avidin (1:1000, 100 μL/well, Sigma-Aldrich, St. Louis, MO, USA, S2890) was added to the wells containing biotin and reacted for 2 hours at 37°C. After three PBS washes for 5 minutes each, the 96-well plate with alkaline phosphatase-labeled avidin was treated with P-nitrophenyl phosphate (2 mg/mL, 100 μL/well, Thermo Scientific, 34045) for 10 minutes. The reaction was then terminated by adding NaOH (0.2 M, 100 μL/well). The optical density at 405 nm of the biotin-labeled peptide-sulfo-SMCC complexes at the bottom of the 96-well plate was measured using a universal microplate spectrophotometer (CMax Plus, Shanghai, China) for quantitative analysis. Each group was set with three parallel holes. Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides. The plate was then shaken at 37°C and 100 r/min. At specific time points (12 and 24 hours), 30 μL of biotin-labeled QK from each well was withdrawn and analyzed using a biotin kit (Abcam, Cambridge, UK, ab185441). The absorbance at 500 nm was measured using a universal microplate spectrophotometer (CMax Plus) for quantitative analysis.

Degradation assays

The degradation of PA-TIMP-QK hydrogels in vitro was evaluated by monitoring the mass loss of the hydrogels in PBS at pH 7.4, according to previous studies (Zhou et al., 2022; Zhang et al., 2023). To perform this assessment, 130 μL of PA-TIMP-QK hydrogel (2 mM) was injected into a 0.5-mL sterile Eppendorf tube and allowed to equilibrate for 10 minutes. This was done with three identical tubes. Then, 100 μL of PBS at pH 7.4 was placed on top of the PA-TIMP-QK hydrogel and incubated at 37°C. The PBS was replaced daily, and the residual mass of the hydrogel eroded by PBS was weighed.

Proliferation assay

Human umbilical vein endothelial cells (HUVECs) were purchased from HyCyte^TM (Suzhou, China, Cat# TCH-C406, RRID: CVCL_B7UP), and were identified by the supplier. In total, 5000 cells were seeded into each well of a 48-well plate and cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin. After the cells had adhered, they were incubated in culture medium containing PA-TIMP-QK and PA-QK at different concentration gradients (0, 0.125, 0.25, 0.5, and 1 μM; 150 μL/well) in a 95% O₂ and 5% CO₂ incubator at 37°C for 48 hours. Then, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (20 μL/well, Solarbio, Beijing, China, M8180) was added to the wells and incubated for 4 hours. Finally, dimethyl sulfoxide (150 μL/well, Solarbio, D8370) was added to the 48-well plates. The absorbance at 492 nm was measured immediately using a light absorption enzyme labeler (CMax Plus).

Oxygen–glucose deprivation and reperfusion model and survival assay

Pheochromocytoma cells (PC12 cells; Kang et al., 2023) were purchased from Pricella (Wuhan, China, Cat# CL-0480, RRID: CVCL_0481) and identified by the supplier. PC12 cells were seeded into a 48-well plate at a density of 3000 cells/well in high-glucose DMEM containing 15% horse serum, 2.5% FBS, and 1% penicillin-streptomycin. The cells were allowed to adhere and grow until the assessment of survival capability. The complete medium (high-glucose DMEM with 15% horse serum and 2.5% FBS) was replaced with low-glucose DMEM (HyClone, Logan, UT, USA) with 1% FBS, or low-glucose DMEM with 1% FBS combined with VEGF165 (2.5 nM, 150 μL/well, Proteintech, Rosemont, IL, USA, Cat# HZ-1038-GMP), PA-QK peptide (1 μM, 150 μL/well), or PA-TIMP-QK peptide (1 μM, 150 μL/well) to establish the oxygen-glucose deprivation and reperfusion (OGD/R) model according to previous studies (Webber et al., 2011; Feng et al., 2020). Immediately thereafter, the PC12 cells were placed in a hypoxic incubator (5% CO₂ and 95% N2, Billups-Rothenberg, San Diego, CA, USA) at 37°C for 6 hours, followed by complete medium in a 95% O₂ and 5% CO₂ incubator at 37°C for 18 hours. At the same time, the normal cell group was cultured in a 37°C incubator as the standard for normal cell growth. The cell survival rate was calculated using the formula: survival rate (%) = number of surviving cells/ number of normal cells × 100. Finally, the PC12 cells surviving after hypoxia were assayed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide method described previously.

Scratch wound migration assay

After cells were plated in a 6-well plate and grown to confluence, they were wounded by scratching a sterile pipette nozzle along the surface of the culture plate. The scraped cells were rinsed and removed with PBS. The remaining adhered HUVECs were incubated with low-glucose culture medium with 2% FBS as control and stimulated with VEGF165 (2.5 nM, 2 mL/well), PA-QK peptide (1 μM, 2 mL/well), or PA-TIMP-QK peptide (1 μM, 2 mL/well) for 24 hours. The cells were then photographed at various time points (0, 6, 12, and 24 hours) using a phase contrast microscope to assess their migration.

Tube formation assay

HUVECs were stimulated with 2% FBS high-glucose DMEM containing VEGF165, PA-QK peptide, or PA-TIMP-QK peptide to assess tubule-like formation capacity, and 2% FBS medium was used as a control. First, a precooled 96-well plate was coated with Matrigel matrix (50 μL/well, Corning, Corning City, NY, USA, 356234) and then placed in a sterile incubator at 37°C for 45 minutes until solidification. The HUVECs in DMEM with 2% FBS (1 × 10⁵/mL, 50 μL/well) were seeded in each Matrigel-coated well and then treated with 2% FBS medium (50 μL/well), VEGF165 (5 nM, 50 μL/well), PA-QK peptide (2 μM, 50 μL/well), or PA-TIMP-QK peptide (2 μM, 50 μL/well). After 12 hours, tube-like structures (Liu et al., 2021) and nodes (Wang et al., 2021) in each group were observed and counted using ImageJ (v.1.52a, National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012).

