Immunoregulation and neurovascular interaction microsphere as well as preparation method and application thereof

Abstract

The invention provides an immunoregulation and nerve and blood vessel interaction microsphere as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. The preparation method of the microspheres for regulating and interacting with the nerve and blood vessels comprises the following steps: firstly, preparing PLGA (Poly (Lactic-co-Glycolic Acid) nanoparticles loaded with interleukin-4; then, through a biotin-avidin reaction, biotin-RAP12 polypeptide is modified to the surfaces of the nano particles, and PLGA nano particles modified by RAP12 and interleukin-4 are prepared; then preparing ROS responsive polyvinyl alcohol microspheres, and loading the obtained nanoparticles into the ROS responsive polyvinyl alcohol microspheres to prepare the immunoregulation and nerve and blood vessel interaction microspheres. The immunoregulation hydrogel microsphere disclosed by the invention only needs to be injected once the first day after a central nervous system is injured, can achieve a long-term treatment effect without being continuously injected for multiple times for a long time, and is extremely remarkable in curative effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and relates to an injectable immune microsphere that can be used for repairing central nervous system damage, and specifically to an immunomodulatory and neurovascular interaction microsphere, and a preparation method and application thereof. Background Art

[0002] Various cell types and tissues communicate through complex signaling networks, often referred to as "crosstalk," which is essential for maintaining normal tissue growth, development, and repair after injury. The immune system interacts closely with the neurovascular unit to maintain neural homeostasis. Immune cells such as microglia maintain neural function by pruning synapses and secreting angiogenic factors to promote blood vessel growth and vascular network formation. Disruption of this crosstalk balance can lead to dysregulated inflammatory responses, abnormal angiogenesis, loss of regenerative capacity, and ultimately hinder tissue remodeling. Therefore, establishing an interactive bridge between damaged tissues through tissue engineering methods is an urgent problem to be solved.

[0003] When the central nervous system (CNS) is injured, the imbalance of the interaction between brain immune cells and neurovascular units is a key factor affecting brain regeneration. The normal dialogue between the immune system and the neurovascular unit in the injured area is disrupted, resulting in an imbalance in the homeostasis of the immune microenvironment, triggering an inflammatory factor storm, leading to blood-brain barrier destruction, glial scar formation, increased oxidative stress, accumulation of neurotoxic substances, neurovascular unit dysfunction, and inhibition of nerve regeneration. Therefore, signal transduction and interaction between immune cells and components of the neurovascular unit (including neurons, glial cells, and endothelial cells) are essential for regulating inflammatory responses and tissue repair.

[0004] Microglia are the main immune effector cells of the central nervous system. After brain injury, they differentiate into anti-inflammatory types and secrete a series of neurotrophic factors, such as insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β), brain-derived neurotrophic factor (BDNF) to promote nerve regeneration, and vascular endothelial growth factor (VEGF) to interact with endothelial cells to stimulate angiogenesis and enhance cerebral blood flow, thereby promoting neurological function recovery. Therefore, after central nervous system injury, promoting microglial polarization to develop an anti-inflammatory phenotype is beneficial to enhance the immune-neurovascular interaction network and promote neural repair.

[0005] Interleukin-4 (IL-4) is a classic therapeutic factor that plays a key role in promoting the transformation of microglia to an anti-inflammatory phenotype. However, current systemic treatments based on IL-4 have limitations due to its very short half-life and extremely low delivery efficiency. The effective time of IL-4 in the body is only 2 to 10 minutes, and it is easily cleared by the body. Therefore, multiple high-dose injections are often required to achieve a certain therapeutic effect. Current studies choose to use nasal injection or intraventricular injection as a method of administration, but it still requires multiple days of continuous drug injection. The administration procedure is extremely cumbersome and causes great pain to patients. At the same time, after multiple days of continuous administration, the efficiency of repairing central nervous system damage is still not high.

[0006] For example, Liu [1] If the intraventricular injection method is chosen, the IL-4 drug concentration needs to be 60 ng / d for 7 consecutive days to show therapeutic effect. At the same time, the drug administration time must be as long as 21 days before a more obvious therapeutic effect can be shown.

[0007] Another example is Zhang [2] The effect becomes gradually apparent after 14-28 days of nasal administration at a drug concentration of 50 μg / kg.

[0008] For example, He [3] Even at a drug concentration of 125 ng / d, it still takes at least 7 days of continuous injection to show efficacy. [4] When the drug is administered through the nasal cavity at a concentration of 50μg / kg, it also requires continuous injection for more than seven days to show therapeutic effect.

[0009] It can be seen that the current treatment of central nervous system damage with IL-4, even if it is administered by nasal injection or intraventricular injection, still requires continuous drug injection for several days to achieve the corresponding therapeutic effect. At the same time, the longer the administration time, the more significant the effect. The drug efficacy level needs to be improved urgently.

[0010] Therefore, for the use of IL-4 in the treatment of central nervous system damage diseases, since IL-4 is effective for a very short time in the body and is easily cleared by the body, the existing administration methods generally require long-term administration for many consecutive days to achieve relatively obvious therapeutic effects, which has the problems of continuous administration, long administration cycle, and low drug efficacy. How to shorten the administration cycle of IL-4, avoid continuous long-term administration, and at the same time improve its efficacy in central nervous system damage repair has become a technical problem that needs to be solved urgently.

[0011] The references cited are as follows:

[0012] [1]Liu X, et al, Interleukin-4is Essential for Microglia / Macrophage M2Polarization and Long-term Recovery after Cerebral Ischemia.Stroke.2016.

