Transplantation of bioengineered Reelin‐loaded PLGA/PEG micelles can accelerate neural tissue regeneration in photothrombotic stroke model of mouse

Abstract Ischemic stroke is characterized by extensive neuronal loss, glial scar formation, neural tissue degeneration that leading to profound changes in the extracellular matrix, neuronal circuitry, and long‐lasting functional disabilities. Although transplanted neural stem cells (NSCs) can recover some of the functional deficit after stroke, retrieval is not complete and repair of lost tissue is negligible. Therefore, the current challenge is to use the combination of NSCs with suitably enriched biomaterials to retain these cells within the infarct cavity and accelerate the formation of a de novo tissue. This study aimed to test the regenerative potential of polylactic‐co‐glycolic acid‐polyethylene glycol (PLGA‐PEG) micelle biomaterial enriched with Reelin and embryonic NSCs on photothrombotic stroke model of mice to gain appropriate methods in tissue engineering. For this purpose, two sets of experiments, either in vitro or in vivo models, were performed. In vitro analyses exhibited PLGA‐PEG plus Reelin‐induced proliferation rate (Ki‐67+ NSCs) and neurite outgrowth (axonization and dendritization) compared to PLGA‐PEG + NSCs and Reelin + NSCs groups (p < 0.05). Besides, neural differentiation (Map‐2+ cells) was high in NSCs cultured in the presence of Reelin‐loaded PLGA‐PEG micelles (p < 0.05). Double immunofluorescence staining showed that Reelin‐loaded PLGA‐PEG micelles increased the number of migrating neural progenitor cells (DCX+ cells) and mature neurons (NeuN+ cells) around the lesion site compared to the groups received PLGA‐PEG and Reelin alone after 1 month (p < 0.05). Immunohistochemistry results showed that the PLGA/PEG plus Reelin significantly decreased the astrocytic gliosis and increased local angiogenesis (vWF‐positive cells) relative to the other groups. These changes led to the reduction of cavity size in the Reelin‐loaded PLGA‐PEG+NSCs group. Neurobehavioral tests indicated Reelin‐loaded PLGA‐PEG+NSCs promoted neurological outcome and functional recovery (p < 0.05). These results indicated that Reelin‐loaded PLGA‐PEG is capable of promoting NSCs dynamic growth, neuronal differentiation, and local angiogenesis following ischemic injury via providing a desirable microenvironment. These features can lead to neural tissue regeneration and functional recovery.


Abstract
Ischemic stroke is characterized by extensive neuronal loss, glial scar formation, neural tissue degeneration that leading to profound changes in the extracellular matrix, neuronal circuitry, and long-lasting functional disabilities. Although transplanted neural stem cells (NSCs) can recover some of the functional deficit after stroke, retrieval is not complete and repair of lost tissue is negligible. Therefore, the current challenge is to use the combination of NSCs with suitably enriched biomaterials to retain these cells within the infarct cavity and accelerate the formation of a de novo tissue. This study aimed to test the regenerative potential of polylactic-co-glycolic acid- angiogenesis following ischemic injury via providing a desirable microenvironment.
These features can lead to neural tissue regeneration and functional recovery.

K E Y W O R D S
functional regeneration, neural stem cells, photothrombotic stroke, PLGA-PEG, Reelin

