Double-fibroin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells as well as preparation method and application of double-fibroin gel platform

Abstract

The invention discloses a double-fibroin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells and a preparation method and application of the double-fibroin gel platform. The double-fibroin gel platform comprises active oxygen response type fibroin gel and single fibroin gel, the active oxygen response type fibroin gel is mainly formed by loading traditional Chinese medicine components on methacrylated silk fibroin modified by active oxygen response groups, and the single fibroin gel is mainly formed by loading mesenchymal stem cells on unmodified methacrylated silk fibroin. The co-delivery gel platform prepared by the invention has the performance of hierarchical functionalization, sequential release of neuroprotective traditional Chinese medicine components and neural repair of mesenchymal stem cells. According to the platform, the reservation of the brain mesenchymal stem cells is improved through a time-space synergistic sequential delivery mechanism, the dual treatment effect of'protection first and repair second 'is realized, and a novel strategy with higher pertinence and high efficiency is provided for precise treatment of cerebral arterial thrombosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nano-preparation, and particularly relates to a double-fibulin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells, and a preparation method and application thereof. BACKGROUND

[0002] Ischemic stroke is caused by occlusion of cerebral arterial blood flow, leading to high morbidity and mortality worldwide. Reestablishing blood supply to the ischemic brain is the gold standard for treatment of ischemic stroke. However, due to the limited therapeutic time window after the onset of ischemic stroke, only less than 5% of patients benefit from clinically approved intravenous thrombolysis and intra-arterial thrombectomy. In the early stage of ischemic stroke, energy supply is insufficient, neurons depolarize, calcium ions flow into, leading to mitochondrial dysfunction, and a large amount of reactive oxygen species are produced. At the same time, the release of excitotoxic glutamate further exacerbates the death of nerve cells and releases damage-associated molecular patterns. A large number of pro-inflammatory glial cells and infiltrating macrophages are recruited to the ischemic peripheral area, reaching a peak at about 2 weeks. They release a variety of pro-inflammatory factors such as interleukin-1β, IL-6, tumor necrosis factor alpha, active oxygen and matrix metalloproteinase, and activate a variety of pro-inflammatory signaling pathways. These interactions of focal oxidative stress, excitotoxicity, and inflammatory response lead to a large number of nerve deaths and ultimately worsen the clinical outcome. In view of the early pathological reaction of ischemia, previous evidence shows that administration of neuroprotective preparations helps to reduce the nerve death caused by pathological reactions. Since the loss of nerve cells is irreversible, for the repair of nerve function in the later stage of ischemia, a large number of clinical trials show that stem cell therapy has good nerve repair capacity and is beneficial to the regeneration and replacement of nerves in the later stage of ischemia. Exogenous stem cells induce endogenous nerve regeneration or directly differentiate into nerve cells to replace dead nerve cells through paracrine effect, thereby playing a function. However, the pathological reaction after stroke is not conducive to the direct delivery of stem cells. SUMMARY

[0003] Invention purposes: In view of the problems existing in the prior art, the present application aims to provide a double silk gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells, and a preparation method and application thereof. The co-delivery platform designed by the present application is composed of two parts: (1) outer gel: formed by a plant vesicle encapsulating traditional Chinese medicine monomers loaded on a methacrylated silk gel modified by an active oxygen response bond, and (2) inner gel: formed by a single unmodified methacrylated silk gel loading mesenchymal stem cells. With the aid of the auxiliary programmed administration (i.e. the outer gel is injected into the stroke cavity first, and then the inner gel is delivered to the core of the outer gel) and the advantage of the primary hierarchical functionalization design, in the early stage of ischemic stroke, the outer gel which contacts more stroke lesion environment first responds to the high active oxygen environment in the lesion area, releases neuroprotective traditional Chinese medicine components, plays a neuroprotective role, and forms a favorable environment for stem cell survival. Subsequently, the inner gel degrades naturally, and the mesenchymal stem cells are gradually released, which rescue the dying neurons through the paracrine effect, and promote the repair of necrotic tissue and the reconstruction of neural function in the subacute and chronic stages. Through the programmed delivery and hierarchical functionalization design of the platform, the phased treatment of "first protection and then repair" is realized, and the synergistic neuroprotective and neurorestorative effects are exerted, thereby providing a multi-target, course-matched combined treatment strategy for ischemic stroke.

[0004] Technical scheme: In order to achieve the above invention purposes, the technical scheme adopted by the present application is as follows:

[0005] A double silk gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells, wherein the double silk gel platform comprises an active oxygen response type silk gel and a single silk gel, the active oxygen response type silk gel is mainly formed by a methacrylated silk protein modified by an active oxygen response group loading traditional Chinese medicine components, and the single silk gel is mainly formed by an unmodified methacrylated silk protein loading mesenchymal stem cells.

[0006] The active oxygen response type silk gel can be used as an outer gel, which has an antioxidant effect, can scavenge active oxygen, reduce oxidative stress, protect mitochondrial function, improve neuronal energy metabolism, up-regulate antioxidant enzyme expression, enhance cell survival ability, and inhibit the expansion of cerebral ischemic infarction core; at the same time, it can reduce neural inflammatory damage and promote the transformation of M1 type pro-inflammatory microglia cells to M2 type anti-inflammatory phenotype. The single silk gel can be used as an inner gel, which has a nerve repair function, promotes nerve cell growth, repairs damaged blood-brain barrier, and promotes angiogenesis through the paracrine effect of mesenchymal stem cells, thereby playing a nerve repair role.

