Optogenetic system promoting targeted homing of mscs to damaged liver, and preparation method therefor

Abstract

An optogenetic system promoting the targeted homing of MSCs to a damaged liver, and a preparation method therefor. On the basis that VUR8 exhibits a dimer structure in a natural state and dissociates following UVB irradiation, an optogenetic system comprising light-controlled CXCR4-releasing plasmids which contain UVR8 and CXCR4 sequences and upconversion nanoparticles which can convert NIR having strong tissue penetration capability into UVB is developed, and the optogenetic system is used for implementing the overexpression of CXCR4 only on MSC cells in the liver region so as to promote the targeted homing effect thereof.

Description

A light genetic system for promoting MSC targeted homing to damaged liver and a preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a light genetic system for promoting MSC targeted homing to damaged liver and a preparation method thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSC) are a kind of multipotent stem cells that can be isolated from various tissues. They have strong in vitro expansion capacity and low immunogenicity, making them an ideal cell source for stem cell therapy. Because MSCs have the ability to differentiate into multiple cell types and can deliver bioactive substances, they are used to treat a variety of diseases, especially immune-related diseases. The main administration method of MSCs is local injection or intravenous injection. However, compared with local injection, intravenous injection often fails to show sufficient therapeutic effect. Studies have shown that the root cause of this situation lies in the limited ability of MSCs to migrate to the injury site. So far, improving the homing efficiency of MSCs remains a key focus of stem cell therapy research.

[0003] In vitro expansion is a common method for preparing a large number of MSCs. However, during this process, the homing ability of MSCs gradually decreases, significantly affecting the therapeutic effect of systemic administration. As a key signaling pathway for regulating homing, the CXC chemokine receptor 4 / matrix-derived factor-1 (CXCR4 / SDF1) axis is an important target for MSC genetic engineering research. In order to achieve high CXCR4 expression in MSCs, researchers have developed various delivery methods, including viral vectors, liposomes, nanotechnology, low-intensity pulsed ultrasound, and surface modification techniques. However, these methods have certain limitations, such as high risk of carcinogenesis, complex operation, and low cost-effectiveness. Most importantly, current modification methods mainly induce overexpression of CXCR4 in MSCs in vitro, lacking the spatiotemporal specificity of in vivo regulation. This defect is particularly prominent in the treatment of systemic diseases. Because MSCs can target other damaged sites, leading to insufficient accumulation of cells in the main or target lesions. Therefore, there is an urgent need to develop an effective method for promoting MSC targeted homing to damaged areas to improve their therapeutic effect. SUMMARY

[0004] Technical problems solved: In order to solve the above technical problems, the present application provides a light genetic system for promoting MSC targeting homing to damaged liver and a preparation method thereof. Based on the characteristics that UVR8 presents a dimer structure in a natural state and dissociates after UVB irradiation, a light-controlled CXCR4 release plasmid containing Uvr8 and Cxcr4 sequences is developed, and up-conversion nanoparticles (UCNPs) capable of converting strong tissue-penetrating NIR into UVB are used to realize overexpression of CXCR4 on the MSC cell membrane in the liver area to promote its targeted homing effect, which can effectively solve the problems of high carcinogenic risk, complex operation and low cost-effectiveness of the above method.

[0005] Technical scheme: In the first aspect, the present application provides a light genetic system for promoting MSC targeting homing to damaged liver, which comprises a light-controlled CXCR4 release plasmid and SiO2 embedded up-conversion nanoparticles. The light-controlled CXCR4 release plasmid is designed by connecting CXCR4 at the N terminus of the light-controlled plasmid, followed by Furin cleavage site (amino acid sequence SARNRQKR) and two UVR8 repeat sequences. The up-conversion nanoparticles are NaYbF4: Tm: Gd UCNP.

[0006] Preferably, the C terminus of CXCR4 in the light-controlled CXCR4 release plasmid is connected with EYFP to track its movement in cells.

[0007] In the second aspect, the present application provides a preparation method of the light genetic system for promoting MSC targeting homing to damaged liver according to the first aspect, which comprises the following steps:

[0008] S1, synthesis of light-controlled CXCR4 release plasmid;

[0009] S2, preparation of SiO2 embedded up-conversion nanoparticles;

[0010] S3, preparation of light genetic system.

