CN111265549B

CN111265549B - Surface PD-L1 molecule over-expressed mesenchymal stem cell membrane coated bionic nanoparticle and preparation and application thereof

Info

Publication number: CN111265549B
Application number: CN202010137688.3A
Authority: CN
Inventors: 汪超; 沈淑芳
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2022-03-01
Anticipated expiration: 2040-03-02
Also published as: CN111265549A; WO2021174738A1

Abstract

The invention relates to a bionic nanoparticle coated by mesenchymal stem cell membranes and with a surface over-expressed by PD-L1 molecules, and preparation and application thereof. The bionic nanoparticle coated by the mesenchymal stem cell membrane with the surface over-expressed by the PD-L1 molecule comprises a nano core and the mesenchymal stem cell membrane coated outside the nano core; the nanometer core comprises a polymer with biocompatibility, and PD-L1 molecules are over-expressed on the membrane surface of the mesenchymal stem cell membrane. The invention also discloses application of the mesenchymal stem cell membrane-coated bionic nanoparticle with the surface over-expressed by the PD-L1 molecule in preparation of an inflammation treatment preparation. The bionic nano-particles have obvious immunosuppressive effect, good biocompatibility and simple and mature preparation process, can be used for preparing inflammation treatment preparations, can be effectively enriched at inflammation parts, and show excellent curative effect in inflammation treatment by inhibiting over-activated immunoreaction of the inflammation parts and cytokine storm.

Description

Surface PD-L1 molecule over-expressed mesenchymal stem cell membrane coated bionic nanoparticle and preparation and application thereof

Technical Field

The invention relates to the field of biomedical materials, in particular to a bionic nanoparticle coated with a mesenchymal stem cell membrane and overexpressed by a surface PD-L1 molecule, and preparation and application thereof.

Background

Inflammation is a pathological reaction mainly preventing the body from harmful stimuli such as various physics, chemistry, biology, and the like, and is a complex reaction in which various cells and factors participate together, wherein the complex reaction comprises various immune cells and cell factors in the immune system. Moderate inflammatory responses help the body to recover health, while excessive inflammatory responses can lead to tissue damage and even death. The nature of this high inflammatory response state is in fact an overactivated immune response, inducing cytokine storms, which in turn leads to severe inflammatory injury. The pathogenesis of cytokine storm is complex, but progresses rapidly and mortality is extremely high. Therefore, in the treatment of critical acute inflammation, the prevention of the production of the cytokine storm as early as possible is the key to relieve the body injury and save the life of the patient. Currently, in the treatment of such diseases, such as SARS-Cov and COVID-19, in addition to supporting therapy clinically, large doses of hormonal drugs, for example, are used to suppress the body's immune response and help the patient to pass the cytokine storm. However, although the hormone medicine with large dose can effectively inhibit immune response, serious sequelae such as femoral head necrosis and the like can be caused, and the life quality of the patient after recovery is greatly influenced. Therefore, the search for an effective new method for treating the inflammation with small toxic and side effects has great significance for promoting the development of modern medicine and benefiting patients.

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells having self-renewal and multipotent differentiation capabilities, and are currently widely used for the treatment of various diseases, such as organ transplantation, tissue repair, and the like. MSCs have been widely used in cell-based therapies, from basic research to clinical trials. Safety and efficacy have been well documented in a number of clinical trials, particularly in immune-mediated inflammatory diseases such as Graft Versus Host Disease (GVHD) and Systemic Lupus Erythematosus (SLE). MSCs play a positive role mainly in two ways: i.e., immunomodulating effect and differentiation ability. MSCs can secrete a number of cytokine types that mediate immune modulation by paracrine secretion or direct interaction with immune cells. The immune modulatory effects of MSCs are further triggered by the activation of TLR receptors in MSCs, which are stimulated by pathogen-associated molecules (e.g., LPS or double-stranded RNA of the HCoV-19 virus). In addition to the properties of stem cells, MSCs have immunomodulatory capacity, which can affect both innate and adaptive immune responses. They can interact with the immune system and exert anti-inflammatory or pro-inflammatory effects. Thus, mesenchymal stem cell therapy is considered to be the most potential treatment for inflammation at present, and has been used for the treatment of various immune-related diseases, including systemic lupus erythematosus, hepatitis, and the like, with good therapeutic effects. However, as a living cell, MSC has a complicated culture method, requires professional personnel, is inconvenient to transport, is not suitable for long-term storage, and has a treatment mechanism which is not particularly clear. Moreover, it is widely believed that MSCs play a major inhibitory role in regulating immunity, and thus their role in the progression of tumor development is also controversial. For example, it is known that MSC promotes the development and metastasis of tumors by secreting bioactive molecules and thus promoting immune tolerance in the tumor microenvironment. These controversies have somewhat limited the clinical use of MSCs in the treatment of inflammation.