Cerebral I/R model and drug delivery procedure

Animal operation procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 23–80, revised 2011). The animal experiments were approved by the Ethics Committee Medical College of Qingdao University (approval No. QDU-AEC-2023365) on May 17, 2023.

Sixty-three adult male Sprague-Dawley rats (6–8 weeks old, 250–280 g) were used in this study. Only male rats were used owing to the established neuroprotective effects of estrogen on cerebral ischemic injury and physiological hormone fluctuations in female rats. All rats were procured from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and housed in an environment that provided ample water and food, standard temperature and humidity, and a 12-hour light/dark cycle. Prior to experimental procedures, rats were anesthetized using an intraperitoneal injection of 50 mg/kg 2% pentobarbital sodium (Sigma-Aldrich, Shanghai, China; Cat# P3761).

After being anesthetized, the rats underwent a paramedian incision, and the right common carotid artery, internal carotid artery, and external carotid artery were sequentially isolated. The common carotid artery was permanently ligated, and both the external carotid artery and internal carotid artery were temporarily ligated. A small hole was made in the common carotid artery, and a silicone thread (MSRC37B200PK50, RWD Life Science, Shenzhen, China) was inserted from the hole into the internal carotid artery to block the middle cerebral artery. After 90 minutes of ischemia, the thread was carefully withdrawn to restore blood flow. The rats were then randomly assigned to four groups: PBS (n = 10, control, 25 μL), VEGF165 (n = 10, 40 μM, 25 μL) (Yang et al., 2014), PA-QK (n = 10, 2 mM, 25 μL), and PA-TIMP-QK (n = 10, 2 mM, 25 μL). The drugs were injected at a location 2.5 mm lateral to the sagittal suture and 0.5 mm anterior to the Bregma (Emerich et al., 2010), and a depth of 3 mm below the cortical surface (Additional Figure 1 ^{(577.5KB, tif)} ).

Behavioral tests

Animal behavioral experiments were executed in a double-blind methodology and performed in a quiet and dimly lit environment. Rats were placed on the test site 1 hour before testing to acclimatize to the environment.

Rotarod test

The rotarod test was used to evaluate the recovery of motor function after cerebral ischemia. Before cerebral I/R (CI/R), Sprague-Dawley rats were trained on the rotarod apparatus (LE8505, Panlab, Madrid, Spain) for 5 consecutive days. The rats were tested at a constant speed of 20 r/min for 5 minutes. Only rats that could stay on the rotarod for at least 180 seconds were selected for the experiments. Seven days after CI/R, the rats were placed on the same rotarod equipment at speeds accelerating from 4 to 40 r/min over 5 minutes. The time and the speed at which the rats fell off the rotarod were recorded. Each rat was given three trials with an interval of 20 minutes, and data from all three experiments for each rat were collected and averaged for analysis.

Grip strength test

The grip strength test is a sensitive indicator of rehabilitation from cerebral ischemia (Sunderland et al., 1989). To evaluate the grip strength of rats after CI/R, we used the BIO-GS3 apparatus (Bioseb, Rome, Italy), which consists of a high-precision force sensor connected to a grid and a monitor. The rat’s tail was lifted and its forelimbs were placed on the grid. The rat was then gently pulled off the grid, and the maximum grip strength was recorded (Zhang et al., 2022). Each rat was tested three times with an interval of 20 minutes, and data from all three experiments for each rat were collected and averaged to evaluate motor function.

Open field test

The open field experiment was performed to assess the locomotor activities of rats after CI/R at 7 and 14 days using the Smart v3.0 system software (Panlab). The open field device used in this study was a square box (100 cm × 100 cm × 40 cm). The area was divided into two zones: the central zone and the periphery zone (with a zone ratio of 1:3). Each rat was allowed to move freely within the box for 5 minutes, starting from a corner on the left side of the box. The route distance and time spent in the central and periphery zones within the 5-minute period were recorded. The open field device was cleaned with sterilized alcohol wipes before each test to eliminate any residual odors (Deng et al., 2023).

Histological analysis

On the 14^th day after ischemia, rats were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (150 mg/kg). Then brain tissue was perfused with physiological saline and 4% paraformaldehyde, and then placed in 4% buffered paraformaldehyde for 72 hours. Subsequently, the brain tissue was cut into a thickness of 5 μm for hematoxylin-eosin (H&E) staining, Nissl staining and Immunofluorescence staining. After dewaxing with xylene and washing with various concentrations of ethanol, the slices were stained with hematoxylin dye for 15 minutes using the HE reagent kit (Solarbio, G1120). After 30 seconds of differentiation, the slices were placed in eosin solution for 2 minutes. After rinsing with tap water, the slices were dehydrated, rendered transparent, and sealed, and their morphology was observed under a microscope (Nikon, Ni-U, Tokyo, Japan). Slices were also stained with a Nissl Staining Kit (LEAGENE, Huaibei, Anhui Province, China, DK0022). The slices were submerged in cresyl violet dye solution and incubated at 56°C for 1 hour. They were then differentiated using Nissl differentiation reagent for 2 minutes. The sections were then examined for counts, morphology, and distribution of Nissl bodies.