[0011] Preferably, the process of SiO2-embedded upconversion nanoparticles is as follows: 1.77 g Triton X-100, 1.6 mL 1-hexanol, 4 mL cyclohexane solution of NaYbF4: Tm: Gd UCNP, 2.5 mL 1-hexanol, 480 μL deionized water and 100 μL ammonia water are added to a 25 mL halogen bottle, which is provided with a Teflon-coated magnetic sub, stirred at 700 rpm for 30 minutes at 25°C to form a reverse microemulsion; 1 mL of cyclohexane containing 50 μL of tetraethoxysilane (TEOS) is added to the reverse microemulsion, and stirred at 700 rpm at 25°C overnight; SiO2@UCNP is released from the microemulsion by adding acetone, centrifuged at 4000 rpm for 30 minutes, and SiO2@UCNP is separated from the mixed solution, and the obtained SiO2@UCNP is washed with ethanol and water three times respectively to obtain washed SiO2@UCNP; then, the washed SiO2@UCNP is dispersed in 10 mL of eluent (acetonitrile: H2O: acetic acid = 50:49:1 (v / v)) at room temperature for 1 hour to remove the surfactant; after removing the surfactant, the solid particles are collected by centrifugation at 4000 rpm for 30 minutes, and washed with water and ethanol three times respectively to obtain SiO2-embedded upconversion nanoparticles.

[0012] Further, the preparation method of the NaYbF4: Tm: Gd UCNP is as follows:

[0013] First, the preparation of NaYF4: Yb: Tm UCNP is carried out according to the method disclosed in the literature (Gnanasammandhan, M. K., Idris, N. M., Bansal, A., Huang, K., and Zhang, Y. (2016). Near-IR photoactivation using mesoporous silica-coated NaYF4:Yb,Er / Tm upconversion nanoparticles. Nature Protocols 11, 688-713. 10.1038 / nprot.2016.035.), specifically:

[0014] 747 mL of 1 M YCl3•6H2O, 250 mL of 1 M YbCl3•6H2O and 100 mL of 0.03 M TmCl3•6H2O aqueous solution are added to a 100 mL three-necked round-bottom flask, heated to 120°C under stirring at 350 rpm, and water is evaporated for about 1.5 hours to obtain mixture A;

[0015] To mixture A, 6 mL of oleic acid and 15 mL of 1-octadecene were added and heated to 160 °C for 3 hours until a clear and homogeneous solution B was formed;

[0016] The heating mantle was removed and to solution B, which was cooled to 60 °C, 5 mL of methanol containing 0.1 g of NaOH and 0.148 g of NH4F was added dropwise and heated to 110 °C for 1 hour to evaporate the residual water and methanol to obtain mixture C;

[0017] The flask was placed in a spherical heating mantle, ensuring a tight fit, and was sealed with a glass stopper, connected to a two-tube condenser, and mixture C was kept under vacuum for 10 minutes; a two-way piston valve cycled the atmosphere of the line between vacuum and argon three times (1 minute each). Then, the flask was filled with argon and the temperature was raised to 300 °C at a heating rate of 10 °C / min for 1 hour to obtain mixture D;

[0018] The heating mantle was removed and mixture D was allowed to cool to room temperature, mixture D was transferred to a 50 mL centrifuge tube and acetone was added to the centrifuge tube to a total volume of 40 mL, the centrifuge tube was centrifuged at 6654 x g for 10 minutes at room temperature and the supernatant was discarded; the precipitate was dissolved in 20 mL of cyclohexane and vortex mixed, then centrifuged at 1000 x g for 5 minutes, the supernatant containing NaYbF4: Tm: Gd UCNP was transferred to a 20 mL glass scintillation vial to obtain a cyclohexane solution of NaYbF4: Tm: Gd UCNP;

[0019] Secondly, the preparation of NaYbF4: Tm: Gd UCNP is based on the introduction of Gd 3+ to a 100 mL three-necked round bottom flask, the subsequent operations were the same as the preparation method of NaYF4:Yb:Tm UCNP.

[0020] Preferably, the preparation process of the optogenetic system is specifically as follows: the light-controlled CXCR4 release plasmid is combined with NaYbF4: Tm: Gd UCNP according to the use requirement, wherein the light-controlled CXCR4 release plasmid is used for MSC transfection, and the NaYbF4: Tm: Gd UCNP is used for in vitro culture (added into culture medium to convert NIR) or in vivo experiment (aggregated in liver after tail vein injection, and the NaYbF4: Tm: Gd UCNP in the liver is activated to emit UVB by irradiating the liver region of the mouse in vitro using NIR).

[0021] In a third aspect, the application provides application of the optogenetic system for promoting MSC homing to damaged liver in the preparation of a product for treating acute severe autoimmune hepatitis.