It is noted that although many scholars believe that inhibition of immune cell proliferation and activation of MSCs is achieved by secretion of soluble inhibitory factors, there are reports that inactivated MSCs still have immunomodulatory properties, suggesting that cellular membranes of MSCs have immunosuppressive ability. Moreover, PD-L1 molecule is expressed on the surface of MSC cell membrane, which can be combined with immunosuppressive molecule PD-1 on the surface of immunocyte to transmit inhibitory signal to immunocyte, so as to achieve the effect of inhibiting the activation of immunocyte.

CN201910740365.0 discloses an anti-inflammatory targeted delivery system, which is formed by using platelet-derived vesicles and anti-inflammatory drugs loaded in the platelet-derived vesicles or attached to the surface of the platelet-derived vesicles. CN201910166781.4 discloses a tumor antigen presenting system prepared from erythrocyte membranes, and discloses that the antigen presenting system is fused with tumor cell membranes to be used for preparing antitumor drugs. In the above techniques, a substance derived from a biological cell is used as a carrier, and the source thereof is limited and the treatment method is complicated. US2012/039411, WO2013/052167 and CN103857387A disclose membrane-encapsulated nanoparticles, in which cell-derived cell membranes or virus-derived membranes are coated on the surface of inner cores of non-cellular substances, but the immunosuppressive effects of these membrane-encapsulated nanoparticles are poor, and methods of use thereof.

Therefore, in order to avoid disputes and reduce side effects, it would be a safer, more convenient and more effective strategy to develop a preparation based on cell membranes of MSCs highly expressed by PD-L1 for use in the treatment of inflammation, but there are few reports of such preparations.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a bionic nanoparticle coated with a mesenchymal stem cell membrane with a surface over-expressed by a PD-L1 molecule, and preparation and application thereof.

The first object of the invention is to provide a bionic nanoparticle coated with mesenchymal stem cell membrane with surface over-expressed PD-L1 molecule, which comprises a nano-core and mesenchymal stem cell membrane coated outside the nano-core; the nano-core comprises a polymer with biocompatibility, and the membrane surface of the mesenchymal stem cell membrane overexpresses PD-L1 molecules.

Further, the polymer having biocompatibility is selected from one or more of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone, polylysine, polyglutamic acid, and Dextran (Dextran). Preferably, the biocompatible polymer is PLGA. The PLGA nanometer core is used as a nanometer carrier which is widely applied, and the biological safety is high.

Further, the molecular weight of the biocompatible polymer is 10-1000 kDa. More preferably, the molecular weight of the biocompatible polymer is 10-100 kDa. By adopting the molecular weight in the range, on one hand, the nano-size structure can be ensured to be generated, and on the other hand, the stroked cell nucleus can be ensured to generate enough supporting effect on the mesenchymal stem cell membrane.

Furthermore, the particle size of the bionic nano-particles is 50-1000 nm. Preferably, the biomimetic nanoparticles have a particle size of 50-300 nm.

Further, the mass ratio of total protein in the nano-core and the mesenchymal stem cell membrane is not more than 1. Preferably, the mass ratio of total protein in the nanonuclear and mesenchymal stem cell membrane is 1: 2.