Immunofluorescence staining

To evaluate the recovery of the brain after cerebral ischemia in rats, immunofluorescence staining was performed. Brain slices were washed with PBS, incubated with 20% FBS, and then reacted with primary antibodies at 4°C overnight. Anti-NeuN antibody (rabbit, 1:100, ABclonal, Wuhan, Hubei, China, Cat# A0951, RRID: AB_2757475) and anti-Tuj1 antibody (rabbit, 1:300, Abcam, Cambridge, UK, Cat# ab18207, RRID: AB_444319) were used to evaluate neuronal survival. Anti-von Willebrand factor (vWF) antibody (rabbit, 1:400, Abcam, Cat# ab6994, RRID: AB_305689) and anti-α-smooth muscle actin (α-SMA) antibody (mouse, 1:100, Abcam, Cat# ab7817, RRID: AB_262054) were used to assess vascular regeneration. Anti-vascular endothelial (VE)-cadherin antibody (rabbit, 1:1000, Abcam, Cat# ab205336, RRID: AB_2891001) and anti-zonula occludens-1 (ZO-1) antibody (rabbit, 1:100, Abcam, Cat# ab221547, RRID: AB_2892660) were used to assess BBB recovery. Anti-CD68 antibody (rabbit, 1:1000, Proteintech, Cat# 28058-1-AP, RRID: AB_2881049) was used to assess inflammation. Anti-CD206 antibody (mouse, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# SC-58986, RRID: AB_2144945) was used to label anti-inflammatory M2-type macrophages. Anti-inducible nitric oxide synthase (iNOS) antibody (mouse, 1:100, Santa, Cat# SC-7271, RRID: AB_627810) was used to label proinflammatory M1-type macrophages. Then, corresponding goat anti-rabbit IgG H&L (Alexa Fluor® 594) (1:500, Abcam, Cat# ab150080, RRID: AB_2650602) and goat anti-mouse IgG H&L (Alexa Fluor® 488) (1:500, Abcam, Cat# ab150113, RRID: AB_2576208) secondary antibodies were added to the slices for 1 hour at 25°C. Finally, 4′,6-diamidino-2-phenylindole (ab104139, Abcam) was used to infiltrate glass slides for nuclear staining. Because CI/R typically induces cell apoptosis (Liu et al., 2023), the TdT-mediated dUTP nick end labeling (TUNEL) assay kit (40307ES20, YEASEN, Shanghai, China) was used to assess cell apoptosis. Six slices were randomly selected from each group, and apoptotic cells were counted using ImageJ.

Western blot assay

The injured brain tissue was lysed using radioimmunoprecipitation assay buffer to extract proteins, which were then assayed using the bicinchoninic acid protein assay kit (WB6501, NCM, Suzhou, China). The proteins were separated via sodium dodecyl-sulfate polyacrylamide gel electrophoresis with different concentrations (7.5%, 10%, 12.5%, and 15%) and transferred onto polyvinylidene fluoride membranes. The nonspecific sites on the polyvinylidene fluoride membranes (IPFL00010, Millipore, new jersey, USA) were blocked with Tris-buffered saline with Tween and 5% skimmed milk powder for 2 hours at 25°C. The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784). The membranes were then incubated with goat anti-rabbit IgG (1:5000, Bioss, Boston, MA, USA, Cat# bs-0295G-HRP, RRID: AB_10923693) at 25°C for 1 hour. The chemiluminescence signal was detected using the Omin-ECLTM Femto Light chemiluminescence Kit (EpiZyme, Shanghai, China, SQ201). Finally, the polyvinylidene fluoride membranes were imaged using an automated chemiluminescence image analysis system (Tanon-5200, Shanghai, China). β-Actin was used as the internal reference and the intensities of bands were analysed using the ImageJ.

Statistical analysis

Although no statistical methods were used to predict sample sizes, our sample sizes were similar to those reported in previous studies (Zuo et al., 2019; Deng et al., 2023). All results were expressed as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0.1 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). To compare data between two groups, the Student’s t-test was used. For comparison between multiple groups, one-way analysis of variance followed by Tukey’s post hoc test was used. A P-value < 0.05 was considered statistically significant.

Results

Preparation and characteristics of PA-TIMP-QK peptide

The supramolecular polymer PA-TIMP-QK (Figure 1A) was designed to self-assemble into nanofibers. These nanofibers incorporate the peptide sequence QK, which mimics VEGF, as well as MMP-2-cleaved peptide TIMP, and PAs made of the tetrapeptide sequence V2A2 (D’Andrea et al., 2005). At concentrations of 4, 2, and 1 mM, PA-TIMP-QK peptides are capable of forming nanofiber gels. According to a previous study (Webber et al., 2011), the most commonly used concentration in vivo is 2 mM. Therefore, 2 mM was selected for subsequent evaluations (Figure 1B). SEM images showed that the PA-TIMP-QK nanofibers had a porous structure, with an average pore diameter of 50 μm. This porous structure facilitated the adhesion and ingrowth of cells (Figure 1C). A previous study showed that the bioactive secondary structure of PA-QK nanofibers resembled an α-helix (D’Andrea et al., 2005), and the high intermolecular cohesion of PAs has been attributed to β-sheets (Álvarez et al., 2021). To confirm the secondary structure of PA-TIMP-QK nanofibers, circular dichroism analysis was conducted, which showed that the peptide exhibited signals typical of both α-helices and β-sheets. This indicated that the addition of the TIMP sequence did not alter the spatial structure of QK (Figure 1D). We also evaluated the stability of PA-TIMP-QK in vitro. As shown in Additional Figure 2 ^{(600.5KB, tif)} , the PA-TIMP-QK hydrogel gradually degraded in simulated physiological conditions (37°C, pH 7.4). After 1 day, the hydrogel retained 96.3% of its initial mass, after 5 days, it retained 83.4%, and after 10 days, it retained 65.1%. The complete degradation time for PA-TIMP-QK was estimated to be approximately 30.6 days, which aligns with previous reports (Zhou et al., 2022; Zhang et al., 2023).

Figure 1.

Preparation and characteristics of self-assembling nanofiber hydrogel PA-TIMP-QK.