| INTRODUCTION
Despite numerous pharmacological and neuro-rehabilitation approaches used to recuperate the neurological disorders, stroke is still the leading neurological disability with a high mortality rate. 1 Statistics have shown near 3.4 million people will suffer a stroke by 2030 in the United States. 2 The lack of efficient medical treatment to restore the function of injured areas in the brain after the stroke imposes considerable economic and health system problems; hence, emerges an urgent need for a novel therapeutic approaches. 3 Regeneration of injured brain tissue via transplanting neural stem cells (NSCs) seems to be effective in patients with ischemic disease. 4 NSCs possess auto self-renewability and target orientation toward main neuroectodermal lineages like oligodendrocytes, neurons, and astrocytes. In the embryonic stage, the existence of NSCs exists in mammalian neural tube which is known as ancestors in the central nervous system (CNS). 5 By contrast, these cells are restricted to specific regions in adults located in the subventricular zone (SVZ) and the subgranular zone of the dentate gyrus (DG). It was suggested that NSCs can be distinguished from other cell lineages based on specific markers such as Nestin and glial fibrillary acidic protein (GFAP). 6,7 The promotion of magnificent neuron death in the ischemic area and consequent pathological remodeling lead to the formation of large blank spaces, which necessitate the application of bulk volume transplants and grafts using stem cells. 3 This area is juxtaposed to periinfarct tissue known as penumbra where both neural plasticity and angiogenesis are prominent. 8 Although many in vitro protocols are effective in the orientation of NSCs toward targeted lineages, the control of NSCs phenotype acquisition is not possible in in vivo conditions. 3 On this basis, several cell delivery approaches have been limited because of the low viability of transplanted stem cells, 9 and the transient activity of growth factors. 10,11 The initiation of inflammatory response and lack of adhesive support, to some extent, have forced researchers to find alternative therapeutic approaches. 12,13 Accordingly, considering the critical role of distinct factors can help the scientists in an efficient NSC-based modality. It is noteworthy to mention that NSCs alone are not potent enough to generate nascent functional neurons and undergo atretic changes soon after transplantation due to the lack of supporting extracellular matrix (ECM). It has been shown that NSCs cannot migrate to the depth of the stroke site due to a lack of signaling ECM. By providing sufficient structural support and substrates, it is applicable to increase NSCs recruitment to the stroke-damaged area. 14,15 To this end, recent findings have developed scaffolds and hydrogels for the transplantation of NSCs with the capability to promote simultaneously the interaction between transplanted NSCs and surrounding matrix. 16 Among different ECM components, Reelin can regulate the migration of neurons during the developmental period of the brain. This protein is touted as a key player in the formation of the cerebral cortex and lamination of the cerebellum, 17 and maintenance of adult synaptogenesis. 18 Likewise, it was suggested that Reelin can reduce pathologies related to cerebral ischemia-reperfusion injury.
In support of this claim, the suppression of Reelin increases vulnerability after cerebral ischemia in mouse models. 19 Moreover, Reelin organizes vascular morphology and orientation and induces vascular sprouting and vascular wall integrity. 20,21 Therefore, the induction of Reelin synthesis in acute CNS injuries can help us to minimize clinically the progression of pathologies after ischemic changes. 22 Although natural ECM components can regulate cell bioactivity the combination of these factors with synthesized scaffolds yielded an appropriate regenerative outcome via supporting structural properties. 23 One key artificial FDA-approved biomaterial is polylactic-coglycolic acid (PLGA) that can be injected directly into the injured sites. Due to certain and unique physicochemical features, PLGA can structurally support stem cells during delivery. 24 Given the role of PLGA nanoparticles as a potentially promising carrier for the treatment of brain disorders, unmodified PLGA possesses numerous weaknesses, such as negative charge, hydrophobic structure, and the existence of free glycolic subunits. Accordingly, the PLGA nanoparticle fails to interact appropriately with the diverse cells due to hydrophobicity. [25][26][27] Along with these comments, self-assembled micellar nanoparticles with hydrophilic shells and hydrophobic cores can increase the solubility of hydrophobic biomolecules. Besides, the existence of nanoparticle corona enclosed by hydrophilic units maintains the structure of target molecules in different microenvironments. 28,29 Hence, surface modification and additional engineering of PLGA nanoparticles are highly demanded. One important avenue for PLGA functionalization is the attachment of polyethylene glycol (PEG) polymer chains known also as PEGylation. 25 Therefore, the combining PLGA copolymer with non-cytotoxic and hydrophilic PEG substrate offers the desired microenvironment for the generation of perineuronal net. 30 Here, we fabricated PLGA-PEG micelles loaded with Reelin for triggering neurogenesis either in in vivo and in vitro conditions. Besides, we explored the regeneration of ischemic brain injury and functional recovery in the photothrombotic stroke model of the mouse.