In the bionic nano-particles, the nano-core is used as a carrier and a bracket of the mesenchymal stem cell membrane, so that the stability of the particle size and the form of the nano-core is maintained, and the aggregation or the breakage of the cell membrane of the bionic nano-particles in the use process is prevented. Secondly, a nano-sized structure can be generated, and the inflammation part can be effectively targeted. The PD-L1 molecule is overexpressed on the membrane surface of the mesenchymal stem cell membrane, so that the bionic nano-particles inherit the membrane biological characteristics of the mesenchymal stem cell and have immunosuppressive capability, and meanwhile, as the PD-L1 molecule is overexpressed on the membrane surface of the mesenchymal stem cell, the PD-L1 molecule is interacted with the PD-1 molecule on the surface of the immune cell, so that the immunosuppressive effect of the bionic nano-particles is obviously enhanced. The bionic nanoparticle has high biocompatibility, uses the inactivated mesenchymal stem cell membrane, avoids using living cells, not only eliminates the safety problem caused by a living cell storage medium, but also avoids the cancer promotion dispute of mesenchymal stem cell inclusion and secretion, is convenient to store, is convenient to use clinically, does not need a complicated separation culture process of the stem cells, can be used in time, has relatively low cost, and has the possibility of long-distance transportation. Secondly, the nano-sized particles are easily enriched on the inflammation part, the targeting effect is improved, and the side effect of the whole body is avoided by effectively aiming at the inflammation part.

The second purpose of the invention is to provide a preparation method of the bionic nanoparticle coated with the mesenchymal stem cell membrane and overexpressed by the PD-L1 molecule on the surface, which comprises the following steps:

mixing the nano-core and the vesicle of the mesenchymal stem cell membrane, and repeatedly extruding by a homogenizer to obtain the bionic nano-particles, wherein the nano-core comprises a biocompatible polymer, and the membrane surface of the mesenchymal stem cell membrane overexpresses PD-L1 molecules.

Further, mixing is performed such that the mass ratio of total protein in the nanonuclear and mesenchymal stem cell membrane is not more than 1. Preferably, the mixing is performed in a mass ratio of total protein in the nanonuclear and mesenchymal stem cell membrane of 1: 2.

Further, a 400nm filter membrane is used in the extrusion process, after the resistance is obviously reduced, the filter membrane with the thickness of 200nm is used for continuous extrusion, and the bionic nano-particles coated by the mesenchymal stem cell membrane and with the surface over-expressed by the PD-L1 molecule can be obtained.

Further, the preparation method of the vesicle of the mesenchymal stem cell membrane comprises the following steps:

(1) resuspending the washed mesenchymal stem cells with the membrane surface over-expressing PD-L1 molecules in a cell disruption solution, performing ultrasonic treatment until no obvious particles exist in the system, and centrifugally separating out precipitates in a cell disruption mixture to obtain cell membranes;

(2) and extruding the cell membrane through the membrane by using a homogenizer to obtain the vesicle of the mesenchymal stem cell membrane. The preparation process for extracting and coating the cell membrane is simple and mature.

Further, in the step (1), the mesenchymal stem cells with the membrane surface over-expressing PD-L1 molecule are washed by PBS for three times.

Further, in the step (1), ice blocks are added during the ultrasonic process to keep the temperature low.

Further, in the step (2), a 400nm filter membrane is used firstly during membrane passing, and after the resistance is obviously reduced, the filter membrane is used for continuous extrusion, so that uniform vesicles of the mesenchymal stem cell membrane can be obtained.

Further, the method for over-expressing the PD-L1 molecule on the surface of the mesenchymal stem cell membrane comprises the following steps:

mesenchymal stem cells were cultured normally for 24 hours in a medium supplemented with 100ng/mL gamma interferon. The method for inducing the PD-L1 overexpression is safe and convenient.