[0007] Therefore, the double silk gel platform described above can be used to prepare an in situ administration preparation. Through programmed delivery, the outer layer gel is first injected into the stroke cavity, and then the inner layer gel is delivered to the core of the outer layer gel, realizing in situ administration, immediate effect, and a hierarchical sequential release mechanism mediated by reactive oxygen species through hierarchical functionalization, improving the brain retention of mesenchymal stem cells for treatment, and matching the treatment process with disease progression, and fully exerting the synergistic treatment advantage.

[0008] As a specific embodiment, the reactive oxygen species response group modified methylacrylated silk fibroin is mainly formed by grafting 3-(propane-2, 2-diylbis(sulfanediyl)) dipropionic acid with methylacrylated silk fibroin.

[0009] As a specific embodiment, the traditional Chinese medicine component is mainly formed by plant vesicles loaded with traditional Chinese medicine monomers; preferably, the plant vesicles are selected from one or more of extracellular vesicles derived from ginseng, fresh ginger, turmeric, lemon and grapefruit; and the traditional Chinese medicine monomers are one or more of gardenoside, astragaloside A, curcumin, tanshinone II A and ginsenoside Rg1.

[0010] As a specific embodiment, the mesenchymal stem cells are selected from one or more of bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells and embryonic mesenchymal stem cells; preferably, the mesenchymal stem cells are Mash1 gene overexpressed mesenchymal stem cells.

[0011] The application also provides a preparation method of the double silk gel platform for co-delivery of the traditional Chinese medicine component and the mesenchymal stem cells, comprising the following steps:

[0012] (1) preparing a reactive oxygen species response group modified methylacrylated silk fibroin precursor solution;

[0013] (2) preparing plant vesicles, mixing the traditional Chinese medicine monomers with the plant vesicles, ultrasonicating under ice bath conditions, incubating after ultrasonicating, removing free drugs, and obtaining neuroprotective nanoparticles loaded with traditional Chinese medicine monomers;

[0014] (3) uniformly mixing the nanoparticles obtained in step (2) with the precursor solution obtained in step (1), crosslinking under ultraviolet light irradiation, and forming a reactive oxygen species response type silk gel;

[0015] (4) uniformly mixing the mesenchymal stem cells with an unmodified methylacrylated silk fibroin solution, and crosslinking to form a single silk gel.

[0016] As a specific embodiment, in step (1), the precursor solution is obtained by grafting 3-(propane-2, 2-diylbis (sulfanediyl)) dipropionic acid and methacrylated silk fibroin through esterification reaction; the mass ratio of 3-(propane-2, 2-diylbis (sulfanediyl)) dipropionic acid and methacrylated silk fibroin is 100:0.25-100:1.

[0017] As a specific embodiment, in step (2), the preparation method of the plant vesicle comprises the following steps: taking fresh plants, juicing, removing large particles and fibers by differential centrifugation, collecting supernatant, ultrafiltration, and collecting filtrate to obtain the plant vesicle; preferably, the differential centrifugation is sequentially performed under the following conditions: 2x10 3 g centrifugation for 15-25 min, 5x10 3 g centrifugation for 25-35 min, and 1.5x10 4 g centrifugation for 55-65 min; the method of ultrafiltration comprises the following: sequentially passing the supernatant through 100 kDa and 10 kDa ultrafiltration membranes.

[0018] The mass ratio of the traditional Chinese medicine monomer to the plant vesicle (based on the protein content) is 1:(1.5-2.5);

[0019] The ultrasonic conditions are 1 s on / 2 s off, the time is 4-6 min, and the power is 120-130 W;

[0020] The incubation conditions are 37±2℃, 55-65 min.

[0021] As a specific embodiment, in step (3):

[0022] The nanoparticle concentration is 300-400 μg / ml (based on the content of gardenoside), the precursor solution concentration is 8%-10% (w / v), and the volume ratio of the two is 1:(0.8-1.2);

[0023] The crosslinking under ultraviolet irradiation requires the addition of a photoinitiator in the system, wherein the ultraviolet light wavelength is 380-420 nm, the photoinitiator concentration is 0.1-0.25% (w / v), and the irradiation time is 20-30 s;

[0024] As a specific embodiment, in step (4), the concentration of the non-methacrylated silk fibroin solution is 4%-5% (w / v); the density of the mesenchymal stem cells is 1x10 6 -1x10 7 cells / mL.

[0025] The application also provides application of the double silk fibroin gel platform of co-delivery of the traditional Chinese medicine component and mesenchymal stem cells in preparation of a drug for treating ischemic stroke. In application, the active oxygen responsive silk fibroin gel and the single silk fibroin gel are respectively used as an outer gel and an inner gel, auxiliary programmed drug delivery (namely, the outer gel is injected into the stroke cavity first, and then the inner gel is delivered to the core of the outer gel) and the advantage of the main hierarchical functional design. The double silk fibroin gel platform can realize co-delivery of the neuroprotective component and the neural repair mesenchymal stem cells, sequentially plays the neuroprotective and neural repair effects in each stage of ischemic stroke, and provides a multi-target, disease course matching combined treatment strategy for treating ischemic stroke.