[0022] Beneficial effects: (1) The application designs a light-controlled CXCR4 release plasmid for overexpressing CXCR4, which not only overcomes the shortcomings of high carcinogenic risk, complicated operation and low cost-effectiveness caused by using virus vectors, liposomes, nanotechnology and low-intensity pulsed ultrasound, but also can realize the spatiotemporal regulation of in vivo protein expression;

[0023] (2) The UCNPs designed in the application have good biocompatibility and low toxicity, and the introduction of Gd 3+ to improve the conversion efficiency of UVB, the method for synthesizing the UCNPs is simple, convenient, strong in repeatability, low in technical requirement, strong in universality, high in flexibility and easy to mass-produce;

[0024] (3) The optogenetic system prepared in the application can effectively enhance the homing effect of MSCs on the CXCR4-SDF1 signal by transfecting the light-controlled CXCR4 release plasmid into MSCs, and then irradiating the liver region using NIR after in vitro UVB irradiation or intravenous injection of UCNPs, thereby further strengthening the ability of MSCs to treat acute liver injury. BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 is a schematic diagram of a light-controlled CXCR4 release plasmid;

[0026] Figure 2 is a schematic diagram of the characterization of improved UCNPs: wherein Figure A and Figure B are the emission spectra of NaYF4:Yb:Tm and NaYbF4:Tm:Gd under 980 nm excitation, respectively, Figure C is the energy transfer process and upconversion mechanism of NaYF4:Yb:Tm and NaYbF4:Tm:Gd, Figure D is a transmission electron microscope (TEM) image of NaYbF4:Tm:Gd, Figure E is a scanning transmission electron microscope (STEM) image of NaYbF4:Tm:Gd (scale bar: 100 nm), Figure F is an X-ray diffraction (XRD) pattern of NaYbF4:Tm:Gd, Figure G to Figure K are energy dispersive X-ray spectroscopy (EDS) element mapping of NaYbF4:Tm:Gd, Figure L is an EDS spectrum of NaYbF4:Tm:Gd (scale bar: 100 nm), and Figure M is a TEM image of SiO2@UCNPs (scale bar: 50 nm);

[0027] Figure 3 is a schematic diagram of the biological safety verification of UCNPs: wherein Figure A is the H&E staining image of the heart, liver, spleen, lung and kidney tissues of mice after intravenous injection of different concentrations of UCNPs for 28 days (scale bar: 100 μm, n=5), Figure B is the body weight change curve of healthy mice treated with different doses of UCNPs during the 28-day observation period (n=5), Figure C and Figure D are the representative results of blood routine and liver and kidney function of healthy mice after treatment with different concentrations of UCNPs for 28 days (n=5), Figure E is the effect of different concentrations of UCNPs on the viability of MSC cells (n=3), Figure F is the dynamic distribution of UCNPs in the liver at different time points (n=5), and Figure G is the distribution of UCNPs in the heart, liver, spleen, lung and kidney of mice (n=5); the data are expressed as mean ± standard error (SEM), *p <0.05, ***p <0.001 (t test);

[0028] Figure 4 is a schematic diagram of functional verification of targeting CXCR4 / SDF1 axis to promote MSC migration, wherein A1 is a graph of SDF1 protein level in liver tissue (n=3), A2 is a quantification of SDF1 protein level in liver tissue (n=3), B1 is a graph of verification of overexpression of CXCR4 in MSC by Western blot (n=3), B2 is a quantification of verification of overexpression of CXCR4 in MSC by Western blot (n=3), C1 is a graph of verification of overexpression of CXCR4 in MSC by immunofluorescence analysis (scale bar: 50 μm, n=4), C2 is a quantification of verification of overexpression of CXCR4 in MSC by immunofluorescence analysis (scale bar: 50 μm, n=4), D1 is an evaluation of the role of CXCR4-SDF1 axis in MSC migration by Transwell migration experiment (scale bar: 100 μm, n=5), D2 is a quantification of the role of CXCR4-SDF1 axis in MSC migration by Transwell migration experiment (scale bar: 100 μm, n=5), E1 is the distribution of MSC in AS-AIH mice (n=4), and E2 is a quantification of the distribution of MSC in AS-AIH mice (n=4); data are expressed as mean ± standard error (SEM), *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 (t test);

[0029] Figure 5 is a schematic diagram of regulation of MSC cell membrane CXCR4 expression by light-controlled CXCR4 release plasmid under in vitro UVB irradiation, wherein A is WB analysis of CXCR4 expression on MSC cell membrane under the condition with or without UVB irradiation (n=3) and a quantification thereof, B is fluorescence detection showing intracellular distribution of EYFP-CXCR4 after UVB irradiation (scale bar: 10 μm) and a quantification thereof, C is verification of overexpression of CXCR4 in MSC by immunofluorescence analysis (scale bar: 50 μm, n=4) and a quantification thereof, D is evaluation of the ability of light-controlled CXCR4 release system to enhance MSC migration toward SDF1 by Transwell migration experiment (scale bar: 100 μm, n=5) and a quantification thereof, E is distribution of MSC transfected with different light-controlled CXCR4 release plasmids in AS-AIH mice under the condition with or without UVB irradiation (n=4) and a quantification thereof; data are expressed as mean ± standard error (SEM), *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 (t test);