(A) (1) The chemical structure of the PA-TIMP-QK created with ChemBioDraw Ultra 14.0. (2) The three-dimensional structure predicted by ChemBioDraw Ultra 14.0. (3) The PA-TIMP-QKs were assembled into cylindrical nanostructures by hydrophobic interaction. The image was created with Adobe Photoshop 2022. (4) The PA-TIMP-QK nanofibers formed a hydrogel after dissolving in water. The image was created with Adobe Photoshop 2022. (B) The PA-TIMP-QK gel in different concentrations. PA-TIMP-QK presented as a semitransparent gel. (C) Scanning electron microscopy of nanofiber gel networks. PA-TIMP-QK nanohydrogel had a porous structure with pore sizes ranging from 50 to 100 μm. Scale bars: 50 μm (left) and 20 μm (right). (D) Circular dichroism analysis of PA-TIMP-QK secondary structure. PA: Peptide amphiphilic molecule; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

Controlled release of QK from MMP2-sensitive PA-TIMP-QK

It has been reported that MMP-2 is gradually upregulated after cerebral ischemia (Yang and Rosenberg, 2015), and MMP-2 expression was detected in the injured brain issue after CI/R. MMP-2 was upregulated at 1 day after the onset of ischemia, and significantly increased in a time-dependent manner (Figure 2A). To detect the release of QK in response to MMP-2 from PA-TIMP-QK peptides, biotin-QK-PA and biotin-QK-TIMP-PA were used. Equal moles of biotin-QK-PA or biotin-QK-TIMP-PA were cross-linked with collagen gels, and the quantification of each peptide was determined by the amount of biotin. The amount of biotin on collagen gels in both the biotin-QK-PA and biotin-QK-TIMP-PA groups increased in a dose-dependent manner, peaking at 200 μM, with no significant difference between PA-QK and PA-TIMP-QK (Figure 2B). Therefore, a dosage of 200 μM was used for each peptide in the subsequent QK release assay. Subsequently, collagen-PA-QK-biotin and collagen-PA-TIMP-QK-biotin were incubated with MMP-2 for 12 or 24 hours. The release of QK-biotin in the supernatant was quantified using a biotin kit. The results indicated that the amount of QK-biotin released by biotin-QK-TIMP-PA was slightly, but not significantly, higher than that of biotin-QK-PA at 12 hours. At 24 hours, the amount of QK released from the biotin-QK-TIMP-PA group was significantly higher than that at 12 hours (P < 0.001) and was also higher than that of biotin-QK-PA at 24 hours (P < 0.01; Figure 2C). These findings suggest that MMP-2 recognized TIMP and released QK from PA-TIMP-QK in a time-dependent manner.

Figure 2.

MMP-2 expression in cerebral ischemia/reperfusion rats and controlled release of QK from self-assembling nanofiber hydrogel PA-TIMP-QK.

(A) Western blot of MMP-2 at 1, 3, 7, and 14 days after injection (n = 3). (B) Collagen binding capacities of PA-TIMP-QK and PA-QK peptides (n = 3). (C) Release capacities of PA-TIMP-QK and PA-QK at 12 and 24 hours (n = 3). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test (A) or Student’s t-test (B, C)). MMP-2: Matrix metalloproteinase-2; OD: optical density; PA: peptide amphiphilic molecule; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

The bioactivity of PA-TIMP-QK in vitro

It was previously reported that VEGF signaling promotes the migration and proliferation of endothelial cells in vitro (Chen et al., 2018). Conventionally, the biological activity of VEGF or its mimic peptide is examined by stimulating the proliferation of HUVECs. Therefore, we evaluated the effects of PA-QK and PA-TIMP-QK on HUVEC proliferation. As the peptide concentration increased, HUVEC proliferation also increased, and the dose-response curves were similar between PA-QK and PA-TIMP-QK, with no significant differences (P > 0.05; Figure 3A). These results were further confirmed by a scratch experiment on HUVECs (Figure 3B). The results indicated that HUVECs in the PA-TIMP-QK group migrated to cover 58.8 ± 7.9% of the scratched area, which was significantly more than the control group ([31.3 ± 6.3]%) and VEGF165 group ([43.8 ± 5.0]%, P < 0.01; Figure 3C).

Figure 3.

Bioactivity evaluation of self-assembling nanofiber hydrogel PA-TIMP-QK in vitro.

(A) MTT assay for detecting the proliferation activity of HUVECs. (B) The scratch assay for detecting the migration ability of HUVECs. Scale bars: 100 μm. (C) Statistical analysis of migration rate in the scratch assay (n = 6). The migration area of the PA-TIMP-QK group was much larger than the control and VEGF165 groups. (D) Assessment of tube formation ability. The ability of the PA-TIMP-QK group to form tubules was stronger than the control group. The PA-QK and VEGF165 groups also had a stronger ability to form tubules than the control group. Scale bar: 50 μm. (E) Statistical analysis of tubes in the tube formation assay (n = 6). (F) Statistical analysis of nodes in the tube formation assay (n = 6). (G) MTT assay by OGD/R for detecting survival activity of PC12 cells. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). HUVECs: Human umbilical vein endothelial cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OGD/R: oxygen-glucose deprivation and reperfusion; PA: peptide amphiphilic molecule; PC12 cells: pheochromocytoma cells; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165.

The in vitro tubule formation assay was used for evaluating the angiogenic effect of growth factors (Figure 3D). The results showed that VEGF165, PA-QK, and PA-TIMP-QK significantly promoted tubule formation compared with the control group (P < 0.01). There was no significant difference between the three experimental groups (P > 0.05; Figure 3E). The PA-TIMP-QK group, the PA-QK group, and the VEGF165 group formed more nodes than the control group (P < 0.01; Figure 3F).

Finally, it has been reported that VEGF has a specific protective effect on hypoxic neurons (Jin et al., 2000; Greenberg and Jin, 2013). Therefore, the OGD/R model was used to assess the neuroprotective effect. Compared with the control group, VEGF165 and PA-TIMP-QK protected PC12 cells against hypoxia (P < 0.05), whereas PA-QK had no significant difference compared with the control group (P > 0.05; Figure 3G). These findings indicated that PA-TIMP-QK exhibited typical angiogenic and neuroprotective effects.