Preferably, the method for overexpressing the PD-L1 molecule on the surface of the mesenchymal stem cell membrane comprises the following steps:

(1) normally culturing the mesenchymal stem cells in an incubator to allow the mesenchymal stem cells to adhere to the wall;

(2) removing cell culture supernatant, changing to a fresh culture medium containing 100ng/mL gamma interferon, and culturing in an incubator for 24 hours;

(3) and after 24 hours, removing cell culture supernatant, and washing the cells for three times by using PBS (phosphate buffer solution), thus obtaining the mesenchymal stem cells with the surface PD-L1 molecule over-expressed.

Further, the extraction method of the mesenchymal stem cell membrane overexpressed by the PD-L1 molecule comprises the following steps: scraping the cells from the culture dish, suspending the cells in a cell disruption solution, and performing ultrasonic treatment to obtain a cell disruption mixture; the mixture was centrifuged at 4000rpm for 10 minutes, the supernatant was collected and centrifuged at 14800rpm for an additional 20 minutes to obtain a cell membrane pellet which was resuspended in clean PBS for use.

Further, the preparation method of the nano-core comprises the following steps:

dissolving a biocompatible polymer in an organic solvent, slowly dripping the obtained solution into a PVA aqueous solution, and sufficiently mixing after ultrasonic treatment so as to solidify the biocompatible polymer into nano particles, thus obtaining the nano core.

Preferably, the biocompatible polymer is PLGA, the organic solvent is DMSO, and the therapeutic fraction of the aqueous PVA solution is 5%.

Preferably, the volume ratio of DMSO to 5 wt% aqueous PVA solution is 1: 3.

Preferably, the mixing is carried out fully by adopting a stirring method, and the mixing time is 12-24 h.

Further, the step of centrifuging to separate the nano-cores is also included after the sufficient mixing.

The third purpose of the invention is to disclose the application of the bionic nanoparticle coated with the mesenchymal stem cell membrane with the surface PD-L1 molecule over-expressed in the invention in preparing an inflammation treatment preparation.

Further, the inflammation includes inflammation of systemic organs, joints or tissues such as brain, heart, liver, spleen, lung, kidney and intestine. The inflammation is hyperacute, acute, subacute or chronic inflammation.

Preferably, the inflammation is hepatitis or pneumonia.

Preferably, the inflammation is caused by an acute or chronic immune response by invasion of the human body by microorganisms such as bacteria, fungi, viruses, spirochetes, mycoplasmas, rickettsias, chlamydia, parasites (protozoa, helminths, medical insects) and all foreign antigens.

Further, the administration form of the inflammation therapeutic agent is intravenous injection.

Further, the inflammation therapeutic agent includes an inflammation therapeutic drug or a drug carrier. Namely, the bionic nano-particles can be directly used as a medicament for treating inflammation, and can also be used as a medicament carrier for treating inflammation, and the bionic nano-particles are loaded with other small molecular medicaments for treating inflammation.

On the basis of the immunosuppressive capability of the mesenchymal stem cell membrane, the large-scale expression of PD-L1 molecules on the membrane surface obviously enhances the immunosuppressive effect of the bionic nanoparticles, and shows excellent curative effect in inflammatory treatment by inhibiting over-activated immunoreaction of inflammatory parts and cytokine storm.

By the scheme, the invention at least has the following advantages:

(1) the bionic nano-particles have the advantages of simple and mature preparation process, high biological safety and good targeting property of inflammation parts, and can be effectively enriched to the inflammation parts.

(2) The extraction method of the mesenchymal stem cell membrane is simple and rapid, and the membrane has an immunosuppressive effect, so that side effects possibly caused by bioactive molecules secreted and intracytoplasmic of the mesenchymal stem cell are avoided.