[0030] Figure 6 is a schematic diagram of the regulation of MSC cell membrane CXCR4 expression by light-controlled CXCR4 release plasmid under the irradiation of rays emitted by UCNPs (converted from NIR) in an in vitro experiment: wherein A is a WB analysis of the expression of CXCR4 on the MSC cell membrane under the premise of the presence or absence of UCNPs converting NIR (n=3) and its quantification chart, B is a fluorescence detection showing the intracellular distribution of EYFP-CXCR4 after UVB irradiation (scale: 10 μm) and its quantification chart, C is a verification of the overexpression of CXCR4 in MSC by immunofluorescence analysis (scale: 50 μm, n=4) and its quantification chart, D is a Transwell migration experiment to evaluate the effect of UCNPs combined with light-controlled CXCR4 release plasmid in enhancing MSC migration towards SDF-1 (scale: 100 μm, n=5) and its quantification chart, E is the distribution of MSC transfected with different light-controlled CXCR4 release plasmids in AS-AIH mice under the condition of with or without NIR irradiation (n=4) and its quantification chart; the data is expressed as mean ± standard error (SEM), *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 (t test);

[0031] Figure 7 is a schematic diagram of the effect of the regulation of cell membrane CXCR4 expression by light-controlled CXCR4 release plasmid under the irradiation of rays emitted by UCNPs (converted from NIR) in an in vivo experiment on the therapeutic effect of MSC on AS-AIH: wherein A1 is a representative image of H&E staining showing the histological morphology of liver tissue (scale: 100 μm, n=4), A2 is a quantification chart of A1, B1 is a TUNEL experiment showing the proportion of apoptotic hepatocytes in liver tissue (scale: 100 μm, n=4), B2 is a quantification chart of B1, C1 is a representative immunofluorescence staining image showing the expression of P-MLKL in liver macrophages (scale: 100 μm, n=4), C2 is a quantification chart of C1, D1 is a representative immunohistochemical staining image showing the proportion of Ki67 positive hepatocytes in liver tissue (scale: 100 μm, n=4), D2 is a quantification chart of D1. DETAILED DESCRIPTION

[0032] The application is described in detail below with reference to the accompanying drawings and specific examples: The MSC used in the following examples was extracted from mouse bone marrow; cyclohexane, 1-hexanol, acetonitrile, ytterbium (III) chloride hexahydrate (YbCl3·6H2O), yttrium chloride hexahydrate (YCl3·6H2O), thulium (III) chloride hexahydrate (TmCl3·6H2O), gadolinium (III) chloride hexahydrate (GdCl3·6H2O), ammonium fluoride (NH4F), and sodium hydroxide (NaOH) were purchased from Aladdin (Shanghai, China); Triton X-100, oleic acid, 1-octadecene, and NIR-797 were purchased from Sigma-Aldrich (St. Louis, MO, USA); tetraethoxysilane (TEOS) was purchased from J&K Scientific (Beijing, China); ammonium hydroxide, acetic acid (HAc), acetonitrile (ACN), methanol, and ethanol were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China).

[0033] Example 1: Synthesis of light-controlled CXCR4 release plasmid

[0034] CXCR4 was designed at the N-terminus of the light-controlled plasmid (VSVG-YFP-2xUVR8, from Chen et al J Cell Biol. 2013 May 13;201(4):631-40. doi), followed by a Furin cleavage site (amino acid sequence SARNRQKR) and two UVR8 repeat sequences in turn. On the basis of this plasmid, EYFP was connected at the C-terminus of CXCR4 to track its movement in cells. The final light-controlled CXCR4 release plasmid map is shown in Figure 1.

[0035] Example 2: Characterization of improved UCNPs

[0036] The preparation method of NaYbF4: Tm: Gd UCNP is as follows:

[0037] First, the preparation of NaYF4: Yb: Tm UCNP was carried out according to the method disclosed in the literature (Gnanasammandhan, M.K., Idris, N.M., Bansal, A., Huang, K., and Zhang, Y. (2016). Near-IR photoactivation using mesoporous silica–coated NaYF4:Yb,Er / Tm upconversion nanoparticles. Nature Protocols 11, 688-713. 10.1038 / nprot.2016.035.), specifically:

[0038] A 100 mL three-necked round bottom flask was charged with 747 mL of a 1 M aqueous solution of YCl3-6H2O, 250 mL of a 1 M aqueous solution of YbCl3-6H2O, and 100 mL of a 0.03 M aqueous solution of TmCl3-6H2O, heated to 120 °C with stirring at 350 rpm to evaporate the water, approximately 1.5 hours, to obtain mixture A;

[0039] To mixture A was added 6 mL of oleic acid and 15 mL of 1-octadecene, and heated to 160 °C for 3 hours until a clear and homogeneous solution B was formed;

[0040] The heating mantle was removed, and to solution B, which was cooled to 60 °C, was added dropwise 5 mL of a methanolic solution containing 0.1 g of NaOH and 0.148 g of NH4F, and heated to 110 °C for 1 hour to evaporate the residual water and methanol to obtain mixture C;