PA-TIMP-QK alleviates brain morphological damage and protects neurons after CI/R

To observe the morphological structure of the injured brain, H&E staining and Nissl staining were performed at 14 days after CI/R. H&E staining showed that compared with the other three groups, the PA-TIMP-QK group had a more intact structure with fewer apoptotic cells and more normal cell morphology, including less nuclear atrophy and membrane regularity (Figure 4A). Furthermore, Nissl staining showed that the PA-TIMP-QK group had a large number of Nissl bodies with regular distribution, whereas the PBS group showed apparent neuronal necrosis, and the VEGF165 and PA-QK groups had moderate neuronal necrosis (Figure 4B and C). These results suggested that PA-TIMP-QK treatment preserved the structural integrity of the injured tissue. To further assess whether PA-TIMP-QK exerts neuroprotective effects after CI/R, the anti-NeuN antibody and anti-Tuj1 antibody were used to label neurons. NeuN immunostaining showed a significantly higher number of surviving neurons in the PA-TIMP-QK group compared with the PBS, VEGF165, and PA-QK groups (P < 0.001; Figure 4D and E). Similar results were found with Tuj1 immunostaining (Figure 4F and G). These results demonstrated that PA-TIMP-QK treatment preserved the morphological and structural integrity of neurons and enhanced neuroprotection compared with the other three groups after cerebral ischemia.

Figure 4.

Histological analysis of cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection.

(A) H&E staining of ischemic brain at 14 days after injection. The PA-TIMP-QK group showed a more complete structure, fewer apoptotic cells, and more normal cell morphology compared with the other three groups. Scale bar: 100 μm. (B) Nissl staining of ischemic brain at 14 days after injection. The number of Nissl bodies in the PA-TIMP-QK group was higher and their distribution was more regular compared with the PBS and VEGF165 groups. The dashed box area shows typical morphological features. Scale bar: 100 μm. (C) Statistical analysis of Nissl bodies (n = 6). (D) Immunofluorescence staining of neurons in ischemic brain (NeuN, red, Alexa Fluor® 594; nuclei, blue). There were more surviving neurons in the PA-TIMP-QK group than in the PBS, VEGF165 and PA-QK groups. Scale bar: 100 μm. (E) Statistical analysis of NeuN⁺ cells (n = 6). (F) Immunofluorescence staining of axons in ischemic brain (Tuj1, red; nuclei, blue). There were more axons in the PA-TIMP-QK group than in the PBS and VEGF165 groups. Scale bar: 100 μm. (G) Statistical analysis of Tuj1⁺ cells (n = 6). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). H&E staining: Hematoxylin and eosin staining; NeuN: neuronal nuclei; PA: peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; Tuj1: beta III tubulin; VEGF165: vascular endothelial growth factor 165.

PA-TIMP-QK promotes angiogenesis after CI/R

QK is one of the most important provascular factors that accelerate the reconstruction of microcirculation in the ischemic hemisphere (Pignataro et al., 2015). Therefore, the vascularization in the injured and surrounding areas was evaluated. The number of vWF-positive capillaries in the PA-TIMP-QK group was significantly higher than that in the PBS and VEGF165 groups at 14 days after treatment (P < 0.01; Figure 5A and B), indicating that PA-TIMP-QK enhanced neovascularization compared with VEGF165 and PBS. Anti-α-SMA staining also showed that PA-TIMP-QK promoted blood vessel regeneration compared with the PBS and VEGF165 groups (P < 0.001; Figure 5C and D). These findings indicated that PA-TIMP-QK promoted angiogenesis and restored blood supply in the injured tissue, which is beneficial for the recovery of brain function.

Figure 5.

Immunofluorescence staining of capillaries and vessels in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection.

(A) Immunofluorescence staining of capillaries in ischemic brain (vWF, red, Alexa Fluor^® 594; nuclei, blue). The number of vWF-labeled capillaries in the PA-TIMP-QK group was greater than the PBS and VEGF165 groups at 14 days after treatment. Scale bar: 100 μm. (B) Statistical analysis of vWF⁺ capillaries (n = 6). (C) Immunofluorescence staining of vessels in ischemic brain (α-SMA, red, Alexa Fluor^® 488; nuclei, blue). PA-TIMP-QK promoted the regeneration of blood vessels compared with the PBS and VEGF165 groups. Scale bar: 100 μm. (D) Statistical analysis of α-SMA⁺ vessels (n = 6). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165; vWF: von Willebrand factor; α-SMA: α-smooth muscle actin.

PA-TIMP-QK alleviates disruption of the BBB after CI/R

In a previous study, it was reported that TIMP alleviated disruption of the BBB (Meng et al., 2021). Therefore, we investigated the effect of PA-TIMP-QK on the BBB, and the integrity of tight junction proteins such as ZO-1, VE-cadherin was a key indicator of disruption of the BBB (Jie et al., 2015; Shi et al., 2023). Immunofluorescence staining was performed and the results showed that as shown in Figure 6A and B, the fluorescence intensity of VE-cadherin was significantly higher in the PA-TIMP-QK group than in the PBS and VEGF165 groups (P < 0.01). Similarly, the level of ZO-1 was significantly upregulated in the PA-TIMP-QK group compared with the PBS, VEGF165, and PA-QK groups (P < 0.05; Figure 6C and D). In addition, enlarged view showed the tight junctions formed with ZO-1 in the PA-TIMP-QK group were more continuous and closer than those in the other three groups (Figure 6C). These results suggested that PA-TIMP-QK had a promising regulatory effect on the function of tight junctions in the BBB.

Figure 6.

Immunofluorescence staining of tight junction protein in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection.