| Preparation of PLGA-PEG polymer
PEG (1 g) with a molecular weight of 4000 was kept at 150 C for 3 ho under a high vacuum nitrogen atmosphere. Concurrently, DL-lactide (0.85 g) and glycolide (0.15 g) with a mole ratio of 85:15 were mixed with PEG and melted under argon flow. Following the addition of stannous 2-ethyl hexanoate, the mixture was maintained at 155 C for 8 h to initiate the reaction. This reaction was performed under a relative vacuum. After the completion of the reaction, we declined the temperature and adjusted to room temperature. The synthesized polymer was dissolved in dichloromethane solution followed by the precipitation in precold diethyl ether solution. The procedure was followed by polymer dehydration at a vacuum oven at RT. Then, the pH of the PVA-Reelin solution pH was set to 7.4. Eventually, the Reelin-loaded micelles were collected using centrifugal filter tubes as above mentioned.

| Hydrogen-1 nuclear magnetic resonance
Hydrogen-1 nuclear magnetic resonance ( 1 H-NMR) analysis was applied to analyze the composition of micelles. For this purpose, the obtained spectra were collected from CDCI3 Brucker AM 300.13 MHz spectrometers (Germany).

| Bradford assay
The releasing content of Reelin-loaded PLGA-PEG micelles was assessed using Bradford assay. To this end, the micelles were incubated for 7 days. A 25 μg Reelin was incubated with 2500 μg PLGA-PEG micelles in 1 ml of fetal bovine serum (FBS)-free culture medium for 7 days. After the completion of incubation time, the micelles were collected and protein content was assessed using Bradford solution.
Data were read at a wavelength of 595 nm using a microplate reader and compared to the PLGA-PEG micelles without Reelin treatment.

| Experimental design
In this study, we performed both in vitro and in vivo experiments to assess neurogenesis, regenerative potential, and functional efficiency of Reelin-loaded PLGA-PEG micelles.

NSCs isolation and expansion
To this end, we isolated NSCs from nine 14-day-old murine embryos in three technical replicates. Pregnant mice were euthanized by cervical dislocation. Next, embryos ganglionic eminences were dissected using fine scissors and forceps under the operating microscope. ODs were read at 570 nm using a microplate reader. The viability was expressed as % of control. 36,37 Measuring proliferation and differentiation capacity of NSCs We investigated the proliferation rate and differentiation capacity of NSCs after being cultured on Reelin-loaded PLGA-PEG micelles using immunofluorescence (IF) assays. In brief, NSCs were allocated into Control; PLGA/PEG; Reelin; and PLGA/PEG + Reelin groups.
In order to evaluate proliferation rate and differentiation capacity, NSCs were cultured at a density of 50,000/ml per well of 24-well plates pre-coated with laminin. On day 7, the proliferation rate was monitored in terms of nuclear Ki-67 protein levels. In line with this assay, we studied differentiation capacity of cultured NSCs on different substrates by monitoring Map-2 levels after 14 days. The proliferation medium consists of basic medium-plus bFGF, and EGF while the differentiation medium was supplemented with 5% FBS in the absence of bFGF, and EGF. After completion of experimental periods, cells were fixed by 4% (w/v) pre-cold paraformaldehyde (PFA) for 20 min and washed with PBS. To reduce non-specific binding, NSCs were blocked with 1% BSA for 20 min followed by permeabilization with 0.1% Triton X100. For proliferation and differentiation assays, cells were incubated overnight with rabbit anti-Ki67 (1:500, Abcam) and anti-MAP-2 (1:500; Sigma-Aldrich) antibodies, respectively. After PBS washes, cells were incubated with Alexa Fluor ® 488-conjugated secondary antibody for 1 h at room temperature. A 1 μg/mL DAPI was used to stain nuclei. In this study, the number of Ki-67 + and MAP-2 + cells was counted per 100 DAPI + cells. 38,39 Neurite formation analysis Neurite formation was also studied in NSCs cultured on three different surfaces including PLGA/PEG; Reeling and PLGA/PEG + Reeling.
Parameters such as the number of primary neurites, branch points, and length of neurite per cell were measured using SEM and brightfield imaging. The cells cultured on plastic surfaces were considered as a control group. The length of neurites was calculated by using ImageJ software version 1.4 (NIH).