[0041] The flask was placed in a spherical heating mantle, ensuring a tight fit, and was sealed with a glass stopper, connected to a two-tube condenser, and mixture C was kept under vacuum for 10 minutes; a two-way piston valve cycled the atmosphere of the tubes between vacuum and argon three times (1 minute each). Then, the flask was filled with argon and the temperature was increased to 300 °C at a heating rate of 10 °C / min for 1 hour to obtain mixture D;

[0042] The heating mantle was removed, and to solution B, which was cooled to 60 °C, was added dropwise 5 mL of a methanolic solution containing 0.1 g of NaOH and 0.148 g of NH4F, and heated to 110 °C for 1 hour to evaporate the residual water and methanol to obtain mixture C;

[0043] Second, the preparation of NaYbF4: Tm: Gd UCNP is based on the introduction of Gd to NaYF4: Yb: Tm UCNP 3+ The preparation of NaYbF4: Tm: Gd UCNP is the same as the preparation of NaYF4: Yb: Tm UCNP, except that the types and proportions of reagents are different, i.e., a 100 mL three-necked round bottom flask was charged with 0.795 μL of a 1 M aqueous solution of YCl3-6H2O, 0.167 μL of a 0.03 M aqueous solution of TmCl3-6H2O, and 0.2 μL of a 1 M aqueous solution of GdCl3-6H2O, and the subsequent operations were the same as those in the preparation of NaYF4: Yb: Tm UCNP.

[0044] Into a 25 mL halogen flask equipped with a Teflon-coated magnetic bar, 1.77 g Triton X-100, 1.6 mL 1-hexanol, 4 mL cyclohexane solution of NaYbF4: Tm: Gd UCNP, 2.5 mL 1-hexanol, 480 μL deionized water and 100 μL ammonia were added, and stirred at 700 rpm for 30 min at 25 °C to form a reverse microemulsion; 1 mL cyclohexane containing 50 μL tetraethoxysilane (TEOS) was added to the reverse microemulsion, and stirred at 700 rpm overnight at 25 °C; SiO2@UCNP was released from the microemulsion by adding acetone, and centrifuged at 4000 rpm for 30 min, and SiO2@UCNP was separated from the mixed solution, and the obtained SiO2@UCNP was washed with ethanol and water for three times respectively to obtain washed SiO2@UCNP; then, the washed SiO2@UCNP was dispersed in 10 mL eluent (acetonitrile: H2O: acetic acid = 50:49:1 (v / v)) at room temperature for 1 h to remove the surfactant; after removing the surfactant, the solid particles were collected by centrifugation at 4000 rpm for 30 min, and washed with water and ethanol for three times respectively to obtain SiO2-embedded upconversion nanoparticles.

[0045] FIG. 2, panels A and B are emission spectra of NaYF4: Yb: Tm and NaYbF4: Tm: Gd under 980 nm excitation, panel C is energy transfer process and upconversion mechanism of NaYF4: Yb: Tm and NaYbF4: Tm: Gd, panel D is a transmission electron microscope (TEM) image of NaYbF4: Tm: Gd, panel E is a scanning transmission electron microscope (STEM) image of NaYbF4: Tm: Gd (scale bar: 100 nm), panel F is an X-ray diffraction (XRD) pattern of NaYbF4: Tm: Gd, panels G-K are energy dispersive X-ray spectroscopy (EDS) element distribution maps of NaYbF4: Tm: Gd, panel L is an EDS spectrum of NaYbF4: Tm: Gd (scale bar: 100 nm), and panel M is a TEM image of SiO2@UCNP (scale bar: 50 nm); indicating that the improved UCNPs (i.e., SiO2-embedded NaYbF4: Tm: Gd UCNP) are successfully synthesized.

[0046] Example 3: verification of biological safety of improved UCNPs (i.e., SiO2-embedded NaYbF4: Tm: Gd UCNP)

[0047] 1) In vivo biological safety experiment: 0, 10, 20 and 30 mg / kg of modified UCNPs were injected into mice through the tail vein, respectively, and 28 days after intravenous injection, H&E staining of heart, liver, spleen, lung and kidney tissues of mice, weight change curve and hematology and biochemical analysis were detected, the results are shown in Figure 3 A-D: modified UCNPs at a concentration of 30 mg / kg still did not cause obvious biological toxicity, therefore, 10 mg / kg was selected as the safe concentration of modified UCNPs for in vivo use.

[0048] 2) In vitro experiment: after co-culturing MSCs with 0, 12.5, 25, 50, 100, 200, 400 and 800 μg / mL of modified UCNPs for 24 h, the effect of modified UCNPs on MSC cell viability was detected by CCK8 method, the results are shown in Figure 3E: the use concentration of modified UCNPs in cell culture is less than 100 μg / mL, which has less toxicity to cells, therefore, 100 μg / mL is selected as the use concentration of modified UCNPs in in vitro culture.