[0026] The application finally provides application of the active oxygen responsive silk fibroin gel and the single silk fibroin gel in preparation of a drug for treating ischemic stroke, wherein the active oxygen responsive silk fibroin gel is mainly formed by loading a traditional Chinese medicine component into methacrylated silk fibroin modified by an active oxygen responsive group, and the single silk fibroin gel is mainly formed by loading mesenchymal stem cells into unmodified methacrylated silk fibroin.

[0027] Ischemic stroke can induce tissue damage and form an infarction stroke cavity, wherein the extracellular matrix is lacking, and physical support for cell infiltration and tissue repair cannot be provided. Meanwhile, the stroke cavity provides a potential implantation site, which can accommodate a filling implant material. A proper amount of filling material is implanted into a regional stroke cavity position, which does not have adverse effects on normal brain tissue, and realizes in-situ effect of the filling material. Hydrogel is a three-dimensional polymer network that simulates the characteristics of natural extracellular matrix, which can provide an exogenous structural scaffold in the stroke cavity to promote cell infiltration to repair damage. Hydrogel can become a potential therapeutic adjuvant in stem cell therapy by improving the survival rate and differentiation ability of stem cells in vivo. The in-situ drug delivery platform of hydrogel can encapsulate drugs or cells for local injection or implantation into the brain, thereby bypassing the blood-brain barrier, achieving effective drug accumulation and slow release at the target site, enhancing the therapeutic effect and reducing toxic side effects. In addition, the exogenous structural scaffold with good cell compatibility is created in the stroke cavity, which supports cell infiltration into the lesion and provides a favorable niche for the survival of exogenous stem cells.

[0028] Methacrylated silk fibroin has been proved to promote nerve repair and has good cell compatibility. Therefore, the present application designs and constructs an active oxygen response modified methacrylated silk fibroin gel as an outer gel to load nerve-protective traditional Chinese medicine components, and a single methacrylated silk fibroin gel as an inner gel to load mesenchymal stem cells, which will sequentially exert therapeutic effects. When the two gels are delivered to the ischemic brain in a sequential in situ programmed manner (i.e., the outer gel is injected into the stroke cavity first, and then the inner gel is delivered to the core of the outer gel), due to the regional accommodation of the ischemic stroke cavity and the sequential administration characteristics, the outer gel will contact more pathological positions of the stroke cavity than the inner gel. At the same time, mainly with the hierarchical functional design, the outer gel has the active oxygen response characteristics, responds to active oxygen at the ischemic site, releases nerve-protective traditional Chinese medicine components, resists pathological reactions in the early stage of ischemia, and at the same time creates a favorable environment for stem cell survival; subsequently, the inner gel naturally degrades, and the mesenchymal stem cells are gradually released to exert therapeutic effects. Therefore, the construction of the double silk fibroin gel platform, the auxiliary programmed drug delivery method and the main hierarchical functional design will be beneficial to the co-delivery of nerve-protective drugs and nerve repair stem cells, combining neuroprotective therapy and nerve repair therapy, and providing a good niche for the survival and differentiation of stem cells, significantly improving the retention of mesenchymal stem cells in the brain, reducing the neurological deficits in the late stage of ischemic stroke, and promoting nerve repair through the paracrine effect of mesenchymal stem cells or the function of differentiating into nerve cells to replace damaged nerve cells.

[0029] The present application realizes the time-sequential delivery of traditional Chinese medicine components through the double silk fibroin gel strategy, and exerts a multi-target, disease course-matched therapeutic effect. After the occurrence of ischemic stroke, in situ drug delivery is achieved through auxiliary programmed delivery (the outer gel is injected into the stroke cavity, and then the inner gel is delivered to the core of the outer gel), so that the outer gel is in full contact with the pathological microenvironment of the stroke. In addition, mainly with the response of the outer gel to endogenous active oxygen, the nerve-protective traditional Chinese medicine components are released to exert a neuroprotective effect and create a favorable survival environment for the survival of mesenchymal stem cells in the inner gel. Subsequently, with the natural degradation of the single methacrylated silk fibroin gel, the mesenchymal stem cells are released, which rescue dying cells through paracrine effect and replace necrotic tissue in the acute, subacute and chronic stages of ischemic stroke, and exert a nerve repair effect. Through the programmed delivery and hierarchical functional design of the double silk fibroin gel platform, a disease course-matched therapeutic effect is achieved.

[0030] The present application prepares active oxygen response bond modified methacrylated silk fibroin through esterification reaction, which is simple compared with traditional modification methods. The active oxygen response bond modified methacrylated silk fibroin is prepared by esterification reaction between the amine group on the methacrylated silk fibroin and the carboxyl group on 3-(propane-2,2-diylbis(sulfanediyl))dipropionic acid.

[0031] The application combines the ultrafiltration method and the differential centrifugation method to prepare plant vesicles, compared with the traditional ultra-high-speed centrifugation method, the preparation method is simple and does not need a long time of ultra-high-speed centrifugation process, the preparation time is significantly shortened, and the integrity of the obtained plant vesicles is guaranteed, which is convenient for long-term preservation.