| Induction of photothrombotic stroke model
For in vivo analyses, a total 50 male Balb/C mice were assigned randomly into five groups (each in 12) as follows; stroke + PBS; stroke + NSCs; stroke + PLGA/PEG + NSCs; stroke + Reelin + NSCs; Stroke + PLGA/PEG + Reelin + NSCs. Mice were anesthetized using 5% isoflurane gas with a rate of 1.5 L/min and carefully placed onto a stereotactic apparatus (Stoelting, USA). was injected per g/body weight and allowed to distribute into the blood circulation. In this study, the light green laser was irradiated on the skull surface for 10 min followed by wound suturing. Thereafter, mice were maintained at temperature-controlled conditions.
To examine the efficiency of our protocol in the induction of ischemic stroke, we selected mice randomly from different groups immediately after laser irradiation. Mice were euthanized by an overdose of ketamine and xylazine. The brains were removed, coronal sections prepared with identical intervals, and stained with 2, 3, 5-triphenyl-tetrazolium chloride (TTC) solution. We monitored general appearance and morphological features.

| Cell labeling
To track transplanted NSCs after injection into the brain tissue, we labeled cells using Cell Tracker™ CM-DiI Dye. To this end, the supernatant medium was removed and NSCs were incubated with 20 μM dye solution for 20 min at 37 C. After three PBS washes, cells were ready for injection.

| Cell transplantation
Seven days after the laser irradiation, mice from different groups were again undergone deep anesthesia and were placed onto a stereotactic apparatus. A small hole of diameter 2 mm was induced at the same location that was previously irradiated.

| Immunohistochemistry analysis used for evaluation of astrocytic gliosis and local angiogenesis
On day 28, the prepared slides (as above-mentioned) were used for Immunohistochemistry (IHC) analysis of GFAP positive astrocytes. In brief, the endogenous activity was inhibited by using 3% H 2 O 2 for 20 min after antigen retrieval. The slides were exposed to anti-GFAP and-von Willebrand factor (vWF) antibodies (Dako) for 1 h at room tem-

| Behavioral performance assessment
Modified neurological severity score (mNSS) One day after stroke induction and 7, 14, 21, and 28 days after transplantation, the mNSS test was performed to assess behavioral performance.
All values correlated with motor and sensory functions such as general movements, balance, and reflex were measured on a scale system consisted of minimal normal (0) and maximal deficit (18) scores. Score 0 stands for a fact that neurological deficit does not exist, whereas score 18 shows the maximum injury of CNS following stroke. Scores between these values were classified as follows; 0-6: mild neurological deficit; 7-12: moderate neurological impairment, and 13-18: the severe neurological deficit. The functionality of the motor system was evaluated by suspending the mice from the tail to monitor head movements and forelimbs flexion in the vertical axis. Mice were placed on a flat surface for assessing gait. 43,44 In addition to motor system function, the function of the sensory system was studied by evaluating both visual, tactile, and proprioception senses. 43,45,46 According to previously published data, the status of balance was assessed as a slim wooden with 100 cm away from the ground. Different reflexes like pinna, corneal, and startle reflexes were scored. We also investigated myodystonia, myoclonus, and seizure in mice from all groups.

| Statistical analysis
Data were represented as mean ± SD. The statistical differences between groups were analyzed using One-way ANOVA with post hoc Tukey (GraphPad Prism Version 8) otherwise mentioned. p < 0.05 was considered statistically significant.

| H-NMR spectrum
H-NMR spectra exhibited peaks at 5.3 and 4.8 ppm correlated with CH and CH 2 groups for lactide and the glycolide, respectively ( Figure 1a). According to our data, the peak at 1.47 ppm is associated with lactide CH 3 while the peak of 3.5 ppm confirms the existence of methylene groups related to PEG (Figure 1a). The data demonstrate successful synthesis of PEG and PEG-PLGA conjugation.