(3) After the mesenchymal stem cell membrane with the immune regulation function in the bionic nano-particles is expressed in a large amount in PD-L1, the immune suppression effect is obviously enhanced, the PD-L1 overexpressed on the surface of the mesenchymal stem cell membrane interacts with PD-1 on the surface of an immune cell, the immune suppression effect of the membrane is obviously enhanced, the over-activated immune reaction of an inflammation part can be obviously suppressed, and the generation of cytokine storm is inhibited, so that the inflammatory injury is effectively relieved.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood and to be implemented according to the content of the specification, the following description is made with reference to the preferred embodiments of the present invention and the detailed drawings.

Drawings

FIG. 1 is a flow analysis result of PD-L1 expression after the mesenchymal stem cells are induced by gamma interferon, wherein 1 is an unstained negative control, 2 is a common mesenchymal stem cell without gamma interferon treatment, and 3 is a mesenchymal stem cell treated by gamma interferon;

FIG. 2 is a graph showing the result of Western blot analysis of the expression amounts of PD-L1 of a PD-L1 overexpressed mesenchymal stem cell membrane and a normal mesenchymal stem cell membrane not treated with gamma interferon used in the present invention, wherein 1 is the normal mesenchymal stem cell membrane not treated with gamma interferon and 2 is the PD-L1 overexpressed mesenchymal stem cell membrane used in the present invention;

FIG. 3 is an appearance of a bionic nanoparticle coated by a mesenchymal stem cell membrane overexpressed by PD-L1 molecule in the invention;

FIG. 4 is a particle size distribution plot of biomimetic nanoparticles of the present invention;

FIG. 5 is a graph showing the particle size change of PD-L1 molecule over-expressed mesenchymal stem cell membrane coated bionic nanoparticles and PLGA nano-core in water (FIG. 5b) and PBS (FIG. 5 a);

FIG. 6 is a graph of the effect of PD-L1 molecule over-expressed mesenchymal stem cell membrane coated biomimetic nanoparticles on macrophage cell polarization phenotype in vitro;

FIG. 7 shows the effect of the PD-L1 molecule over-expressed mesenchymal stem cell membrane coated biomimetic nanoparticles of the present invention on inflammatory factors TNF-alpha (FIG. 7a) and IL-6 (FIG. 7b) secreted by macrophage cells in vitro;

FIG. 8 is a graph of the effect of the PD-L1 molecule over-expressed mesenchymal stem cell membrane coated biomimetic nanoparticles of the present invention on the immunophenotype of a mixture of macrophage and T cells in vitro;

FIG. 9 shows the effect of the PD-L1 molecule over-expressed mesenchymal stem cell membrane-coated biomimetic nanoparticles of the present invention on the secretion of inflammatory factors TNF- α (FIG. 9a) and IL-6 (FIG. 9b) in a mixture of in vitro Multimacrophage cells and T cells;

FIG. 10 shows the fluorescence imaging analysis results of the distribution of PD-L1 molecule over-expressed mesenchymal stem cell membrane coated bionic nanoparticles in hepatitis mice;

FIG. 11 is a confocal analysis of the interaction of biomimetic nanoparticles coated with cell membranes of mesenchymal stem cells overexpressed by PD-L1 molecule of the present invention with lymphocytes in the liver of hepatitis mice;

FIG. 12 is a liver immune flow cytometry of PD-L1 molecule over-expressed mesenchymal stem cell membrane coated biomimetic nanoparticles of the invention after intravenous injection into hepatitis mice for 5 hours;

FIG. 13 shows the results of the phenotypic analysis of the liver immunocytes of FIG. 12;

FIG. 14 shows the levels of inflammatory factors TNF- α (FIG. 14a), IL-6 (FIG. 14b) and IL-1 β (FIG. 14c) in liver tissue after 5 hours of intravenous injection of the mesenchymal stem cell membrane-coated biomimetic nanoparticles overexpressing PD-L1 molecule in hepatitis mice according to the present invention;

FIG. 15 shows the results of HE liver slices of a liver of a bionic nanoparticle coated with a PD-L1 molecule overexpressed mesenchymal stem cell membrane, which is intravenously injected into a hepatitis mouse for 5 hours;