[0032] The application selects traditional Chinese medicine monomer compounds as therapeutic drugs, and promotes the loading of traditional Chinese medicine components into plant vesicles through ultrasonic crushing, thereby improving the low encapsulation efficiency of traditional vesicle drug loading technology.

[0033] The application can effectively improve the drug loading capacity and bioavailability of the traditional Chinese medicine components, enrich the neuroprotective substances in the ischemic lesion area, and resist the pathological reaction of stroke by using the neuroprotective substances in the active oxygen response outer gel, and create a favorable survival environment for the survival and differentiation of the inner mesenchymal stem cells. Therefore, the platform can deliver therapeutic substances in different stages of the disease, realize continuous treatment and synergistic effect. The specific advantages are as follows:

[0034] (1) Biological safety: the traditional Chinese medicine component and mesenchymal stem cell co-delivery double silk fibroin gel platform of the application has good biological safety, and uses methacrylated silk fibroin as a delivery carrier, which has low immunogenicity and high biological safety.

[0035] (2) Hierarchical response: the application constructs an outer layer of active oxygen response type methacrylated silk fibroin gel and an inner layer of non-active oxygen response type methacrylated gel, and releases the substances through active oxygen mediated hierarchical response.

[0036] (3) Intelligent time sequence effect: in the acute stage of stroke, the active oxygen response key modified methacrylated silk fibroin outer gel carries the neuroprotective traditional Chinese medicine components, starts the neuroprotective effect, slows down the rapid expansion of the infarct core and resists the pathological reaction, and creates a favorable survival environment for the mesenchymal stem cells in the inner gel. In the subacute and chronic stages of stroke, the inner gel naturally degrades to release mesenchymal stem cells, which promote nerve repair and remodeling through the paracrine effect and replacement effect of mesenchymal stem cells.

[0037] (6) Synergistic effect: plant vesicles contain a variety of plant active substances, which have significant efficacy. When the plant vesicles load traditional Chinese medicine monomer compounds, they can play a synergistic effect. Both of them intervene in the pathological microenvironment of stroke first, create a favorable microenvironment for mesenchymal stem cell implantation, and significantly improve the retention rate and therapeutic efficacy of mesenchymal stem cells in brain tissue.

[0038] Advantages: Compared with the prior art, the application has the following advantages:

[0039] (1) The double silk fibroin gel platform prepared by the application has the characteristics of rapid photo-crosslinking and active oxygen response. The double silk fibroin gel platform can gel within 20-30s, and the hierarchical functionalization of the active oxygen response group realizes controllable active oxygen response advantage.

[0040] (2) The plant-derived extracellular vesicle is used in the traditional Chinese medicine component nanoparticles provided by the application, which reduces the toxicity and side effects of drugs, improves the biological safety and biocompatibility, and ensures the safety of the carrier.

[0041] (3) The double silk fibroin gel platform provided by the application realizes the co-delivery of the traditional Chinese medicine component composed of plant extracellular vesicles loaded with traditional Chinese medicine monomers and mesenchymal stem cells, and combines the neuroprotective therapy and the nerve repair therapy. The double silk fibroin gel platform is composed of an outer gel formed by the traditional Chinese medicine component loaded on the active oxygen response group modified methacrylated silk fibroin, and an inner gel formed by the mesenchymal stem cells loaded on the unmodified methacrylated silk fibroin.

[0042] (4) The double silk fibroin gel provided by the application has a low elastic modulus and a low storage modulus, and is soft in nature, which is suitable for in situ implantation in brain tissue, reduces the mechanical stimulation or inflammatory reaction caused by the mismatch between the material hardness and the brain tissue; at the same time, the double silk fibroin gel has good self-healing performance, realizes state transformation in the stage strain test, repairs itself, maintains structural integrity, and improves the stability of the gel system. Moreover, due to the complex structure and limited space of the brain, the plasticity of the gel enables it to adapt to irregular brain surfaces or lesion areas, realizing local precise drug delivery.

[0043] (5) The double silk fibroin gel platform is designed and constructed, realizing the sequential delivery of the traditional Chinese medicine component and the mesenchymal stem cells, and improving the brain retention of the mesenchymal stem cells. The double silk fibroin gel platform assists the programmed delivery (injecting the outer gel into the stroke cavity, and then delivering the inner gel to the core of the outer gel) for in situ drug delivery, so that the outer gel first contacts the stroke pathological microenvironment. In addition, mainly by means of hierarchical functionalization design, the active oxygen response outer silk fibroin gel first delivers the traditional Chinese medicine component to play a neuroprotective role, and creates a favorable environment for the survival of stem cells. Then, through the natural degradation of the inner gel, the mesenchymal stem cells are released to play a therapeutic effect. Overall, the double silk fibroin gel realizes sequential delivery for the complex pathological process of ischemic stroke, and improves the brain retention of the therapeutic mesenchymal stem cells.