[0013] [2]Zhang Q, et al, The interleukin-4 / PPARγ signaling axis promote soligodendrocyte differentiation and remyelination after brain injury.PLoSBiol.2019.

[0014] [3]He Y,et al,Neuroscience.2020Jun;437:161-171.

[0015] [4]Xu J,et al,Proc NatlAcad Sci US A.2020;117:32679-32690. Summary of the invention

[0016] The present invention is to solve the above technical problems, thereby providing an immunomodulatory and neurovascular interaction microsphere and its preparation method and application. The technical purpose of the present invention is to solve the problem that when IL-4 is used to treat central nervous system damage diseases, the existing administration method requires long-term administration for at least 7 consecutive days to obtain relatively obvious therapeutic effects, and it is impossible to achieve the therapeutic effect with only one administration; on the other hand, it solves the problem that the existing drug efficacy of IL-4 is still low even after continuous administration for many days, and it is impossible to achieve extremely obvious therapeutic effects on the 14th day of administration.

[0017] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0018] One of the purposes of the present invention is to provide a method for preparing immunomodulatory and neurovascular interaction microspheres, comprising the following steps:

[0019] (1) using avidin to modify PLGA nanoparticles, and then encapsulating interleukin-4 in the nanoparticles by a double emulsion method to prepare avidin-modified PLGA nanoparticles loaded with interleukin-4;

[0020] (2) modifying the biotin-RAP12 polypeptide onto the surface of the nanoparticles obtained in step (1) through a biotin-avidin reaction to prepare PLGA nanoparticles modified with RAP12 and interleukin-4;

[0021] (3) PVA microsphere precursors were prepared by air-microfluidics method, and then cross-linked with TSPBA linker to prepare ROS-responsive PVA microspheres;

[0022] (4) The RAP12 and interleukin-4 modified PLGA nanoparticles obtained in step (2) are absorbed into freeze-dried ROS-responsive polyethanol microspheres by negative pressure to prepare immunomodulatory and neurovascular interaction microspheres.

[0023] The interaction between immune cells and neurovascular units plays a key role in neural regeneration after central nervous system injury. Maintaining brain immune homeostasis is the key to restoring neurovascular function. The present invention has developed an immunomodulatory hydrogel microsphere (MP / RIL4) composed of interleukin-4 (IL-4) nanoparticles modified with RAP12 peptide targeting microglia and boron ester functionalized hydrogel through biotin-avidin reaction and air-microfluidics technology. It has been experimentally confirmed that the above-mentioned immunomodulatory hydrogel microspheres provided by the present invention only need to be injected once (400ng / mouse, 20μg / kg) on ​​the first day after central nervous system injury (transient middle cerebral artery occlusion mice), without the need for long-term multiple continuous injections, to achieve the effect of long-term treatment, and the neurobehavioral score has been significantly improved from the 3rd day compared with the control group or the single microsphere group, and the brain atrophy volume has shown a significant decrease at 14 days, which is more significant than the efficacy on the 14th day after continuous multiple administrations.

[0024] The above-mentioned immune functionalized microspheres MP / RIL4 provided by the present invention serve as an interactive bridge for regulating the immune microenvironment, connecting the interactive network between microglia and neurovascular units, and promoting the recovery of neurological function. In vitro and in vivo studies have confirmed that such immunomodulatory microspheres reduce the expression of proinflammatory factors such as IL-1β, iNOS and CD86 in microglia, while upregulating the levels of anti-inflammatory factors such as Arg-1, IL-10 and CD206. In addition, injection of MP / RIL4 can significantly reduce the brain atrophy volume in the transient middle cerebral artery occlusion model of stroke mice, promote neurobehavioral recovery, enhance crosstalk between immune cells and neurovascular units, thereby promoting angiogenesis and neurogenesis in stroke mice. In summary, immunomodulatory microspheres can coordinate the interaction between immune cells and neurovascular units, and have considerable therapeutic potential in ischemic stroke and other central nervous system diseases.

[0025] Furthermore, the reaction steps of step (1) are: firstly reacting avidin with palmitic acid-N-hydroxysuccinimide ester to synthesize avidin-palmitate, then using PLGA and dichloromethane as the oil phase and interleukin-4 as the external aqueous phase to prepare a primary emulsion by ultrasound, then mixing the primary emulsion with an internal aqueous phase solution of avidin-palmitate and polyvinyl alcohol and then ultrasound to form a double emulsion, and after volatilizing the solvent, obtaining avidin-modified PLGA nanoparticles loaded with interleukin-4.

[0026] Furthermore, in step (1), the weight ratio of avidin to palmitic acid-N-hydroxysuccinimide ester is 13-15:1.

[0027] Furthermore, in step (1), the concentration ratio of PLGA to interleukin-4 is 25 mg:10 μg.

[0028] Furthermore, the concentration of biotin-RAP12 polypeptide in step (2) is 2.5 mg / ml.

[0029] Furthermore, in step (3), the concentration of TSPBA is 5-10wt%, the concentration of polyvinyl alcohol is 5-10wt%, and the volume ratio of the two is 1:0.25-2.

[0030] Furthermore, the negative pressure load in step (4) is provided by vacuuming.

[0031] The second object of the present invention is to provide immunomodulatory and neurovascular interaction microspheres prepared by the method described above.

[0032] The third object of the present invention is to provide the use of the above-mentioned immunomodulatory and neurovascular interaction microspheres in the preparation of drugs for treating central nervous system damage and repair.