FIG. 16 shows the results of four indicators of liver function in serum of PD-L1 molecule overexpressed mesenchymal stem cell membrane-coated biomimetic nanoparticles of the present invention after intravenous injection into a hepatitis mouse for 5 hours;

FIG. 17 shows the effect of PD-L1 molecule over-expressed mesenchymal stem cell membrane coated biomimetic nanoparticles on survival rate of hepatitis mice;

in FIGS. 6-9, 1 represents a negative control group without any treatment, 2 represents a positive control group stimulated with LPS but without treatment, 3 represents a PLGA group (PLGA nanonuclear treatment after LPS stimulation), 4 represents a normal membrane group (bionic nanoparticle coated with normal mesenchymal stem cell membrane after LPS stimulation), and 5 represents a PD-L1 overexpression membrane group (bionic nanoparticle coated with PD-L1 overexpression mesenchymal stem cell membrane after LPS stimulation); in fig. 10 and 11, 1 represents the free cy5.5 group, 2 represents the cy5.5 labeled PD-L1 overexpressed mesenchymal stem cell membrane coated biomimetic nanoparticles group; in FIGS. 12 to 17, 1 indicates a negative control group (healthy mice), 2 indicates a positive control group (LPS/D-GalN-stimulated, hepatitis mice), 3 indicates a PLGA group (PLGA nanocore was administered after LPS/D-GalN-stimulated), 4 indicates a common membrane group (ordinary mesenchymal stem cell membrane-coated biomimetic nanoparticles were administered after LPS/D-GalN-stimulated), and 5 indicates a PD-L1 overexpression membrane group (PD-L1-overexpressed mesenchymal stem cell membrane-coated biomimetic nanoparticles were administered after LPS/D-GalN-stimulated).

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The material sources in the embodiments of the invention are:

mesenchymal stem cells are extracted from mouse bone marrow and purified and expanded. Macrophages were extracted from the abdominal cavity of mice and T cells were extracted from the spleen of mice.

C57BL/6 mice, 5-8 weeks old, were purchased from Calvens laboratory animals, Inc., Changzhou. Mice were treated according to the protocols of the institute for Biochemical and cellular laboratory animal Care (IACUC).

Gamma interferon IFN-. gamma.315-05-100 was purchased from PeproTech. Polylactic-co-glycolic acid Polymer (PLGA) was purchased from Sigma-Aldrich.

Example 1: induction of overexpression of PD-L1 molecule on surface of mesenchymal stem cell

(1) And (5) normally culturing the purified and amplified mesenchymal stem cells in an incubator to adhere to the walls.

(2) The cell culture supernatant was removed and replaced with fresh medium containing 100ng/mL gamma interferon, and the culture was continued in an incubator for 24 hours.

(3) After 24 hours, the cell culture supernatant was removed, and the cells were washed three times with PBS and digested with pancreatin to obtain cells.

The expression level of PD-L1 in the obtained cells was analyzed by flow cytometry, and the results showed that the expression level of PD-L1 in the gamma interferon-treated mesenchymal stem cells was significantly increased (fig. 1).

Example 2: extraction and characterization of PD-L1 overexpressed mesenchymal stem cell membranes

(1) And (3) harvesting the mesenchymal stem cells treated by the gamma interferon, suspending the mesenchymal stem cells in a cell disruption solution, and performing ultrasonic treatment to obtain a cell disruption mixture.

(2) And (3) centrifuging the mixture in the step (1) at 4000rpm for 10 minutes, collecting supernatant, and continuing to centrifuge at 14800rpm for 20 minutes to obtain cell membrane precipitates for later use.

(3) The protein precipitated by the membrane in the step (2) is extracted by using a membrane protein extraction reagent, and is subjected to western blotting analysis, and the result shows that compared with the mesenchymal stem cell membrane without being treated by the gamma interferon, the signal of PD-L1 on the mesenchymal stem cell membrane treated by the gamma interferon is obviously stronger, which indicates the overexpression of PD-L1 (figure 2).