| Reelin-loaded PLGA-PEG micelles can release Reelin in aqueous phase
Here, we measured the releasing capacity of Reelin after blending with PLGA-PEG micelles using the Bradford assay (Figure 2a,b).
We blended 25 μg Reelin peptide with 2500 μg PLGA-PEG micelles and releasing capacity was measured after seven-day incubation inside culture medium. Here, we found that the released content of total protein reached 20 ± 3 μg compared to the Reelin-free PLGA-PEG micelles. This protocol yielded an 80% loading rate which seems suitable for Reelin peptide delivery to the target cells and tissues.

| NSCs morphology and characterization
In this study, we isolated NSCs from ganglionic eminences of E14 brains (Figure 2a). Bright-field imaging revealed that small-sized colonies were generated on Day 5 after plating. These structures were enlarged to perform mature neurospheres in which Days F I G U R E 3 Isolation of mouse embryonic NSCs from ganglionic eminence (a). Bright-field imaging revealed the formation of neurospheres during the first 7 days. Data showed that isolated cells can form distinct neurospheres 6 days after plating in vitro (b). Flow cytometry analysis confirmed that about 85% of cultured NSCs were positive for nestin, indicating the efficiency of our protocol in the isolation of mouse NSCs (c). MTT assay (d). NSC viability was significantly increased after exposure to 100, 250, and 500 ng/mL PLGA-PEG micelles for 21 days compared to the nontreated control NSCs. MTT assay showed that Reelin can increase NSC survival rate in a dose-dependent manner after 21 days. One-way ANOVA and Tukey post hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. NSC, neural stem cells; PLGA-PEG, polylactic-coglycolic acid-polyethylene glycol 3.7 | Reelin-loaded PLGA-PEG micelles increased proliferation and differentiation of mouse NSCs Both proliferation and differentiation were studied in NSCs treated with the combination of Reelin and PLGA-PEG micelles on days 7 and 14, respectively (Figure 4a,b). Data showed that 7-day incubation of mouse NSCs with Reelin (200 nM), PLGA-PEG micelles, and Reelin plus PLGA-PEG micelles increased proliferation rate (Ki-67 positive NSCs) as compared to the non-treated control NSCs (Figure 4a).
Based on our data, we found that Reelin alone can increase the proliferation of NSCs compared to the PLGA-PEG micelles and control groups (p < 0.05). Noteworthy, the combination of Reelin and PLGA-PEG micelles yielded maximum effect to enter mouse NSCs proliferation in comparison with other groups (Figure 4a), showing the synergistic effect of Reelin and PLGA-PEG micelles in dynamic growths.
We also found that the numbers of Map-2 + cells were significantly increased in PLGA-PEG, Reelin, and Reelin-loaded PLGA-PEG groups after 14 days compared to the NSCs (p < 0.05; Figure 4b). Compared to the PLGA-PEG and control groups, Reelin increased the number of Map-2 + cells (p < 0.05). As expected, PLGA-PEG micelles plus Reelin led to prominent differentiation of mouse NSCs toward Map-2 + neurons ( Figure 4b).
These data showed that the loading of Reelin on PLGA-PEG micelle surface increased the proliferation and differentiation of mouse NSCs.

| Reelin-loaded PLGA-PEG micelles improved neurite outgrowth
The number of primary neurites, branch points, and neurites length were analyzed per cell 14 days after plating (Figure 5a