[0033] Specifically, the central nervous system damage diseases described in the present invention include stroke.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) The present invention provides an immunomodulatory microsphere, which acts as an interface between microglia and neurovascular units, and can precisely regulate immune-neurovascular crosstalk to achieve neurological function repair. In addition, the system can continuously release IL-4 nanoparticles in response to the microenvironment, regulate the immune niche for a long time, prolong the action time and therapeutic efficiency of IL-4 in the brain injury area, thereby increasing angiogenesis and neurogenesis, and ultimately promoting neurobehavioral recovery in ischemic stroke mice.

[0036] (2) The immunomodulatory microspheres prepared by the present invention only require one administration, without the need for multiple consecutive administrations, to achieve a long-term therapeutic effect. Moreover, after one administration, the volume of cerebral infarction can be significantly reduced on the 14th day, and the neurobehavioral recovery and neurovascular remodeling of stroke mice can be significantly improved. The efficacy level is significantly improved compared to the existing administration method on the 14th day. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of PVA microparticles loaded with RAP12-PLGA / IL-4 and their effects in vitro and in vivo; PVA microparticles loaded with RAP12-PLGA / IL-4 (MP / RIL4) promote the polarization of microglia toward an anti-inflammatory phenotype in vitro and in vivo, and promote angiogenesis and neurogenesis in ischemic stroke mice.

[0038] Figure 2 Preparation and characterization of RAP12-PLGA / IL-4 nanoparticles PVA microspheres; (A) Representative TEM images of IL-4-loaded nanoparticles (IL4-NP) and RAP12-modified IL-4 / NP (RIL4); (B) Quantitative size distribution analysis of IL4-NP and RIL4; (C, D) Bright field images and quantitative size distribution analysis of PVA microspheres (MPs); (E, F) Representative SEM images of PVA MPs and MPs loaded with RIL4 (MP / RIL4); (G) Drug release of naked NPs in PBS, PVAMP loaded with RIL4 in PBS and H2O2; n=3; All data are expressed as mean ± SEM.

[0039] Figure 3 Microspheres loaded with RAP12-PLGA / IL-4 had no effect on the proliferation and survival of microglia; (A) Cell viability of BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 for 24 hours, n=4; (B) Representative images of CalceinAM / PI staining of BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 for 24 hours, scale bar=100μm; (C) Cell viability of BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 for 48 hours, n=4; (D) Representative images of CalceinAM / PI staining of BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 for 48 hours, scale bar=100μm, n=4; All data are expressed as mean±SEM.

[0040] Figure 4In vitro immunomodulatory effect of "immunomodulator"; (A, B) Quantitative RT-PCR analysis of the expression of pro-inflammatory markers (IL-1β, iNOS) and anti-inflammatory markers (Arg-1, IL-10) in BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 and LPS after 24h, n=3-4; (C, D) Western blot and quantitative analysis of Arg-1 expression in BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 and LPS-stimulated BV2 microglia for 24h, n=3; (EH) Representative immunostaining images of BV2 microglia after co-culture with PBS, MP, RIL4 and MP / RIL4 and LPS and quantification of iNOS, Agr-1 and IBA-1, scale bar = 100μm, n=4; all data are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001.

[0041] Figure 5 "Immunomodulator" promotes neurobehavioral function and reduces cerebral infarction volume; (A) Experimental plan; (B) Body weight analysis of each group; (C) mNSS analysis of ischemic mice; (D) EBST; (E) Hanging wire test, n=7-15; (F,G) Cresyl violet-stained brain sections and quantification of brain atrophy volume 14 days after ischemic stroke, n=3; All data are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001.

[0042] Figure 6 In vivo immunomodulatory effect of "immunomodulator"; (A) Experimental scheme; (B) Schematic diagram of the peri-infarct area, fluorescence imaging and quantitative analysis of areas 1, 2, and 3; (C, E) Representative co-immunostaining images of CD86 and IBA-1 and quantitative analysis of the CD86+ / IBA-1+ cell ratio in IBA-1+ cells, scale bar = 50 μm, n = 3; (D, F) Representative co-immunostaining images of CD206 and CD11b and quantitative analysis of the CD206+ / CD11b+ cell ratio in CD11b+ cells, scale bar = 50 μm, n = 3, quantitative RT-PCR analysis of the expression of pro-inflammatory markers (IL-1β) and anti-inflammatory markers (Arg-1) in MP, RIL4 and MP / RIL4 injection after ischemic stroke, n = 3, all data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

[0043] Figure 7"Immunomodulator" improves angiogenesis and neurogenesis in mice with ischemic stroke; (A) Experimental scheme; (B) Schematic diagram of the peri-infarct area, fluorescence imaging and quantitative analysis of C1, C2, C3, E, and F areas of the corresponding brain slices; (C) Representative images of CD31+ vessels and (D) CD31+ / Ki67+ vessels, scale bar = 50 μm, n = 3; (E) Representative images of SVZ and (F) striatal DCX+ cells, scale bar = 50 μm, n = 3; Quantitative analysis of (G) vessel density and (H) number of CD31+ / Ki67+ vessels; (I) DCX area in SVZ; (J) DCX migration, n = 3; All data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

[0044] Figure 8 Quantitative RT-PCR analysis; (A) Arg-1, (B) IL-10 expression in BV2 microglia co-cultured with PBS, MP, RIL4 and MP / RIL4 after 24 hours, n=3-4; all data are expressed as mean±SEM, *p<0.05, **p<0.01, ***p<0.001.