[0049] 3) The distribution of modified UCNPs in vivo was detected by using a live imaging instrument, NIR797 was used to label modified UCNPs to detect the distribution of modified UCNPs in mice after tail vein injection and the time of reaching the liver. The results are shown in Figure 3F-G: after injection, modified UCNPs mainly accumulated in the liver, and reached the peak at about 24 h.

[0050] Example 4: Verification of whether targeting CXCR4 / SDF1 axis can promote MSC migration

[0051] 1) MSC pretreatment: divided into three groups, namely NC group, AMD3100 treatment group and OE-CXCR4 group, MSC was cultured in low glucose (1 g / L) DMEM containing 10% FBS (26010074, Gibco), supplemented with penicillin-streptomycin mixture (100 U / mL, 100 μg / mL; 15070063, Gibco), cells were cultured in a humidified environment at 37 ℃, 5% CO2 to about 50% confluence, as NC group; AMD3100 treatment group: to inhibit the binding ability of CXCR4 and SDF1, part of the MSC in the NC group was pretreated with AMD3100 (10 μM, HY-10046, MedChemExpress) for 6 hours; OE-CXCR4 group: by lentivirus transfection to induce overexpression of CXCR4 in part of the MSC in the NC group and using WB (shown in Figure B1-B2 in Figure 4) and immunofluorescence (immunofluorescence, IF) (shown in Figure C1-C2 in Figure 4) methods to detect confirmed lentivirus transfection success, and achieved overexpression of CXCR4 in MCS.

[0052] 2) Transwell co-culture system was used to evaluate the role of CXCR4-SDF1 axis in regulating MSC migration: 5 × 10⁵ MSCs were seeded in the upper chamber (8 μm pore size membrane, 3428, Corning), and SDF1 (200 ng / mL, HY-P7285, MedChemExpress) was added to the lower chamber medium. After 24 hours of culture, the MSCs on the reverse side of the filter membrane were stained with crystal violet, and the cell migration efficiency was evaluated by observing six randomly selected fields under a microscope (Leica DMi8). The results are shown in Figure D1-D2 in Figure 4: AMD3100 inhibition or lentivirus overexpression of CXCR4 significantly affected the migration ability of MSCs targeting SDF1.

[0053] 3) Detection of MSC homing ability to liver by small animal imaging: AS-AIH model was induced by intravenous injection of Concanavalin A (ConA) via tail vein, which is the most commonly used method to establish acute immune-mediated liver injury model in mice. In addition, to ensure the disease to occur rapidly and cause high mortality within a few hours, a higher dose of ConA (30 mg / kg, C2010, Sigma-Aldrich) than that commonly used in chronic or mild acute liver injury models was used. After the model was established, WB was used to detect the synthesis of SDF1 in liver tissue during AS-AIH. To study whether targeting the CXCR4-SDF1 axis can enhance the homing of MSCs to the damaged liver and thus improve the therapeutic effect, 2 × 106 MSCs were injected into mice via tail vein at 1 hour after ConA injection. The distribution of MSCs in vivo was detected by NIR-labeled MSCs on the live imaging instrument to analyze their homing ability to the liver. The results are shown in Figures A1-A2 and E1-E2 in Figure 4: the synthesis of SDF1 in liver tissue during AS-AIH increased, and overexpression of CXCR4 significantly improved the homing ability of MSCs into the liver.

[0054] Example 5: Detection of the effect of the UVB-UVR8-based optogenetic system on the expression of CXCR4 on the cell membrane of MSCs

[0055] 1) Light-controlled CXCR4 release plasmid transfection: according to the use method of Lipo8000 TM The corresponding dose of Lipo and the light-controlled CXCR4 release plasmid synthesized in Example 1 (Lipo:DNA = 2 μL:1 μg) was added to the MSC culture medium for transfection according to the use method of Lipo8000

[0056] 2) After 24h transfection, MSCs transfected with the light-controlled CXCR4 release plasmid were irradiated by a UVB light source (ENB-280C, Spectronics) for a total of 10s. After 2h irradiation, the cell membranes of MSCs transfected with the light-controlled CXCR4 release plasmid were isolated and the protein content of CXCR4 was detected by WB method, and the results are shown in Figure 5A: it is confirmed that the UVB irradiation successfully induced the activation of the light-controlled protein and the release of CXCR4 in MSCs. The migration of EYFP-CXCR4 at 0, 60min and 120min was detected by confocal microscope, respectively, and the results are shown in Figure 5B: the dynamic migration of YFP further indicates that under non-irradiation conditions, CXCR4 mainly accumulates in the intracellular, and irradiation can trigger the dissociation of the light-controlled protein and the release of CXCR4. The expression of CXCR4 in MSCs transfected with the light-controlled CXCR4 release plasmid after UVB irradiation was detected by IF method, and the results are shown in Figure 5C: UVB irradiation successfully induced the activation of the light-controlled protein and the release of CXCR4 in MSCs. The migration of MSCs transfected with the light-controlled CXCR4 release plasmid after UVB irradiation was evaluated by Transwell co-culture system and small animal imaging, and the method was the same as Example 4, and the results are shown in Figure 5D and Figure 5E: after UVB activation, the in vitro and in vivo migration ability of MSCs targeting SDF1 was greatly improved.