| Reelin-loaded PLGA-PEG micelles reduced cavity size
In this study, we used focal photothrombotic stroke model of the mouse (Figure 6a,b). Gross appearance exhibited focal ischemia immediately after laser irradiation at the right hemispheres ( Figure 6C). To confirm ischemic changes, we performed TTC staining to monitor the existence of lesion sites in brain sections after 7 days (Figure 6d).
The ischemic changes were indicated with a pale appearance while the intact sites were red. These data showed degenerative changes in sites exposed to green laser irradiation. Besides, cavity size was evaluated by stereological examination at 7, and 28 days in different groups (Figure 6e,f). Noteworthy, we did not find statistically significant differences in cavity size in all groups 7 days after therapeutic intervention (p > 0.05). On day 28, the transplantation of NSCs alone did not alter cavity size compared to the PBS-treated stroke mice (p > 0.05).
Based on our data, the application of Reelin alone or in combination with PLGA-PEG micelles can reduce cavity size in comparison with the PBS + Stroke group (p < 0.05). Interestingly, we found that the reduction of cavity size was at the maximum levels in stroke mice that received Reelin-loaded PLGA-PEG micelles (p < 0.05; Figure 6e,f).
These data showed that NSCs cannot alter the cavity size that occurred after the ischemic change. Simultaneous application of NSCs with PLGA-PEG micelles or Reelin seems an appropriate strategy to F I G U R E 5 Measuring neurite growth in embryonic NSCs after 14 days culture on PLGA-PEG micelles, Reelin, and Reelin-loaded PLGA-PEG micelles (a-c). SEM imaging revealed robust neurite growth in NSCs plated on Reelin-loaded PLGA-PEG micelles compared to other groups (a). Consistently, bright-field imaging showed that the mean number of primary neurites, branch points, and neurite length was significantly increased per neuron after the combination of Reelin-loaded PLGA-PEG micelles compared to the control NSCs (b,c). Reelin had more effects to induce neurite formation when compared to the PLGA-PEG and control groups. Yellow arrows and blue arrowheads: primary neurites. One-way ANOVA and Tukey post hoc analysis. *p < 0.05; **p < 0.01; and ***p < 0.001. NSC, neural stem cells; PLGA-PEG, polylactic-co-glycolic acid-polyethylene glycol F I G U R E 6 Induction of ischemic changes in mouse brain using PT (571 nm-cold light green laser illumination) under stereotactic surgery (a,b). Macroscopic findings revealed prominent local hemorrhagia (ecchymosis) on the right hemisphere immediately after the completion of PT stroke induction (c). Longitudinal TTC staining on day 7 post-PT stroke induction revealed a distinct ischemic area. Measuring cavity size using crystal violet staining in brain sections on days 7 and 28 after PT and after injection of PLGA-PEG, Reelin, and Reelin-loaded PLGA-PEG micelles (e-f). On Day 7, no statistically significant differences were found in lesion sites between groups while the injection of PLGA-PEG, Reelin, and Reelinloaded PLGA-PEG micelles decreased cavity size compared to the control stroke group. The injection of NSCs did not alter cavity size compared to the Stroke + PBS group. One-Way ANOVA and Tukey post hoc analysis. *p < 0.05; and ****p < 0.0001. NSC, neural stem cells; PLGA-PEG, polylactic-co-glycolic acid-polyethylene glycol; PT, photothrombotic; TTC, 2, 3, 5-triphenyl-tetrazolium chloride diminish the extent of the lesion. The concurrent use of Reelin and PLGA-PEG micelles heightens the therapeutic effect of NSCs.