[0045] Fig. 9 The original data of WB. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0047] Example 1

[0048] 1. Experimental Materials and Methods

[0049] 1. Animal experiments

[0050] Animal experiments followed the regulations of the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University. 8-week-old specific pathogen-free (SPF) grade wild-type (WT) male ICR mice (weight, 24-26 g) were purchased from Shanghai Lingchang Biotechnology Co., Ltd., China. The mice were placed under 22°C, 60% humidity, 12 h light / 12 h dark cycle conditions, and food and water were supplied on time.

[0051] 2. Preparation and characterization of IL-4 loaded RAP12-PLGA nanoparticles

[0052] (1) Avidin-palmitate was synthesized by reacting 5 mg of avidin with 0.37 mg of palmitic acid-N-hydroxysuccinimide ester in a 2% sodium deoxycholate solution at 37°C in phosphate buffered saline (PBS) for 12 h. The resulting product was then dialyzed overnight in PBS containing 0.15% sodium deoxycholate (Mw = 8,000-12,000).

[0053] (2) PLGA nanoparticles (PLGA-NPs) were prepared by double emulsion method. PLGA (polylactic acid-polyglycolic acid copolymer, 50 mg / ml) was dissolved in 500 μL of dichloromethane to form an oil solution. Then, 200 μL of an aqueous solution containing 10 μg of IL-4 (interleukin-4, Novoprotein, Shanghai, China) protein was added to the oil solution. The solution was sonicated at 70 W for 1 min using SCIENTZ-IID (Scientz, Ningbo, China). The emulsion was added to 7.5 mL of a 0.5% polyvinyl alcohol (PVA) solution containing avidin-palmitate and sonicated at 70 W for 3 min to form a double emulsion. The mixture was then added to 12 mL of a 0.5% PVA solution and magnetically stirred for 3 hours to evaporate and eliminate the dichloromethane, thereby obtaining uniform nanoparticles. PLGA-NPs were collected by evaporation and centrifugation (12,000 rpm, 4°C, 25 min). To remove unencapsulated IL-4, PLGA-NPs were centrifuged with double distilled water (ddH2O). The product was resuspended in 1 ml PBS, 1 mg RAP12-biotin (Apeptide Co., Ltd, Shanghai, China) was added and magnetically stirred for 0.5 h, and the excess RAP12-biotin was removed by centrifugation again. Finally, the IL-4-loaded RAP12-PLGA-NPs (RAP12-PLGA / IL-4, abbreviated as RIL4) were resuspended in 1 ml PBS containing 0.5% trehalose, and stored at -20°C after freeze-drying for later use.

[0054] After the construction was completed, the size distribution and Zeta potential of the nanoparticles were measured by dynamic light scattering (DLS, Malvern Instruments Ltd., UK). Before reacting with RAP12-biotin, 50 μL of PLGA-NPs loaded with IL-4 (PLGA / IL-4) and 50 μL of RAP12-PLGA / IL-4 were placed in DLS for size distribution and then diluted with ddH2O for Zeta potential testing. The morphology of PLGA / IL4 and RAP12-PLGA / IL4 was detected by scanning electron microscopy (SEM).

[0055] 3. Preparation and characterization of polyvinyl alcohol microspheres

[0056] PVA microspheres were prepared using air-microfluidics technology: a 7.5% PVA solution dissolved in dimethyl sulfoxide (DMSO) was sheared into independent droplets by replacing petroleum with gas and collected in anhydrous ethanol. The PVA droplets were rapidly dehydrated in anhydrous ethanol and formed into PVA microsphere precursors by solvent exchange between DMSO and ethanol. The precursor microspheres were then added to N 1 -(4-Borophyll)-N 3 -(4-Borophyll)-N 1 ,N 1 ,N 3 ,N 3 -tetramethylpropane-1,3-diamine (TSPBA) linker to form reactive oxygen species (ROS)-responsive PVA hydrogel microspheres (MPs) and freeze-dried. RIL4 NPs were mixed with freeze-dried MPs to prepare MP / RIL-4. The morphologies of PVA microsphere precursors, MPs, and MP / RIL4 were observed by optical microscopy and scanning electron microscopy.

[0057] 4. BV2 microglial cell culture and stimulation

[0058] The BV2 microglial cell line was purchased from the National Authentication Cell Culture Bank in Shanghai, China. BV2 microglial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Meilunbio) supplemented with 10% heat-inactivated fetal bovine serum (FBS, BioVision, Shanghai, China) and 1% penicillin / streptomycin antibiotics (Meilunbio). BV2 microglial cells were cultured at 5×10 4 Cells were seeded at 400 μg / mL in 24-well plates for 12 h and co-cultured with PVA microspheres (MP), RAP12-PLGA / IL-4 (RIL4), or PVA microspheres loaded with RAP12-PLGA / IL-4 (MP / RIL4) for 1 h. Subsequently, cells were stimulated with LPS (200 ng / mL, Sigma-Aldrich, St. Louis, MO) or PBS for 24 h to assess whether RAP12-PLGA / IL-4 pretreatment affects LPS-induced neuroinflammation in vitro. Neuroinflammation was assessed by real-time quantitative polymerase chain reaction (RT-PCR), immunoblotting (WB), and immunostaining.

[0059] 5. Identification of cell proliferation and survival

[0060] BV2 microglia were inoculated in 24-well plates for 12 hours, co-cultured with PBS, MP, RIL4 or MP / RIL4 for 1 hour, and then stimulated with 200 ng / mL LPS or PBS for 24 hours or 48 hours. The culture medium was discarded, and 350 μL Cell Counting Kit-8 (CCK-8) solution (Meilunbio) was added to each well for 30 minutes. 300 μL of each group was extracted to 96-well plates, 100 μL per well, and the OD value was detected by microplate reader. 1 μL PI and 1 μL Calcein-AM were added to 2 ml PBS, and live and dead cells were double-stained with PI (red) and Calcein-AM (green) to observe cell survival. After 20 minutes, the live and dead cells of the cells were detected under a microscope.