[0057] Example 6: Detection of the effect of improved UCNPs combined with light-controlled CXCR4 release plasmid on the expression of CXCR4 in the cell membrane of MSCs

[0058] 1) Light-controlled CXCR4 release plasmid transfection: according to the use method of Lipo8000 TM According to the use method of Lipo8000 (Biyuntian, C0533), the corresponding dose of Lipo and the light-controlled CXCR4 release plasmid synthesized in Example 1 (Lipo:DNA=2μL:1μg) were added to the MSC culture medium for transfection, and after 24h, the fresh culture medium was replaced and the concentration of 100 μg / mL improved UCNPs was added and mixed.

[0059] 2) MSCs were irradiated with 980 nm NIR laser (Leopetics). After 2h irradiation, MSC cell membranes were isolated and the protein content of CXCR4 was detected using WB method, the results are shown in Figure 6A: it is confirmed that the improved UCNPs conversion light also successfully induced the activation of light-controlled proteins and the release of CXCR4 in MSCs. The migration of EYFP-CXCR4 at 0, 60min, 120min was detected using confocal microscope, the results are shown in Figure 6B: the dynamic migration of YFP further indicates that under non-irradiation conditions, CXCR4 mainly aggregates in the intracellular, and the improved UCNPs conversion light can also trigger the dissociation of light-controlled proteins and the release of CXCR4. The expression of CXCR4 in MSC cells after UVB irradiation was detected by IF method, the results are shown in Figure 6C: the improved UCNPs conversion light irradiation successfully induced the activation of light-controlled proteins and the release of CXCR4 in MSCs. The migration of MSCs after UVB irradiation was evaluated by Transwell co-culture system and small animal imaging, the method is the same as Example 4, the results are shown in Figure 6D and Figure 6E: the improved UCNPs conversion light irradiation can also greatly improve the in vitro and in vivo migration ability of MSCs targeting SDF1.

[0060] Example 7: Detection of the effect of UCNPs combined with light-controlled CXCR4 release plasmid on promoting MSC homing to treat AS-AIH

[0061] The improved UCNPs (10 mg / kg) were injected into the mice through the tail vein, and ConA and MSC were injected into the tail vein 23 h and 24 h after the injection of the improved UCNPs, respectively, which can ensure that most of the improved UCNPs have reached the liver at the time of NIR irradiation. After most of the improved UCNPs reached the liver (about 24 h), the mouse liver area was irradiated in vitro using a 980 nm NIR laser, with a single irradiation time of 20 min, an interval of 1 h, and a total of 3 times. The mice were sacrificed 12 h after the injection of MSC, and H&E staining was used to detect the tissue morphology of the liver tissue (results shown in FIG. 7, panels A1-A2). TUNEL experiments showed the proportion of apoptotic hepatocytes in the liver tissue (results shown in FIG. 7, panels B1-B2). IF detected the expression of P-MLKL in macrophages in the liver (results shown in FIG. 7, panels C1-C2). Immunohistochemical staining detected the proportion of Ki67-positive hepatocytes in the liver tissue (results shown in FIG. 7, panels D1-D2). The results showed that the homing ability of MSCs regulated by targeting CXCR4 significantly affected its therapeutic effect; AMD3100 pretreatment significantly interfered with the ability of MSCs to inhibit liver tissue necrosis, hepatocyte apoptosis, macrophage necrotic apoptosis, and promote liver regeneration, while overexpression of CXCR4 showed enhanced therapeutic effect. In addition, the improved UCNPs combined with light-controlled CXCR4 release plasmid showed stronger therapeutic effect than direct overexpression of CXCR4 in vitro through the spatiotemporal regulation effect.

[0062] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An optogenetic system to promote MSCs homing to the injured liver as a target, characterized in that: The light-controlled CXCR4 release plasmid comprises a CXCR4 at the N-terminal of the light-controlled plasmid, a Furin cleavage site and two UVR8 repeat sequences connected in sequence after the CXCR4; and the upconversion nanoparticle is NaYbF4: Tm: Gd UCNP.

2. The optogenetic system for facilitating MSCs homing to the damaged liver of claim 1, wherein: The C-terminal of the CXCR4 in the light-controlled CXCR4 release plasmid is connected with EYFP to track the movement of the CXCR4 in the cell.