Example 3: preparation and characterization of PD-L1 overexpressed mesenchymal stem cell membrane coated biomimetic nanoparticles

(1) And (3) resuspending the harvested mesenchymal dry cell membrane overexpressed by the PD-L1 molecule in PBS, performing ultrasonic treatment until no obvious particles exist in the system, and adding ice blocks to keep the temperature low in the ultrasonic treatment process.

(2) Repeatedly extruding the membrane suspension obtained in the step (1) by using a homogenizer, firstly using a 400nm filter membrane, and continuously extruding by using a 200nm filter membrane after the resistance is obviously reduced to obtain uniform nano plasma membrane vesicles.

(3) And (3) uniformly mixing the nano plasma membrane vesicle obtained in the step (2) with the prepared PLGA nano core according to the mass ratio of the total amount of the membrane protein to the PLGA of 2: 1.

(4) Repeatedly extruding the mixture obtained in the step (3) by using a homogenizer, firstly using a 400nm filter membrane, and continuously extruding by using a 200nm filter membrane after the resistance is obviously reduced to obtain the PD-L1 overexpressed mesenchymal stem cell membrane coated bionic nanoparticles.

Wherein, in the step (3), the preparation method of the PLGA nano-core is as follows:

dissolving 10mgPLGA polymer (molecular weight is 44kDa) powder in 1mL DMSO, slowly dropping the DMSO solution dissolved with PLGA into the 5 wt% PVA aqueous solution according to the volume ratio of 1:3 of the DMSO to the 5 wt% PVA aqueous solution, stirring overnight after ultrasonic treatment, and performing high-speed centrifugation to obtain the PLGA nano core.

Microscopic imaging of the biomimetic nanoparticles obtained in step (4) was performed using Transmission Electron Microscope (TEM), demonstrating successful coating of the membrane of PD-L1-overexpressed mesenchymal stem cells on PLGA nanocore (fig. 3).

The particle size distribution of the biomimetic nanoparticles obtained in step (4) was measured using Dynamic Light Scattering (DLS) and the results are shown in fig. 4. The result shows that the particle size of the bionic nano-particles prepared by the method is mainly distributed between 70 nm and 200 nm.

And (4) testing the stability of the bionic nanoparticles and the single PLGA nano cores obtained in the step (4), and respectively re-suspending the bionic nanoparticles and the single PLGA nano cores in water and PBS solution. Fig. 4 shows that within two weeks of standing, the bionic nanoparticles and the PLGA nanocore have no obvious particle size mutation in two mediums, which indicates that no obvious agglomeration occurs in the two mediums and the stability is good.

Example 4: in vitro immunosuppressive effect of PD-L1-overexpressed mesenchymal stem cell membrane-coated biomimetic nanoparticles

(1) After C57BL/6 mice were sacrificed, peritoneal macrophages and spleen T cells were aseptically extracted and cultured in vitro in an incubator.

(2) Firstly, uniformly paving the macrophages extracted in the step (1) in a 12-pore plate for culture, and setting 5 groups, namely a negative control group, a positive control group, a PLGA group, a common membrane group and a PD-L1 overexpression membrane group. After the adhesion, all the original culture solution except the negative control group was discarded, and a fresh medium containing 1. mu.g/mL LPS was added to continue the culture. After 24 hours, removing the original culture solution, adding a fresh culture medium, respectively adding 10 mu g of PLGA nano-core, 20 mu g of bionic nano-particles coated by common mesenchymal dry cell membranes with the total protein amount and 20 mu g of bionic nano-particles coated by mesenchymal dry cell membranes with the total protein amount, and adding the bionic nano-particles coated by the mesenchymal dry cell membranes with the total protein amount of PD-L1 into the PLGA group, the common membrane group and the PD-L1 overexpression membrane group, and co-incubating the bionic nano-particles with macrophages. Macrophage and cell culture supernatants were collected after 24 hours.