[0044] (6) The double silk fibroin gel provided by the application can load active drugs for different treatment methods, different lesion sites for combined treatment, realize combined drug delivery and time sequence delivery, provide a reference for the time sequence delivery system construction of safe targeted treatment, multi-drug combination therapy, and multi-lesion simultaneous treatment for ischemic stroke and other cardiovascular and cerebrovascular diseases, and has broad application prospect and clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Inverted Vial and Rheology Investigation of Shell and Core Gels in Example 6.1.1;

[0046] Figure 2 Scanning Electron Microscopy Investigation of Shell and Core Gel Morphology in Example 6.1.2;

[0047] Figure 3 In Vitro Antioxidant Performance Investigation of Shell and Core Gel Carriers in Example 6.1.3;

[0048] Figure 4 In Vitro Release of Traditional Chinese Medicine Components from Shell Gels in Example 6.1.4;

[0049] Figure 5 In Vitro Antioxidant Activity Investigation of Shell Gels in Example 6.1.5;

[0050] Figure 6 In Vitro Mitochondrial Function Improvement Investigation of Shell Gels in Example 6.1.6;

[0051] Figure 7 Polarization of Microglial Cells Phenotype Investigation of Shell Gels in Example 6.1.7;

[0052] Figure 8 In Vitro Promotion of hCMEC / D3 Cell Migration Investigation of Core Gels in Example 6.1.8;

[0053] Figure 9 In Vitro Promotion of hCMEC / D3 Tube Formation Investigation of Core Gels in Example 6.1.9;

[0054] Figure 10 In Vivo Antioxidant Activity Investigation of Shell-Core Gels in Example 6.1.10;

[0055] Figure 11 Polarization of Microglial Cells Phenotype Investigation of Shell-Core Gels in Example 6.1.11;

[0056] Figure 12 In Vivo Mesenchymal Stem Cell Retention Improvement Investigation of Shell-Core Gels in Example 6.1.12. DETAILED DESCRIPTION

[0057] The present application is further illustrated by the following examples. These examples are purely illustrative and are not intended to limit the present application. The present application is further described below with reference to the accompanying drawings and examples:

[0058] Example 1: Preparation of active oxygen response bond modified methacrylated silk fibroin

[0059] A 10 mg / mL solution of 3-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK) was mixed with EDC (TK:EDC molar ratio = 1:4) in an ice bath for 30 min, and then NHS (TK:NHS molar ratio = 1:2) was added to continue the reaction for 30 min. A methacrylated silk fibroin (SFMA) aqueous solution was mixed with 20% of 2-(N-morpholino)ethanesulfonic acid by mass of SFMA and slowly stirred in an ice bath for 10 min. The activated TK was slowly added to the SFMA solution to graft TK into the SFMA macromolecular chain at a mass ratio of SFMA:TK = 100:1, and slowly stirred for 2 h. Finally, the composite solution was loaded into a dialysis bag (MWCO 8-14 kDa) and dialyzed at 4°C for 24 h to obtain the final SFMA-TK solution. Finally, the SFMA-TK solution was frozen at -80°C for 12 h and freeze-dried for 48 h.

[0060] Example 2: Extraction of ginseng vesicles

[0061] Ginseng vesicles (GDNPs) were extracted by differential centrifugation combined with ultrafiltration: Fresh ginseng was peeled and juiced, and differential centrifugation (2x10 3 g for 20 min, 5x10 3 g for 30 min, and 1.5x10 4 g for 60 min) was performed to remove large particles and fibers, and the supernatant was collected. The supernatant was ultrafiltered through 100 kDa and 10 kDa filters in sequence, and the filtrate was collected.

[0062] Example 3: Preparation of ginseng vesicle-loaded geniposide nanoparticles (G / G NPs)

[0063] G / G NPs were prepared by ultrasonic fragmentation. GDNPs (400 μg in terms of protein content) were added with 200 μg of geniposide, and ultrasonication was performed at 120 W power, 1 s on / 2 s off mode for 5 min under ice bath conditions to load the drug into the vesicles. Subsequently, incubation was performed at 37°C for 1 h to promote the closure of the membrane structure, and free drugs were removed by dialysis to obtain G / G NPs.

[0064] Example 4: Preparation of active oxygen response shell gel

[0065] The Shell gel was formed by mixing G / G NPs (400 pg / ml, calculated by the content of geniposide) and 8% (w / v) SFMA-TK solution at a volume ratio of 1:1, adding 0.25% (w / v) of the photoinitiator LAP, mixing well, and then irradiating under 405 nm ultraviolet light for 30 s.

[0066] Example Five: Preparation of the Core gel

[0067] According to the literature method, bone marrow mesenchymal stem cells were transfected with lentivirus carrying the Mash1 gene to construct Mash1 gene overexpressing bone marrow mesenchymal stem cells (M-BMSCs). A final concentration of 4% (w / v) SFMA solution was mixed with 1 x 10 7 cells / mL of M-BMSCs, 0.25% (w / v) of LAP photoinitiator was added, mixed well, and then irradiated under 405 nm ultraviolet light for 30 s to form the Core gel.

[0068] Example Six: Investigation of the properties of the co-delivery double-fibulin gel platform Shell and Core

[0069] 1.1 Inverted vial and rheological investigation of Shell and Core gels

[0070] The Shell and Core obtained in Examples Four and Five were used to investigate the formation and rheological properties of the gels by inverted vial experiments and rheometers. Parallel plate method was used to determine the storage modulus and loss modulus of the Shell and Core gels. As shown in Figure 1 , the Shell and Core gels both showed gel state in the inverted vial experiment without flow phenomenon. Rheological detection showed that the storage modulus (G') of both was greater than the loss modulus (G"), indicating the successful formation of a stable gel network. With the increase of shear rate, the viscosity decreased, showing shear thinning characteristics, and the step strain experiment showed that it had good self-healing performance.