| Reelin-loaded PLGA-PEG micelles promoted NSCs differentiation in vivo
To assess the effect of Reelin-loaded PLGA-PEG, micelles on NSCs migration and differentiation, we performed IF analysis (Figure 7a-d).
On Day 14, three mice were selected for the evaluation of DCX positive cells, migrating neuroblast marker, at the site of injection (Figure 7a-b). Data showed that the number of Dil-labeled NSCs expressed DCX was increased in all groups compared to the Stroke + PBS group. Transplantation of NSCs in PLGA-PEG micelles loaded with Reelin increased cell neuroblast-like phenotype acquisition compared to all groups at the site of injection (p < 0.05). Compared to the Stroke + PLGA-PEG + NSCs, we found significant increase in DCX + cells in group was found in the Stroke + Reelin + NSCs group (p < 0.05; Figure 7a F I G U R E 7 Measuring protein levels of DCX and NeuN in lesion site after transplantation of PLGA-PEG, Reelin, and Reelin-loaded PLGA-PEG micelles in brain tissue (a-d). According to data, the Reelin-loaded PLGA-PEG micelles can efficiently increase the percent of DCX and NeuN positive cells in the sites of injection compared to the Reelin, PLGA-PEG, and other groups. One-way ANOVA and Tukey post hoc analysis. *p < 0.05; **p < 0.01; and ***p < 0.001. PLGA-PEG, polylactic-co-glycolic acid-polyethylene glycol 3.11 | Co-administration of NSCs with Reelinloaded PLGA-PEG micelles reduced astrocytic gliosis and increased local angiogenesis Data showed that the administration of Reelin, PLG-PEG, and Reelinload PLGA-PEG micelles with NSCs can alter the levels of astrocytic gliosis and local angiogenesis in the ischemic areas compared to the Stroke group received PBS (Figure 8a,b). According to our findings, the promotion of ischemic changes and necrosis recruited numerous astrocytes in the periphery of lesion sites. Of note, the administration of Reelin or PLGA-PEG in combination with NSCs can significantly reduce the number of recruited astrocytes (GFAP + cells) in the periphery and inside the necrotic sites compared to the control stroke group (Figure 8a,b). These features were more evident in groups after transplantation of Reelin-load PLGA-PEG micelles with NSCs. By contrast, we found that the reduction of astrocyte recruitment coincided with enhanced vascularization (vWF + cells) when Reelin, PLG-PEG and Reelin-load PLGA-PEG micelles with NSCs transplanted into the lesion sites (Figure 8c). Of note, the combination of Reelin-load PLGA-PEG micelles yielded significant differences in the number of vWF cells compared to groups that used Reelin and PLGA-PEG micelles alone with NSCs ( Figure 8c). These data showed that the reduction of cavity size in the ischemic brain after injection of Reelinloaded PLGA-PEG micelles and NSCs occurred with induction of local F I G U R E 8 Monitoring astrocytic gliosis and angiogenesis at the site of injection using IHC analysis of GFAP and vWF factors (a,b). Data showed enhanced angiogenesis rate and inhibition of astrocytic gliosis in the ischemic region with the combination of Reelin and PLGA-PEG micelles with NSCs (red arrows). mNSS test revealed a neurological deficit in all animals during the first 7 days after PT stroke induction (c). The injection of NSCs with PLGA-PEG, Reelin, and Reelin-loaded PLGA-PEG micelles improved mNSS and reached control levels 14, 21, and 28 days after stroke induction. Data showed the superiority of Reelin-loaded PLGA-PEG micelles plus NSCs to alleviate mNSS. Two-way ANOVA. ***p < 0.001. GFAP, glial fibrillary acidic protein; mNSS, modified neurological severity score NSC, neural stem cells; PLGA-PEG, polylactic-coglycolic acid-polyethylene glycol; PT, photothrombotic angiogenesis and suppression of astrocytic gliosis into the infarct areas.

| Reelin-load PLGA-PEG micelles plus NSCs improved neurological outcomes
The mean mNSS score of mice from different groups was measured 1 day after stroke and 7, 14, 21, and 28 days after hydrogel transplantation (Figure 8c). According to our data, mNSS indices were increased (between 11 and 12 score), showing the existence of functional deficits following stroke and these conditions last for 7 days. On Day 7, we transplanted micelles plus NSCs into the stroke area. As shown by the mNSS analysis, the stroke-related neurological deficits were