(A) Immunofluorescence staining of endothelial cell-specific intercellular adhesion molecules with VE-cadherin in ischemic brain (red, Alexa Fluor^® 594; nuclei, blue). The fluorescence intensity of VE-cadherin was significantly increased in the PA-TIMP-QK group compared with the PBS and VEGF165 groups. Scale bar: 100 μm. (B) Statistical analysis of mean fluorescence intensity of VE-cadherin (n = 6). (C) Immunofluorescence staining of intercellular junction protein ZO-1 in ischemic brain (red, Alexa Fluor^® 594; nuclei, blue). The TJ formed with ZO-1 in the PA-TIMP-QK group was closer to that in the other three groups. Scale bars: 20 μm (upper) and 5 μm (lower). (D) Statistical analysis of mean fluorescence intensity of ZO-1 (n = 6). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; TJ: tight junction; VE-cadherin: vascular endothelial-cadherin; VEGF165: vascular endothelial growth factor 165; ZO-1: zonula occludens-1.

PA-TIMP-QK attenuates inflammation and cell apoptosis and activates downstream AKT and ERK pathways after CI/R

Neuroinflammation in cerebral ischemia increases neuronal damage and hinders neural tissue regeneration, which aggravates the ischemic injury. Macrophages play a key role in inflammatory response, and they are categorized into M1 type, which is proinflammatory, and M2 type, which is anti-inflammatory. Recently, it was reported that QK-encapsulated silk fibroin hydrogels or nanoliposomes promoted M2 polarization, which facilitated wound repair and nerve reconstruction (Chen et al., 2018; Xu et al., 2023). To assess macrophage polarization, antibodies against CD68 and iNOS were used to identify M1-type macrophages, and antibodies against CD68 and CD206 were used identify M2-type macrophages. The ratio of CD68 and iNOS-colabeled M1 macrophages in the PA-TIMP-QK group was significantly lower than that in the VEGF165 and PBS groups (P < 0.01; Figure 7A and B), which suggested that PA-TIMP-QK reduced neuroinflammation. Conversely, the ratio of CD68 and CD206-colabeled M2 macrophages in the PA-TIMP-QK group was significantly higher than that in PBS and VEGF165 groups (P < 0.01), which suggested an anti-inflammatory effect. However, there was no significant difference between the PA-TIMP-QK and PA-QK groups (P > 0.05; Figure 7C and D). Furthermore, to investigate the anti-apoptotic effect of PA-TIMP-QK in CI/R, apoptotic cells were assessed by the TUNEL assay. The PA-TIMP-QK group had notably fewer TUNEL-positive cells compared with the PBS and VEGF165 groups (P < 0.001; Figure 7E and F). TUNEL-positive cells were also decreased in the VEGF165 and PA-QK groups compared with the PBS group (P < 0.0001; Figure 7E and F). In addition, previous studies have shown that VEGF promotes proliferation and neuroprotection through both the AKT and ERK pathways (Chang et al., 2023; Guo et al., 2023; Yang et al., 2023). Therefore, we validated the downstream pathways of VEGF activation. PA-TIMP-QK increased phosphorylated AKT and ERK expression after CI/R (Figure 7G and H). These findings suggest that PA-TIMP-QK improved the repair of cerebral ischemia, which may involve the activation of AKT and ERK pathways during cerebral ischemic injury.

Figure 7.

Evaluation of cell inflammation and apoptosis and the activation of downstream signaling pathways in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection.

(A) Immunofluorescence staining of M1-type macrophages colabeled with CD68 and iNOS in the ischemic brain (CD68, red, Alexa Fluor^® 594; iNOS, green, Alexa Fluor^® 488; colocalization, yellow; nuclei, blue). CD68 and iNOS-colabeled M1 macrophages ratio in the PA-TIMP-QK group was much lower than that in the VEGF165 and PBS groups. Scale bar: 100 μm. (B) Statistical analysis of iNOS⁺/CD68⁺ macrophages (n = 5). (C) Immunofluorescence staining of M2-type macrophages colabeled with CD68 and CD206 in the ischemic brain (CD68, red, Alexa Fluor^® 594; CD206, green, Alexa Fluor^® 488; colocalization, yellow; nuclei, blue). CD68 and CD206-colabeled M2 macrophages ratio in the PA-TIMP-QK group was much higher than that in the PBS and VEGF165 groups, but there was no statistical difference between the PA-TIMP-QK and PA-QK groups. Scale bar: 100 μm. (D) Statistical analysis of CD206⁺/CD68⁺ macrophages (n = 5). (E) TUNEL staining (TUNEL, green; nuclei, blue). The PA-TIMP-QK group had the fewest TUNEL-positive cells compared with the VEGF165 and PBS groups. Scale bar: 100 μm. (F) Statistical analysis of TUNEL⁺ cells (n = 6). (G) Western blot of p-AKT and AKT at 14 days after injection (n = 4). (H) Western blot of p-ERK and ERK at 14 days after injection (n = 4). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). AKT: The serine/threonine kinase; ERK: extracellular signal-regulated kinase; iNOS: inducible nitric oxide synthase; PA: peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling; VEGF165: vascular endothelial growth factor 165.

PA-TIMP-QK promotes the recovery of motor function after CI/R

To evaluate the effect of PA-TIMP-QK hydrogel on motor function recovery, the rotation test, grip strength test, and open field test were used. The PA-TIMP-QK group had significantly higher rotation speed and longer latency compared with the VEGF165 and PBS groups at 7 days after surgery during the rotation test (P < 0.001; Figure 8A and B). This result indicated that the rats in the PA-TIMP-QK group had improved balance and motor function recovery compared with rats in the PBS and VEGF165 groups at 7 days post-surgery. However, there was no significant difference in the rod rotation test between the PA-TIMP-QK group and the PA-QK group (P > 0.05). In the grip strength experiment at 7 days after injury, the PA-TIMP-QK group had significantly stronger grip strength compared with the PBS and VEGF165 groups (P < 0.01; Figure 8C). There was no significant difference between the VEGF165 and PBS groups (P > 0.05). These results suggested that compared with the VEGF165 and PBS groups, the PA-TIMP-QK group improved motor function after CI/R.

Figure 8.

Animal behavior testing for rotarod test, grip strength test, and open field test at 7 and 14 days in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection.