[0061] 6. Transient middle cerebral artery occlusion (tMCAO) surgery

[0062] The surgical procedure for tMCAO was as follows: adult mice (25 per group) were anesthetized with 4–5% isoflurane and maintained with 1.5–2% isoflurane in O2 (RWD Life Sciences Co., Ltd., Shenzhen, China). The carotid artery was carefully isolated and ligated, and then a 6-0 silicone-coated suture (Covidien, USA) was inserted into the external carotid artery and reversed into the internal carotid artery until it reached the origin of the middle cerebral artery. After 90 min, the suture was removed, the ligation was released, and reperfusion was achieved. Laser Doppler flowmetry (Moor Instruments, Devon, UK) was used to monitor the blood flow in the middle cerebral artery to ensure successful occlusion and reperfusion. Occlusion was confirmed when the blood flow was less than 20% of the baseline and the blood flow recovered to more than 80% of the baseline. The mice in the sham group underwent the same surgical procedure except for the insertion and occlusion of the suture. The animals were sacrificed 14 days after tMCAO for immunostaining, RT-PCR, and WB analysis.

[0063] 7. Immunostaining

[0064] Cells were collected using 24-well cell slides and fixed with 4% paraformaldehyde (PFA, Macklin, Shanghai, China) for 10 min before immunostaining. Mice were anesthetized with isoflurane and perfused with PBS and 4% PFA. Brain tissue was fixed in 4% PFA at 4°C for 4-6 h, then replaced with 30% sucrose for 2 days to fully dehydrate it, and then quickly frozen in -80°C isopentane for later use. Slices were sliced ​​from the anterior commissure to the hippocampus with a thickness of 20 μm. Before immunostaining, the slides were removed from -80°C and dried under a fan. After fixation with 4% PFA for 10 min, they were rinsed with PBS three times for 5 min each. Antigen retrieval was performed using pH 9.0 EDTA (Servicebio, Wuhan, China) and PBS rinses. Brain sections or cells cycled with an immunohistochemical pen were incubated with 0.3% Triton X-100 (Sigma-Aldrich) for 10 min, blocked with 2% bovine serum albumin (BSA, Meilunbio) for 1 h at room temperature (RT), and then incubated with primary antibodies overnight at 4°C, including goat anti-IBA1 (1:200, Novusbio, Centennial, CO), rabbit anti-inos (1:200, ab clone, Wuhan, China), rabbit anti-Arg1 (1:200, ab clone), rabbit anti-IBA1 (1:200, WAKO, Osaka, Japan), rat anti-CD86 (1:100, Thermo Fisher, Waltham, MA), rabbit anti-CD206 (1:100, Abcam, Cambridge, UK), rat anti-CD11b (1:100, Biolegend, San Diego, CA). Diego, CA), goat anti-CD31 (1:200, R&D, Emeryville, CA), rabbit anti-Ki67 (1:200, Abcam), rabbit anti-DCX (1:200, Abcam). On the second day, the slides were rinsed three times in PBS for 10 min each time and incubated with Alexa Fluor 488-conjugated donkey anti-goat (1:400, Invitrogen, Waltham, MA), Alexa Fluor 555-conjugated donkey anti-goat (1:400, Invitrogen), Alexa Fluor 488-conjugated donkey anti-rabbit (1:400, Invitrogen), Alexa Fluor 594-conjugated donkey anti-rabbit (1:400, Invitrogen), Alexa Fluor 488-conjugated donkey anti-mouse (1:400, Invitrogen), Alexa Fluor 488-conjugated donkey anti-rat (1:400, Invitrogen), and Alexa Fluor 488-conjugated donkey anti-mouse (1:400, Invitrogen). After being rinsed three times in PBS, the brain sections or cells were covered and sealed with mounting medium containing DAPI (Beyotime, Jiangsu, China).Fluorescence images were collected by confocal microscopy (Leica, Wetzlar, Germany). Three fields of view were imaged per slice, with a 200 μm interval between slices of each mouse brain.

[0065] 8. Real-time quantitative polymerase chain reaction (RT-PCR)

[0066] TRIzol (Invitrogen) was added to the cells to extract RNA. Mice were killed 14 days after tMCAO and perfused with PBS. Brain tissue was quickly removed and placed in cold mouse brain matrix (on ice). The brain was then cut into coronal sections 2 mm before and after the Willis circle, and the damaged tissue was taken and placed in a microtube containing TRIzol to extract RNA. Total RNA was extracted from lysed cells or target brain areas. RNA concentration was detected using a Nanodrop 1000 spectrophotometer (Thermo Fisher,) and then reverse transcribed into cDNA using the SynthesisSuperMix Kit (Yeason, Shanghai, China). RT-PCR was performed using the SYBR Green Master Mix Kit (season). Semi-quantitative analysis (2-ΔΔCT) was performed using the comparative CT method. The mRNA expression level was normalized to the control group GAPDH, and the results are shown as broken line changes.