[0071] 1.2 Scanning electron microscope investigation of the morphology of Shell and Core gels

[0072] The freeze-dried Shell and degraded Core hydrogels were sputtered with gold, and the cross-section structure of the hydrogels was analyzed using a scanning electron microscope. As shown in Figure 2 , the freeze-dried hydrogel samples all showed a porous and dense network structure.

[0073] 1.3 Investigation of the antioxidant performance of Shell and Core gel carriers

[0074] To the same volume of Shell and Core gel, add DPPH solution, incubate at 37℃ for 30 min, measure the absorbance at 517 nm, and calculate the DPPH clearance rate according to the absorbance of the control group. The results are shown in Figure 3 As shown in the figure, only the Shell gel carrier showed DPPH free radical scavenging ability within 1 h, while the Core gel carrier had no significant scavenging effect. After 24 h, both gel carriers showed certain antioxidant activity, but the DPPH scavenging efficiency of the Shell gel carrier was the highest.

[0075] 1.4 Investigation of the release of traditional Chinese medicine components of Shell gel in vitro under PBS or H2O2 environment

[0076] Place the Shell gel in PBS or 1 mM H2O2 solution and detect the release amount of gardenoside. The results are shown in Figure 4 As shown in the figure, compared with the PBS environment, the Shell gel has a higher release rate of gardenoside in the H2O2 environment, indicating that the Shell gel responds to the active oxygen in the environment and accelerates the release of the drug.

[0077] 1.5 Investigation of the anti-active oxygen effect of Shell gel in vitro

[0078] Establishment of oxidative stress cell model: Take PC12 cells in logarithmic growth phase and inoculate them in the well plate. After overnight adhesion, discard the culture solution and add DMEM single culture solution containing 1 mM H2O2. Place it in the incubator at 37℃ for oxidative stress culture for 12 h.

[0079] Use 2', 7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe to detect the intracellular active oxygen after Shell gel treatment. Culture PC12 cells in a 12-well plate and incubate to the appropriate cell density. After oxidative stress exposure, co-culture PC12 cells with hydrogel for 24 h with or without treatment agent. Then, stain the cells with 10 μM DCFH-DA at 37℃ for 30 min. Finally, measure the fluorescence intensity of dichlorofluorescein (DCF) in each group by flow cytometry. The results are shown in Figure 5 As shown in the figure, compared with the oxidative stress H2O2 induction group, Shell gel significantly inhibits the intracellular active oxygen level and has certain antioxidant effect.

[0080] 1.6 Investigation of the effect of Shell gel on improving mitochondrial function in vitro

[0081] Establishment of OGD cell model: Take PC12 cells in logarithmic growth phase and inoculate them in the well plate. After overnight adhesion, discard the culture solution and add DMEM sugar-free culture solution. Place it in the anoxic chamber and place it in the incubator at 37℃ for oxygen-glucose deprivation culture for 6 h.

[0082] JC-1 fluorescent probe labeling method was used to detect the effect of Shell gel on mitochondrial membrane potential of PC12 cells after OGD injury. PC12 cells were inoculated in 12-well plates containing confocal climbing sheets, with a cell density of 1 x 10 5 cells per well, and cultured at 37°C in 5% CO2. After OGD modeling treatment, PC12 cells were co-cultured with hydrogel with or without therapeutic agents for 24 h. The culture medium was discarded, the cells were washed with PBS, and JC-1 working solution was added, mixed well, and incubated at 37°C in a cell incubator for 20 min in the dark. After incubation, the supernatant was discarded, and JC-1 staining buffer was added for washing. The cells were imaged by laser confocal instrument. As shown in Figure 6 , compared with the OGD group, Shell gel significantly reversed the decrease in mitochondrial membrane potential, maintained normal mitochondrial membrane potential, and improved mitochondrial function.

[0083] 1.7 Investigation of the in vitro polarization of microglial cell phenotype by Shell gel

[0084] Establishment of a lipopolysaccharide (LPS) inflammatory cell model: Logarithmic growth period BV2 cells were inoculated in the well plate, and after overnight adhesion, 1 μg / mL of LPS solution was added for induction for 24 h, and the modeling was completed.

[0085] The effect of Shell gel on polarized microglial cells was investigated by immunofluorescence staining of nitric oxide synthase (iNOS) and arginine-1 (Arg-1). BV2 cells were inoculated in a 24-well plate, with a cell density of 2 x 10 5 cells per well, and cultured at 37°C in 5% CO2. After the cells were incubated overnight, an LPS-induced inflammatory cell model was established. After modeling was completed, the culture medium was discarded, and the cells were washed with PBS. Shell gel was added for 24 h. Fluorescent primary antibodies were used for staining, and the nuclei of BV2 cells were stained with DAPI. The cells were imaged by laser confocal instrument. As shown in Figure 7 , compared with the LPS group, the Shell gel group significantly enhanced the expression of M2-type BV2 cell marker Arg-1 and reduced the expression of M1-type marker iNOS, indicating that Shell gel could effectively promote the polarization of microglial cells to M2 phenotype.