| DISCUSSION
The delivery of autologous and allogeneic NSCs can support the restoration of injured areas in CNS in studies targeting different animal models. 47,48 Despite these advantages, most of the previously conducted clinical trials failed to achieve the therapeutic outcome in human counterpart which tempered enthusiasm in the understanding of the underlying reparative mechanisms after NSCs transplantation. 49 On this basis, the promotion of regeneration after ischemic changes will necessitate certain tissue engineering strategies like the selection of suitably modified substrates (native ECM components or ECM-like composites) along with NSCs to support the integration of transplanted cells, protect the neighboring host tissue, and promote revascularization into the affected areas. 50 Here, we examined the ability of PLGA-PEG micelles loaded with Reelin in the modulation of mouse NSCs dynamic growths, differentiation, neurite growth, and neuroregenerative potential in in vitro condition and in vivo ischemic stroke model. In this study, PLGA blocks were functionalized with the addition of PEG to increase hydrophilicity which is indicated by H NMR and FTIR spectra. SEM imaging and DLS analysis revealed the synthesis of relatively homogenous spherical PLGA-PEG micelles with appropriate zeta potential and diameter sizes. Our data showed that PLGA-PEG micelles can harbor appropriate Reelin content indicated by Bradford analysis. The existence of electrostatic interactions between the Reelin and PLGA-PEG micelles seems to be the main cause in the formation of Reelin-PLGA-PEG micelles. 51 The previous experiments noted the fact that PLGA nanoparticles have been confirmed as the appropriate carriers for transferring the macromolecules such as genes and proteins. 52 Meanwhile, the addition of PEG to the PLGA backbone can increase the bioavailability of nanostructure and resistance against enzymatic degradation. 52 In vitro analysis indicated that PLGA-PEG micelles significantly Neuronal proliferation and differentiation are essential elements for providing a neural network in vitro. 30 We indicated that three con-  30 It has been shown that Reelin phosphorylates S6 kinase 1 in mTORC1/PI3K-dependent manner, which leads to dendritic growth and branching. 58 These data showed that substrate com- The induction of angiogenesis along with the reduction of pathological remodeling (uncontrolled astrocytic gliosis) is an appropriate strategy to restore the function of injured cells inside the ischemic sites. 64,65 Here, we found that NSCs can promote local angiogenesis and inhibit statistical correction for multiple comparisons astrocytes recruitment into the ischemic site when co-administrated with Reelinloaded PLGA-PEG micelle. Previously, the critical role of Reelin has been proved in association with neuro-glia-vascular regeneration. 66 The activity of Reelin is done on the target cells via an engaging Dab-1 signaling pathway, leading to the activation of vascular endothelial growth factor receptor 2). 60,66,67 Besides, the increase of NSCs migration into the injured area occurs in the presence of Reelin possibly by the promotion of the Akt/Ras axis. 59,68,69 The promotion of angiogenesis inside the micelles can provide the vascular bed that is essential for neuronal migration, cortical development, and neurogenesis. 70 Moreover, Reelin-mediated signaling cascades can regulate certain adhesion molecules in neuronal cells and their differentiation of NSCs to functional motor neurons. 59 Therefore, Reelin might be an effective candidate for the restoration of brain damage and patients with hemiplegia. 59 Based on the great body of experiments, three-dimensional biomaterials not only offer a supportive environment for the transplanted cells, but also is capable of reducing glial scar formation. 71 It was suggested that Reelin can stimulate the regeneration of dorsal root ganglia following axotomy. 72 Of note, the injection of purified Reelin increases the regional density of the dendritic spine and hippocampal CA1 long-term potentiation coincides with spatial learning and memory, showing Reelin activity on synaptic function and cognitive performance in rodents. 73 Therefore, it can be proposed that the reduction of astrocytes in the injured sites correlates with the promotion of neurogenesis, leading to a reduction of astrocyte recruitment into the injured sites. Accordingly, mNSS scores exhibited higher functional recovery in stroke mice that received Reelin-loaded PLGA-PEG micelles compared to the other groups. Therefore, the ori-

| CONCLUSION
This study provides a deeper understanding of the three conditions (PLGA-PEG micelle, Reelin, Reelin-loaded PLGA-PEG micelles) that dictate the differentiation of neural progenitors into the mature neurons. All conditions induced cell differentiation to some extent, suggesting that the presence of PLGA-PEG micelles with Reelin promoted efficiently NSCs proliferation, survival, and differentiation in vitro and in vivo conditions. Taken together, our study presents eligibility and suitability of Reelin-loaded PLGA-PEG micelles on neural tissue regeneration and functional recovery following ischemic stroke, leading to the introduction of novel approaches in the field of neural tissue engineering and regenerative medicine.

CONFLICT OF INTEREST
The authors declare that they have no conflict oF interests.

DATA AVAILABILITY STATEMENT
Any data associated with this study are available from the authors upon reasonable request.