[0067] The primer sequences are as follows:

[0068] iNOS forward primer: 5′-CAGCTGGGCTGTACAAACCTT-3′, reverse primer: 5′-CATTGGAAGTGAAGCGTTTCG-3′;

[0069] IL-1β forward primer: 5′-TCCAGGATGAGGACATGAGCAC-3′, reverse primer: 5′-GAACGTCACACACCAGCAGGTTA-3′;

[0070] Agr-1 forward primer: 5′-GAACACGGCAGTGGCTTTAAC-3′, reverse primer: 5′-TGCTTAGCTCTGTCTGCTTTGC-3′;

[0071] IL-10 forward primer: 5′-GGTTGCCAAGCCTTATCGGA-3′, reverse primer: 5′-ACCTGCTCCACTGCCTTGCT-3′;

[0072] GAPDH forward primer: 5'-GGCAAATTCAACGGCACAGTCAAG-3', reverse primer: 5'-TCGCTCCTGGAAGATGGTGATGG-3'.

[0073] 9. Western blot (WB) analysis

[0074] Cells were collected by cell scraper in protein lysis buffer (RIPA, protease cocktail inhibitors, phosphatase inhibitors). Mice were killed 14 days after tMCAO and perfused with PBS. Brain tissue was quickly removed and placed in cold mouse brain matrix (on ice). The brain was then cut into coronal sections 2 mm before and after the Willis circle, and the injured tissue was taken and placed in a microtube containing protein lysis buffer to extract protein. The protein concentration was determined by the BCA method. Equal amounts of protein (40 μg) were loaded on 10% (W / V) SDS-PAGE for electrophoresis. The protein was then transferred to a PVDF membrane (Millipore) and incubated with primary antibodies against Arg1 (1:500, Abclonal) and β-actin (1:100, Invitrogen) in a shaker at 4°C overnight. The protein-loaded PVDF membrane was rinsed three times in TBST buffer (Epizyme, Shanghai, China) for 10 min each time and incubated with enzyme-labeled anti-rabbit and anti-mouse IgG (1:4000, Invitrogen) at room temperature for 1 h. After being rinsed three times in TBST buffer, it was reacted with enhanced chemiluminescent substrate (Meilunbio). Semi-quantification was performed using ImageJ software (NIH, Bethesda, MD).

[0075] 10. Cresyl violet staining

[0076] Cresyl violet staining was used to evaluate the volume of brain atrophy. Mice were killed 14 days after surgery and frozen sections were prepared. Brain sections were stained with 0.05% acetocresyl violet (Sigma-Aldrich). The areas of the contralateral and ipsilateral cerebral hemispheres were measured using ImageJ. The atrophy area (ΔS) was calculated by subtracting the area of ​​the contralateral hemisphere from the area of ​​the ipsilateral hemisphere. The thickness between two adjacent brain sections was expressed as height (H), and the atrophy area of ​​two adjacent brain sections was expressed as ΔSn and ΔSn+1.

[0077] The calculation formula for the total volume of brain atrophy (V) is: V = ∑H / 3×[ΔSn+(ΔSn×ΔSn+1)1 / 2+ΔSn+1].

[0078] 11. Neurobehavioral testing

[0079] Neurobehavioral testing was performed by randomized investigators 1, 3, 7, and 14 days after tMCAO surgery.

[0080] Modified Neurological Severity Score (mNSS). The mNSS includes assessment of motor, sensory, and balance functions and the score ranges from 0 to 14. A score of 0 indicates normal and higher scores indicate more severe impairment. Elevated Body Swing Test (EBST). The EBST is used to assess motor asymmetry in mice. The mice are lifted by their tails and their turning behavior is observed. This is repeated 20 times for each mouse. The results are recorded as the number of rightward deflections / total number of repetitions. Hanging Wire Test. To assess the upper limb strength and coordination of mice. The mouse is placed in the middle of a wire and a timer is started. Each mouse is initially given a score of 10. One point is deducted for each fall from the wire. The test continues until the score reaches 0 or the mouse remains on the wire for 180 seconds.

[0081] 12. Statistical analysis

[0082] All data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using Student's t test for two groups and one-way or two-way ANOVA followed by Dunnett or Tukey's multiple comparisons for multiple groups. p < 0.05 was considered statistically significant.

[0083] 2. Experimental Results and Analysis

[0084] 1. Preparation and characterization of immunomodulatory microspheres (MP / RIL4)

[0085] Preparation and characterization of immunomodulators Figure 1 As shown. The double emulsion method was first used to encapsulate the immunomodulatory factor IL-4 in avidin-modified PLGA nanoparticles, and then the biotin-RAP12 peptide was modified on the surface of the nanoparticles by biotin-avidin reaction. TEM and DLS data showed that both nanoparticles were uniformly spherical with a hydrodynamic diameter of about 180nm. The size of the free PLGA nanoparticles loaded with IL4 (IL4-NP) was about 183nm, and after modification with RAP12-biotin (RIL4), its average size was 186nm ( Figure 2 AB), indicating that the modification of RAP12 on the surface of IL4-NP has no effect on its particle size.

[0086] Then, PVA-based microsphere precursors (MP precursors) were prepared by air-microfluidics and DMSO solvent exchange. The MP precursors were then cross-linked by adding TSPBA linkers to form ROS-responsive PVE microparticles (MPs) via boron ester reactions. Bright field images showed that the size distribution of both MP precursors and MPs was around 240 μm ( Figure 2 Finally, RIL4 nanoparticles were sucked into the freeze-dried MP microspheres by negative pressure to obtain MP / RIL4. Scanning electron microscopy showed that MP / RIL4 had a uniform spherical structure and a rougher surface than MP ( Figure 2 RIL4 encapsulated with PVA microparticles can be slowly released in PBS, with the release reaching 40% at 72 h, but the release rate is very fast in H2O2 solution ( Figure 2 (G) The results showed that the immunomodulatory microspheres MP / RIL4 were ROS responsive and could achieve sustained release of IL-4 in PLGA nanoparticles.