[0086] 1.8 Investigation of the in vitro promotion of vascular migration by Core gel

[0087] hCMEC / D3 cells were inoculated in a 6-well plate and cultured overnight. After the cells were grown and adhered, a gun head was used to vertically scratch the cells, and PBS was used to wash and remove the scratched cells and establish an OGD model. Then, hCMEC / D3 cells were co-cultured with Core gel with or without Core gel for 24 h, and the migration of hCMEC / D3 cells was observed by inverted fluorescence microscope. The results are shown inFigure 8 As shown, compared with the OGD model group, Core gel significantly reduced the gap of hCMEC / D3 cell scratch area, indicating that it has the ability to promote the migration of vascular endothelial cells.

[0088] 1.9 Investigation of the pro-angiogenic effect of Core gel in vitro

[0089] Matrigel was seeded in a 24-well plate and left to stand overnight at 4°C to flatten the matrigel. hCMEC / D3 cells were seeded on the matrigel to construct an OGD model. Then the hCMEC / D3 cells were co-cultured with or without Core gel for 24 h, Calcein AM staining was used to stain the cells, and Thunder wide-field imaging system was used to observe the tube formation performance of hCMEC / D3 cells. The results are shown in Figure 9 As shown, compared with the OGD group, Core gel significantly promoted the tube formation of hCMEC / D3, showing a pro-angiogenic effect.

[0090] 1.10 Investigation of the anti-active oxygen effect of Shell-Core gel in vivo

[0091] Construction and administration of photothrombosis (PT) model: isoflurane was used to anesthetize rats, and rat head dorsal hair was removed using a hair clipper and sterilized with 70% ethanol. The rat's head skin was cut about 1.5 cm along the midline using surgical scissors, and the skull soft tissue was bluntly dissected with forceps to expose the anterior and posterior fontanelles. The skull surface was disinfected again, and the drill point was marked 0.5 mm behind the anterior fontanelle and 1.5 mm to the right, with a depth of 4.0 mm. A high-speed skull drill was used to drill a small hole with a diameter of about 1 mm at this point. The photosensitizer rose bengal (20 mg / kg) was injected into the tail vein. A laser beam with a wavelength of 561 nm was used to stereotactically position the middle cerebral artery for 10 minutes, and the wound was sutured. Three days after modeling, the PT rats were divided into PT model group, M-BMSCs group, Core group, and Shell-core group. Different treatment substances were injected in situ in combination with a stereotactic instrument. The PT model group was injected with PBS, the M-BMSCs group was injected with M-BMSCs suspension, the Core group was injected with Core gel, and the Shell-core gel group was injected with Shell-core gel by program injection (Shell gel was injected first, and then Core gel was injected into Shell gel). After injection, the syringe needle was left in the brain for 5 minutes, and then the syringe was slowly pulled out. The blank (Ctrl) group of rats did not undergo modeling surgery, and the same volume of PBS solution was injected into the brain at the same position as a control, and the relevant operations were the same as those of the other groups of rats.

[0092] The Shell-Core gel in vivo anti-active oxygen effect was investigated by dihydroethidium (DHE) immunofluorescence staining. After 4 days of treatment, the brain tissues of rats in each group were collected, frozen sectioned after embedding with OCT embedding agent, and DHE staining was performed to analyze the active oxygen level of brain tissue. The results are shown in Figure 10 PT and M-BMSCs group, the Core gel group and the Shell-core gel group had more significant anti-active oxygen ability, and the brain DHE fluorescence was significantly reduced. Moreover, compared with the Core gel group, the DHE fluorescence of the Shell-core gel group was weaker, indicating that the Shell gel in the outer layer played its antioxidant role, synergistically promoted the overall antioxidant effect of the Shell-core gel, and was beneficial to improve the severe oxidative stress microenvironment of ischemic stroke.

[0093] 1.11 Investigation of Shell-Core gel in vivo polarization of microglial cell phenotype effect.

[0094] The Shell-Core gel in vivo polarization of microglial cell phenotype effect was investigated by Arg-1 / iNOS immunofluorescence staining. After 4 days of treatment, the brain tissues of rats in each group were collected, frozen sectioned after embedding with OCT embedding agent, and Arg-1 / iNOS staining was performed to analyze the phenotype of brain microglial cells. The results are shown in Figure 11 PT and M-BMSCs group, the Core gel group and the Shell-core gel group Arg1 green fluorescence was stronger, and iNOS red fluorescence was weaker, effectively promoting the polarization of brain microglial cells to M2 phenotype, and the Shell-core gel group induced stronger M2 polarization of microglial cells. The results showed that the Shell-core gel played a role in inducing the transformation of microglial cells to M2 anti-inflammatory phenotype, which helped to reduce the inflammatory level of ischemic stroke.

[0095] 1.12 Investigation of Shell-Core gel improving mesenchymal stem cell retention in vivo;

[0096] M-BMSCs were labeled with live cell dye PKH26 to form Core gel and Shell-core gel. Brain implantation was performed by stereotactic microsyringe, and the brain retention of M-BMSCs was observed at 3 days and 14 days. The results are shown in Figure 12 compared with the M-BMSCs group without gel, the Shell-core gel group achieved high retention of M-BMSCs in the brain at 14 days, indicating that the construction of Shell-core gel provided a favorable niche for the survival of M-BMSCs.

[0097] The above embodiments are the best mode of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement modes and should be included in the protection scope of the present application.