(A) Statistical analysis of latency during the rotarod test (n = 6). (B) Statistical analysis of speed during the rotarod test (n = 6). (C) Statistical analysis of grip strength test (n = 6). (D) Representative paths of rats in the open field test, tracked by the SMART 3.0 system. The total distances of the PA-TIMP-QK group at 7 days were longer than those in the PBS and VEGF165 groups. The total distances of the PA-TIMP-QK group at 14 days were longer than those in the PBS, VEGF165 and PA-QK groups. There was no significant difference in anxiety assessment after 7 days. On the 14^th day, the PA-TIMP-QK group spent significantly longer time in the central zone of the open field compared with the PBS, PA-QK groups. (E) Statistical analysis of total distance at 7 and 14 days (n = 6). (F) Statistical analysis of time in the center zone at 7 and 14 days (n = 6). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165.

The open field test was performed at 7 and 14 days after injury. There was a significant difference in total distance traveled between the four groups at both 7 and 14 days (Figure 8D). The motor trajectories of rats in the PA-TIMP-QK group were significantly longer than those in the PBS and VEGF165 groups at 7 days (P < 0.01). At 14 days, the total distance traveled by the PA-TIMP-QK group exceeded that of the PBS, VEGF165, and PA-QK groups (all P < 0.05; Figure 8E). These findings suggest that the sustained release effect of PA-TIMP-QK is beneficial for motor function recovery in rats. There was no significant difference in time spent in the central zone of the open field after 7 days (P > 0.05). However, on the 14^th day, the PA-TIMP-QK group spent a significantly longer time in the central zone compared with the PBS and PA-QK groups (P < 0.05; Figure 8F). This result suggested that PA-TIMP-QK reduced anxiety.

Discussion

Cerebral ischemia remains a major cause of mortality and disability worldwide. In clinical settings, recombinant tissue plasminogen activator and surgical vascular interventions are effective treatment options (Sena et al., 2010; Roaldsen et al., 2021). However, the typical time window for these treatments is limited to within 6 hours of ischemia onset. Consequently, many patients with cerebral ischemia miss the optimal window for treatment, leading to debilitating conditions such as muscle weakness, limb dysfunction, ataxia, or language disorders. This creates a great demand for rehabilitation services, which poses a significant social and economic burden.

Currently, novel regenerative therapies, such as stem cells and angiogenic growth factors, hold promise as therapeutic options for treating cerebral ischemia. VEGF, in particular, is the most commonly used angiogenic growth factor in ischemic disease therapy. However, compared with traditional VEGF application, angiogenic peptide QK, derived from the helix sequence 17–25 of VEGF, has the ability to bind and activate VEGF receptors, thereby enhancing angiogenesis (D’Andrea et al., 2005). Previous research has also shown that QK has a neuroprotective effect in neurological diseases (Verheyen et al., 2013; Pignataro et al., 2015) and can trigger the transformation of proinflammatory M1 macrophages into anti-inflammatory M2 macrophages (Chen et al., 2023b). Additionally, the QK peptide exhibits relatively high thermal stability (Diana et al., 2008; Finetti et al., 2012). Therefore, self-assembly peptide nanofiber scaffolds incorporating VEGF mimic peptide QK present a suitable biomaterial with optimal biocompatibility, degradability, and safety. These scaffolds have the potential to not only prevent rapid diffusion but also ensure the effective concentration of angiogenic peptides in the ischemic region. In the present study, PA-TIMP-QK nanofiber hydrogels were developed, incorporating both angiogenic peptide QK and MMP-2-cleaved peptide TIMP.

A recent study compared the effect of eight different PA sequences on the formation of nanofiber hydrogels, including V2A2, A2G2, AVG2, VAG2, G4, VA3, AVA2, and A4 (Álvarez et al., 2021). The results showed that V2A2 formed the least mobile supermolecule module, resulting in the most stable nanofiber structure. This nanofiber structure was completely degraded within 12 weeks in a spinal cord injury animal model in vivo. Therefore, in the present study, the nanocarrier in the supramolecular polymer PA-TIMP-QK was composed of PAs containing the tetrapeptide sequence V2A2 and a C16 alkyl chain. PA-TIMP-QK formed hydrogels at different concentrations, indicating that the PA-TIMP-QK gel induced by V2A2 and a C16 alkyl chain was stable in nature. Furthermore, SEM showed that the nanohydrogel was interlaced by nanofibers, which formed a porous structure that promotes cell adhesion and ingrowth. Circular dichroism analysis indicated that the nanohydrogels had a rich α-helix structure, which serves as the foundation for imitating VEGF. These microstructural findings demonstrated that the synthesized PA-TIMP-QK did not alter the spatial structure of QK, ensuring its VEGF mimic peptide functionality. Additionally, the biodegradation assay in vitro showed that the PA-TIMP-QK hydrogel was relatively stable in a simulated physiological environment, satisfying the temporal requirement of MMP-2 for responsive release.

In recent years, there has been a focus on the introduction of stimuli responsiveness into self-assembly peptide hydrogels, which would enable the programmable release of bioactive peptides in targeted tissues and organs. After cerebral ischemia, MMP-2 is upregulated in response to hypoxic injury and plays an important role in both physiological and pathological conditions. It maintains the integrity of the basement membranes to prevent overgrowth of the extracellular matrix in a steady state. However, MMP-2 is typically overexpressed in diseased or inflammatory tissues (Wen et al., 2011; Yang and Rosenberg, 2015; Li et al., 2016). Previous studies have shown that MMP-2 is activated and expressed extensively in different stages of CI/R, leading to disruption of the basal lamina and tight junction proteins (Yong et al., 2001; Yang and Rosenberg, 2015; Mi et al., 2016). It has been observed that MMP-2 is activated several hours after the onset of CI/R, and this process leads to the initial opening of the BBB, which is reversible (Yang and Rosenberg, 2015). After 24–48 hours of cerebral ischemia, activated MMP-2 further exacerbates BBB injury, and the BBB disruption may continue for several days, leading to hemorrhage and vasogenic edema (Rosenberg, 2002). Additionally, excessive levels of MMP-2 are destructive to nerve repair (Kumar and Patnaik, 2018), and promote the accumulation of inflammatory cells in the brain after cerebral ischemia (Victoria et al., 2020). In a rat reperfusion model, TIMP was found to interact with MMP-2, and thus indirectly reduce BBB disruption, promote neuroprotection, and inhibit neuroinflammation in reperfusion injury. Therefore, TIMP is an attractive target for the construction of self-assembly angiogenic peptide nanofiber hydrogels that can control the release of QK after cerebral ischemia. This approach not only promotes angiogenesis but also minimizes BBB damage caused by MMP-2. The release of QK in response to MMP-2 from PA-TIMP-QK was evaluated in vitro, and the results showed that PA-TIMP-QK stably released the angiogenic peptide QK in the presence of MMP-2.