[0087] 2. Biocompatibility identification of “immunomodulators”

[0088] To identify the biocompatibility of “immunomodulators”, we used CCK8 to detect cell viability and Calcein-AM / PI staining to determine cell survival / death, where dead cells are PI positive and live cells are Calcein-AM positive. We found that even after 48 hours of culture ( Figure 3 CD), MP, RIL4 or MP / RIL4 treatment for 24 h did not affect the viability of BV2 microglia ( Figure 3 These data indicate that the immunomodulator has good biocompatibility.

[0089] 3. In vitro immunomodulatory effects of “immunomodulators”

[0090] Microglia exhibit a wide range of phenotypes from pro-inflammatory to anti-inflammatory. IL-4 can induce microglia to differentiate into an anti-inflammatory phenotype and has a protective effect on a variety of central nervous system diseases. First, we tested the anti-inflammatory effects of “immunomodulators” compared with naked microparticles and found that the expression of Arg-1 and IL-10 was upregulated in the RIL4 and MP / RIL4 groups. As a classic in vitro neuroinflammation model, we used lipopolysaccharide (LPS)-stimulated BV2 microglia to simulate neuroinflammation in vitro. BV2 microglia were co-cultured with PVA microparticles (MPs), RIL4, and MP / RIL4 for 1 h, and then stimulated with LPS (200 ng / mL) or PBS to observe whether RAP12-PLGA / IL-4 pretreatment changed LPS-induced neuroinflammation. After 24 h, BV2 cells were collected and pro-inflammatory and anti-inflammatory factors were detected. RT-PCR results showed that pro-inflammatory factors such as IL-1β and iNOS were significantly increased in the LPS-stimulated group compared with the control group. RAP12-PLGA / IL-4 pretreatment can reduce the expression of IL-1β and iNOS, and increase the levels of anti-inflammatory factors Arg-1 and IL-10 ( Figure 4 AB in the middle). Western blot verified the anti-inflammatory effect of RAP12-PLGA / IL-4 on BV2 cells at the protein level. Compared with the LPS group, the anti-inflammatory factor Arg-1 in the RAP12-PLGA / IL-4 treatment group increased significantly ( Figure 4CD). BV2 cells pretreated with RAP12-PLGA / IL-4 showed a significant anti-inflammatory effect. In addition, immunofluorescence analysis showed that RAP12-PLGA / IL-4 pretreatment decreased iNOS and increased the immunoreactivity of Arg-1 ( Figure 4 Collectively, these results suggest that RAP12-PLGA / IL-4 has a superior anti-inflammatory effect in vitro.

[0091] 4. Injection of “immunomodulators” can promote neurobehavioral function and reduce brain atrophy volume in ischemic mice

[0092] Ischemic stroke often leads to irreversible brain damage and subsequent brain atrophy. Figure 5 The experimental scheme shown in Figure A was used to detect the neurorepair effect of the "immunomodulator" we designed. Eight-week-old mice were randomly divided into five groups: (1) sham group (Sham group, no suture insertion surgery, no use of any drugs); (2) IS group (exposed to tMCAO surgery); (3) IS-MP group (PVA microspheres were stereotactically administered 1 day after tMCAO); (4) IS-RIL4 group (RAP12-PLGA / IL-4 was stereotactically administered 1 day after tMCAO); (5) IS-MP / RIL4 group (PVA microspheres combined with RAP12-PLGA / IL-4 were stereotactically administered 1 day after tMCAO). Neurobehavioral function was evaluated using mNSS, EBST, rotating rod, and hanging wire methods. Cresyl violet staining was used to detect cerebral infarction. There was no statistically significant difference in body weight among the five groups of rats ( Figure 5 Compared with the IS and IS-MP groups, the neurological deficits in mice injected with RIL4 and MP / RIL4 were significantly reduced ( Figure 5 MP / RIL4 treated mice showed reduced brain atrophy volume ( Figure 5 These results suggest that “immunomodulators” have promising neuroprotective effects in promoting neurobehavioral function and reducing brain atrophy volume.

[0093] 5. Immunomodulatory effects of “immunomodulators” in vivo

[0094] Modulating the activation and polarization of microglia, the resident immune cells in the brain, is crucial for alleviating inflammatory responses and promoting tissue repair after ischemic stroke. To test the immunomodulatory effects of our “immunomodulators,” we sacrificed mice on day 14 after tMCAO and performed immunostaining and RT-PCR ( Figure 6 CD86 is a pro-inflammatory marker that is co-expressed by IBA-1+ microglia and is elevated after tMCAO, which is reversed by RIL4 and MP / RIL4 ( Figure 6In contrast, CD206, an anti-inflammatory marker, was co-expressed with IBA-1+ microglia and was increased in the RIL4 and MP / RIL4 groups compared with the is and is-MP groups ( Figure 6 D, F), the MP / RIL4 group performed better than the RIL4 group in increasing anti-inflammatory factors. RT-PCR analysis results confirmed the anti-inflammatory effect of MP / RIL4 ( Figure 6 This study showed that MP / RIL4 has significant anti-inflammatory effects and can potentially improve microglial function, making it a promising approach for treating brain pathologies involving neuroinflammation.