Claims

1. A dual-silk fibroin gel platform for co-delivering traditional Chinese medicine components and mesenchymal stem cells, characterized in that, The dual-silk fibroin gel platform includes reactive oxygen species-responsive silk fibroin gel and single-silk fibroin gel. The reactive oxygen species-responsive silk fibroin gel is mainly formed by loading traditional Chinese medicine components onto methacryloyl fibroin modified with reactive oxygen species-responsive groups. The single-silk fibroin gel is mainly formed by loading mesenchymal stem cells onto unmodified methacryloyl fibroin.

2. The dual-silk fibroin gel platform for co-delivering traditional Chinese medicine components and mesenchymal stem cells according to claim 1, characterized in that, The methacrylamide silk fibroin modified with reactive oxygen species is mainly formed by grafting 3-(propane-2,2-diylbis(thionyl))dipropionic acid with methacrylamide silk fibroin.

3. The dual-silk fibroin gel platform for co-delivering traditional Chinese medicine components and mesenchymal stem cells according to claim 1, characterized in that, The herbal components are mainly formed by loading herbal monomers onto plant vesicles; preferably, the plant vesicles are selected from one or more extracellular vesicles derived from ginseng, ginger, turmeric, lemon, and grapefruit; the herbal monomers are one or more selected from geniposide, astragaloside A, curcumin, tanshinone IIA, and ginsenoside Rg1.

4. The dual-silk fibroin gel platform for co-delivering traditional Chinese medicine components and mesenchymal stem cells according to claim 1, characterized in that, The mesenchymal stem cells are selected from one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and embryonic mesenchymal stem cells; preferably, the mesenchymal stem cells are mesenchymal stem cells overexpressing the Mash1 gene.

5. The method for preparing the dual-silk fibroin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of a methacrylamide silk fibroin precursor solution modified with reactive oxygen species response groups; (2) Prepare plant vesicles by mixing the Chinese herbal monomer with the plant vesicles, sonicating under ice bath conditions, incubating after sonication, removing free drugs, and obtaining neuroprotective nanoparticles loaded with Chinese herbal monomers. (3) Mix the nanoparticles obtained in step (2) with the precursor solution obtained in step (1) and crosslink them under ultraviolet light to form reactive oxygen species responsive silk fibroin gel. (4) Mix mesenchymal stem cells with unmodified methacrylamide silk fibroin solution evenly and cross-link to form a monofilament gel.

6. The method for preparing the dual-silk fibroin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells according to claim 5, characterized in that, Step (1) includes grafting 3-(propane-2,2-diylbis(thioalkyldiyl))dipropionic acid with methacrylated silk fibroin through an esterification reaction to obtain a precursor solution; the mass ratio of 3-(propane-2,2-diylbis(thioalkyldiyl))dipropionic acid to methacrylated silk fibroin is 100:0.25-100:

1.

7. The method for preparing the dual-silk fibroin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells according to claim 5, characterized in that, In step (2), the method for preparing the plant vesicles includes the following steps: taking fresh plants, juicing them, centrifuging at differential speed to remove large particles and fibers, collecting the supernatant, ultrafiltration, and collecting the filtrate to obtain the plant vesicles; preferably, the differential centrifugation is performed sequentially under the following conditions: 2×10 3 Centrifuge for 15-25 minutes, 5×10⁻⁵ min. 3 Centrifuge for 25-35 minutes at 1.5 × 10⁻⁶ g. 4 Centrifuge for 55-65 minutes; the ultrafiltration method includes the following: passing the supernatant sequentially through 100kDa and 10kDa ultrafiltration membranes; The mass ratio of the herbal monomers to plant vesicles (based on protein content) is 1:(1.5-2.5); The ultrasound conditions are 1s on / 2s off, duration 4-6 minutes, and power 120-130W; The incubation conditions were 37±2℃ for 55-65 minutes.

8. The method for preparing the dual-silk fibroin gel platform for co-delivery of traditional Chinese medicine components and mesenchymal stem cells according to claim 5, characterized in that, In step (3): The concentration of the nanoparticles is 300-400 μg / ml (based on geniposide content), the concentration of the precursor solution is 8%-10% (w / v), and the volume ratio of the two is 1:(0.8-1.2). The crosslinking under ultraviolet light irradiation requires the addition of a photoinitiator to the system, wherein the ultraviolet light wavelength is 380-420nm, the photoinitiator concentration is 0.1-0.25% (w / v), and the irradiation time is 20-30s; In step (4), the final concentration of the unmethacrylated silk fibroin solution is 4%-5% (w / v); the density of the mesenchymal stem cells is 1×10⁻⁶. 6 -1×10 7 cells / mL.

9. The use of the dual-silk fibroin gel platform for co-delivering traditional Chinese medicine components and mesenchymal stem cells as described in any one of claims 1-4 in the preparation of drugs for treating ischemic stroke.

10. The application of reactive oxygen species-responsive silk fibroin gel and monofilament silk fibroin gel in the preparation of drugs for treating ischemic stroke, characterized in that, The reactive oxygen species responsive silk fibroin gel is mainly formed by loading traditional Chinese medicine components onto methacrylamide silk fibroin modified with reactive oxygen species responsive groups, while the single silk fibroin gel is mainly formed by loading mesenchymal stem cells onto unmodified methacrylamide silk fibroin.

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