In the present study, PA-TIMP-QK demonstrated similar biological activity to VEGF, promoting proliferation, migration, and tubular formation of HUVECs in vitro. Additionally, VEGF has been reported to protect PC12 cells against hypoxia (Mo et al., 2016), so the neuroprotective effect of PA-TIMP-QK was also evaluated in an OGD/R model. The results showed that PA-TIMP-QK significantly protected PC12 cells against damage in hypoxic environments. In vivo experiments with PA-TIMP-QK were also conducted, and the results were consistent with the in vitro experiments. There were significant differences between the PA-TIMP-QK and VEGF165 groups in measures of vessel regeneration, neuroprotection, and BBB repair. These differences may be related to the different release profiles of growth-stimulating factors or peptides in the injured area, with PA-TIMP-QK providing stable on-demand release compared with the burst release of VEGF165. It has been reported that the use of high-dose VEGF in early CI/R increases vascular leakage and causes brain edema (Wu et al., 2018). This issue has limited the clinical use of VEGF. In the present study, the findings suggest that the controllable release of QK and the blocking effect of TIMP on MMP-2 in PA-TIMP-QK alleviated BBB destruction to a certain extent, reducing the unwanted side effects of VEGF in the treatment of cerebral ischemia. Recently, an injectable hydrogel encapsulating QK-loaded nanoliposomes was shown to trigger macrophage polarization from M1 to M2, providing an optimized microenvironment for nerve regeneration (Xu et al., 2023). Our study also demonstrated that PA-TIMP-QK promoted macrophage polarization to the M2 phenotype, further exerting neuroprotective effects. Additionally, we explored the downstream pathway of the PA-TIMP-QK hydrogel. AKT and ERK signaling pathways were investigated by western blotting, which indicated that AKT and ERK signaling may be involved in PA-TIMP-QK activation. Finally, investigation of the effect of PA-TIMP-QK on animal behavior showed that PA-TIMP-QK administration significantly facilitated the recovery of motor function in rats after CI/R.

The regenerative effect of the PA-TIMP-QK hydrogel was evaluated at 14 days after hydrogel implantation based on previous studies (Zhou et al., 2022; Zhang et al., 2023). This is a relatively short observation period, which is a limitation of the current study. Additionally, although the regenerative effect of PA-TIMP-QK was superior to that of the PBS, VEGF165, and PA-QK groups, the difference between PA-TIMP-QK and a sham group was not compared, which is another limitation of our experiments. In future work, long-term observation will be conducted to better determine the recovery efficiency of the PA-TIMP-QK hydrogel, and the difference in functional recovery between PA-TIMP-QK and a sham group will also be emphasized. Furthermore, the combination of multiple functional motifs for tissue regeneration was also investigated. For instance, a nanofiber hydrogel dual-functionalized with laminin-derived motif IKVAV (IKV) and a BDNF-mimetic peptide epitope RGIDKRHWNSQ (RGI) was demonstrated to enhance axonal remodeling with remyelination and motor function recovery (Yang et al., 2020). The laminin signal IKV and a fibroblast growth factor-2 mimetic peptide YRSRKYSSWYVALK connected with different PA molecules were shown to promote vascular growth and axonal regeneration (Álvarez et al., 2021). Finally, ongoing research should combine the present self-assembling MMP-2-responsive angiogenic peptides with other biofunctional peptides, such as IKV, or with stem cells. This approach may enhance angiogenesis and neuron regeneration more efficiently in the repair of cerebral ischemia.

In conclusion, the self-assembling nanofiber hydrogel PA-TIMP-QK was constructed with angiogenic peptide QK and MMP-2-cleaved TIMP peptides. These functional hydrogels released QK in response to upregulated MMP-2 and exhibited similar biological properties to PA-QK. The findings showed that PA-TIMP-QK hydrogels promoted angiogenesis, neuroprotection, and BBB integrity in the CI/R rat model. Our study suggests that these specific self-assembling nanofiber hydrogels have the potential to control QK release in regenerative environments in response to upregulated MMP-2, and therefore may provide a therapeutic approach for cerebral ischemia.

Additional files:

Additional Figure 1 ^{(577.5KB, tif)} : Timeline of the in vivo experiment.

Additional Figure 1

Timeline of the in vivo experiment.

tMCAO: Transient middle cerebral artery occlusion.

Additional Figure 2 ^{(600.5KB, tif)} : Evaluation of PA-TIMP-QK stability in vitro.

Additional Figure 2

Evaluation of self-assembling nanofiber hydrogel PA-TIMP-QK stability in vitro.

Degradation assays of PA-TIMP-QK when PA-TIMP-QK hydrogel in PBS with a pH value of 7.4 at 37C for different time points. PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

Funding Statement

Funding: This work was supported by the Natural Science Foundation of Shandong Province, No. ZR2023MC168; the National Natural Science Foundation of China, No. 31670989; and the Key R&D Program of Shandong Province, No. 2019GSF107037 (all to CS).