3. A method of preparing the optogenetic system for promoting MSCs to target and home to a damaged liver according to claim 1 or 2, characterized in that, The method comprises the following steps: S1, synthesis of the light-controlled CXCR4 release plasmid; S2, preparation of the SiO2-embedded upconversion nanoparticle; S3, preparation of the optogenetic system.

4. The method of claim 3, wherein: The process of the SiO2-embedded upconversion nanoparticle is as follows: 1.77 g of Triton X-100, 1.6 mL of 1-hexanol, 4 mL of a cyclohexane solution of NaYbF4: Tm: Gd UCNP, 2.5 mL of 1-hexanol, 480 μL of deionized water and 100 μL of ammonia water are added to a 25 mL halogen bottle provided with a Teflon-coated magnetic stirrer, and stirred at 700 rpm at 25°C for 30 minutes to form a reverse microemulsion; 1 mL of cyclohexane containing 50 μL of tetraethoxysilane is added to the reverse microemulsion, and stirred at 700 rpm at 25°C overnight; SiO2@UCNP is released from the microemulsion by adding acetone, and centrifuged at 4000 rpm for 30 minutes to separate SiO2@UCNP from the mixed solution; the obtained SiO2@UCNP is washed with ethanol and water three times to obtain washed SiO2@UCNP; then, the washed SiO2@UCNP is dispersed in 10 mL of an eluent (acetonitrile:H2O:acetic acid=50:49:1 (v / v)) at room temperature for 1 hour to remove the surfactant; after removing the surfactant, the solid particles are collected by centrifugation at 4000 rpm for 30 minutes, and washed with water and ethanol three times, respectively, to obtain the SiO2-embedded upconversion nanoparticle.

5. The preparation method according to claim 4, characterized in that, The preparation method of the NaYbF4: Tm: Gd UCNP is as follows: First, the preparation of NaYF4: Yb: Tm UCNP is carried out according to the existing method, specifically as follows: 747 mL of 1 M YCl3·6H2O, 250 mL of 1 M YbCl3·6H2O and 100 mL of 0.03 M TmCl3·6H2O aqueous solution are added to a 100 mL three-necked round-bottom flask, heated to 120°C under stirring at 350 rpm to evaporate water, about 1.5 hours, to obtain a mixture A; 6 mL of oleic acid and 15 mL of 1-octadecene are added to the mixture A, and heated to 160°C for 3 hours until a transparent and uniform solution B is formed; The heating mantle was removed, 5 mL of methanol containing 0.1 g of NaOH and 0.148 g of NH4F was added dropwise to solution B cooled to 60°C, and heated to 110°C for 1 hour to evaporate the residual water and methanol to obtain mixture C; The flask was placed in a spherical heating mantle, ensuring a tight fit, and was sealed with a glass stopper, connected to a double-tube condenser, and mixture C was kept under vacuum for 10 minutes; the two-way piston valve cycled the atmosphere of the line between vacuum and argon three times (1 minute each). Then, the flask was filled with argon and the temperature was raised to 300°C at a heating rate of 10°C / min for 1 hour to obtain mixture D; The heating mantle was removed, mixture D was allowed to cool to room temperature, mixture D was transferred to a 50 mL centrifuge tube, and acetone was added to the centrifuge tube to a total volume of 40 mL, the centrifuge tube was centrifuged at 6654 x g for 10 minutes at room temperature, and the supernatant was discarded; the precipitate was dissolved in 20 mL of cyclohexane and vortexed, then centrifuged at 1000 x g for 5 minutes, and the supernatant containing NaYbF4: Tm: Gd UCNP was transferred to a 20 mL glass scintillation vial to obtain a cyclohexane solution of NaYbF4: Tm: Gd UCNP; Secondly, the preparation of NaYbF4: Tm: Gd UCNP is based on NaYF4: Yb: Tm UCNP with the introduction of Gd 3+ The preparation method of NaYbF4: Tm: Gd UCNP is the same as that of NaYF4: Yb: Tm UCNP, except that the types and proportions of reagents are different, i.e. 0.795 μL of 1 M YCl3•6H2O, 0.167 μL of 0.03 M TmCl3•6H2O and 0.2 μL of 1 M GdCl3•6H2O aqueous solution are added into a 100 mL three-necked round-bottom flask, and the subsequent operations are the same as those in the preparation method of NaYF4: Yb: Tm UCNP.

6. The preparation method according to claim 3, characterized in that, The preparation process of the optogenetic system is as follows: the light-controlled CXCR4 release plasmid is combined with NaYbF4: Tm: Gd UCNP according to the use requirement, wherein the light-controlled CXCR4 release plasmid is used for MSC transfection, and the NaYbF4: Tm: Gd UCNP is used for in vitro culture or in vivo experiment.

7. Use of the optogenetic system for promoting MSC targeted homing to damaged liver according to claim 1 or claim 2 in the preparation of a product for treating acute severe autoimmune hepatitis.

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