[0095] 6. “Immunomodulators” promote angiogenesis and neurogenesis in ischemic mice

[0096] In the chronic stage of stroke, it initiates repair mechanisms including angiogenesis and neurogenesis, both of which play a key role in promoting functional recovery after stroke. However, angiogenesis and neurogenesis after stroke are limited due to the lack of effective communication between neurotrophic factors and neurovascular components. The immunomodulators we prepared can also provide structural support in the infarct area and may play a bridge role between polarized microglia and neurovascular units. We then went on to detect angiogenesis and neurogenesis in ischemic mice by immunostaining ( Figure 7 AB). Figure 7 CD and Figure 7 As shown in Figure GH, the density of CD31+ blood vessels in the perilesional area of ​​mice treated with RIL4 and MP / RIL4 increased compared with the control group. At the same time, the number of CD31+ / Ki67+ endothelial cells increased in MP / RIL4-treated mice, indicating that the microparticles loaded with RIL4 nanoparticles can achieve sufficient angiogenesis. We also found that there were more DCX+ neuroblasts in the SVZ of ischemic mice treated with MP / RIL4 ( Figure 7 In addition, the MP / RIL4 group showed better migration potential to the injured area than the other groups ( Figure 7 (F, J). Figure 8 The results of quantitative RT-PCR analysis are shown in Figure 2. Fig. 9 The data are original WB data. These data indicate that injection of MP / RIL4 increases angiogenesis and neurogenesis in ischemic stroke mice.

[0097] Conclusion

[0098] The present invention combines PLGA nanoparticles with the microglia targeting peptide RAP12 and loads the immunomodulatory factor IL-4, which is then integrated with PVA microspheres prepared by air-microfluidics technology to make injectable microspheres. This system acts as a bridge to regulate the local immune microenvironment, promoting angiogenesis and neurogenesis by inducing microglia to transform into an anti-inflammatory phenotype.

[0099] The immunomodulatory hydrogel microspheres provided by the present invention only need to be injected once (400 ng / mouse, 20 μg / kg) on ​​the first day after central nervous system injury (transient middle cerebral artery occlusion mice), without the need for long-term multiple continuous injections, to achieve long-term treatment effects. The neurobehavioral score was significantly improved compared with the control group or the microsphere group alone from the 3rd day, and the brain atrophy volume showed a significant decrease at 14 days, which was more significant than the efficacy of the administration method mentioned in the background technology after multiple continuous administrations on the 14th day.

[0100] This immunomodulator not only down-regulated the expression of pro-inflammatory factors (IL-1β, iNOS, CD86) in in vitro cell models and in vivo stroke models, but also up-regulated anti-inflammatory markers (Arg-1, IL-10, CD206) and improved the local immune environment. It also reduced the volume of brain atrophy in stroke mice, enhanced neurobehavioral function, and precisely coordinated immune cells with neurovascular units to promote angiogenesis and neurogenesis. In short, this targeted microglia-loaded immunomodulatory system provides a new way to improve the abnormal immune microenvironment after stroke, enhance the interaction between the immune system and the neurovascular network, and thus promote neurovascular regeneration.

Claims

1. A method for preparing immunomodulatory and neurovascular interaction microspheres, characterized in that: The following steps are involved: (1) using avidin to modify PLGA nanoparticles, and then encapsulating interleukin-4 in the nanoparticles by a double emulsion method to prepare avidin-modified PLGA nanoparticles loaded with interleukin-4; (2) modifying the biotin-RAP12 polypeptide onto the surface of the nanoparticles obtained in step (1) through a biotin-avidin reaction to prepare PLGA nanoparticles modified with RAP12 and interleukin-4; (3) PVA microsphere precursors were prepared by air-microfluidics method, and then cross-linked with TSPBA linker to prepare ROS-responsive PVA microspheres; (4) The nanoparticles obtained in step (2) are loaded into ROS-responsive polyvinyl alcohol microspheres by negative pressure to prepare immunomodulatory and neurovascular interaction microspheres.

2. The preparation method according to claim 1, characterized in that: The reaction steps of step (1) are: firstly reacting avidin with palmitic acid-N-hydroxysuccinimide ester to synthesize avidin-palmitate, then using PLGA and dichloromethane as the oil phase and interleukin-4 as the external aqueous phase to prepare a primary emulsion by ultrasonication, then mixing the primary emulsion with an internal aqueous phase solution of avidin-palmitate and polyvinyl alcohol and ultrasonication to form a double emulsion, and after volatilizing the solvent, obtaining avidin-modified PLGA nanoparticles loaded with interleukin-4.

3. The preparation method according to claim 2, characterized in that: The weight ratio of avidin to palmitic acid-N-hydroxysuccinimide ester in step (1) is 13-15:

1.

4. The preparation method according to claim 2, characterized in that: In step (1), the concentration ratio of PLGA to interleukin-4 is 25 mg:10 μg.

5. The preparation method according to claim 1, characterized in that: The concentration of biotin-RAP12 polypeptide in step (2) is 2.5 mg / ml.

6. The preparation method according to claim 1, characterized in that: In step (3), the concentration of TSPBA is 5-10wt%, the concentration of polyvinyl alcohol is 5-10wt%, and the volume ratio of the two is 1:0.25-2.

7. The preparation method according to claim 1, characterized in that: The negative pressure load in step (4) is provided by vacuuming.

8. Immunomodulatory and neurovascular interaction microspheres prepared by the method according to any one of claims 1 to 7.

9. Use of the immunomodulatory and neurovascular interaction microspheres according to claim 8 in the preparation of drugs for treating central nervous system damage and repair.

10. The use according to claim 9, characterized in that: The central nervous system damage includes stroke.

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