(3) Measuring the polarized phenotype of the macrophage in the step (2) by using flow cytometry, and as a result, as shown in fig. 6, the stimulation of the LPS leads to the increase of the expression of CD80 on the surface of the macrophage, which indicates that the macrophage is polarized to M1 type and shows a proinflammatory phenotype; the PLGA nano core has little influence on macrophage phenotype, the bionic nano particles coated by the common mesenchymal stem cell membrane inhibit the increase of CD80 to a certain extent, and the bionic nano particles coated by the mesenchymal stem cell membrane overexpressed by PD-L1 obviously inhibit the polarization of macrophage cells to M1 type, which shows that the immunosuppression effect of the mesenchymal stem cell membrane is greatly enhanced by the overexpression of PD-L1.

(4) And (3) detecting the levels of inflammatory factors TNF-alpha and IL-6 in the cell culture supernatant in the step (2) by using an ELISA kit. As shown in FIG. 7, stimulation with LPS causes macrophages to secrete large amounts of inflammatory factors such as TNF- α and IL-6; the PLGA nanophase core has little influence on the level of inflammatory factors, the bionic nanoparticles coated by the common mesenchymal stem cell membrane reduce the secretion amount of the two inflammatory factors to a certain extent, and the bionic nanoparticles coated by the mesenchymal stem cell membrane overexpressed by PD-L1 obviously inhibit the secretion of the two factors, which shows that the overexpression of PD-L1 greatly enhances the immune suppression and anti-inflammatory capability of the mesenchymal stem cell membrane.

(5) And (2) uniformly paving the macrophages in the step (1) in a 12-pore plate for culture, and setting 5 groups, namely a negative control group, a positive control group, a PLGA group, a common membrane group and a PD-L1 overexpression membrane group. After their adherence, equal amounts of T cells were added to all groups and co-cultured. Meanwhile, the stock culture was completely discarded except for the negative control group, and fresh medium containing 1. mu.g/mL of LPS was added to continue the culture. After 24 hours, removing the original culture solution, adding a fresh culture medium, respectively adding 10 mu g of PLGA nano-core, 20 mu g of bionic nano-particles coated by a common mesenchymal stem cell membrane with the total protein amount and 20 mu g of bionic nano-particles coated by a mesenchymal stem cell membrane with the total protein amount, and adding PD-L1 overexpressed bionic nano-particles coated by a mesenchymal stem cell membrane with the total protein amount into the PLGA group, the common membrane group and the PD-L1 overexpression membrane group, and co-incubating the bionic nano-particles with two types of immune cells. Macrophages, T cells and cell culture supernatant were collected after 24 hours.

(6) Detecting immunophenotype of macrophages and T cells in the step (5) by using flow cytometry, and as a result, as shown in FIG. 8, the stimulation of LPS leads to the increase of the expression of CD80 on the surfaces of the macrophages, which indicates that the macrophages are polarized to M1 type and show proinflammatory phenotype, and leads to the increase of the expression of CD69 and PD-1 on the surfaces of the T cells, which indicates that the T cells are activated; the PLGA nanophase core hardly influences the phenotypes of two cells, the bionic nanoparticles coated by the common mesenchymal stem cell membrane inhibit the macrophage from being activated to a pro-inflammatory phenotype, namely M1 type, and inhibit the activation of T cells to a certain extent, but the bionic nanoparticles coated by the PD-L1 overexpressed mesenchymal stem cell membrane significantly inhibit the macrophage from being polarized to M1 type, and also strongly and effectively inhibit the activation of the T cells, which shows that the overexpression of the PD-L1 greatly enhances the immunosuppressive effect of the mesenchymal stem cell membrane.

(7) And (3) detecting the levels of inflammatory factors TNF-alpha and IL-6 in the cell culture supernatant in the step (5) by using an ELISA kit. As shown in FIG. 9, stimulation with LPS dramatically increased the levels of inflammatory factors such as TNF-. alpha.and IL-6; similarly, the PLGA nanophase core has little influence on the level of inflammatory factors, the bionic nanoparticles coated by the common mesenchymal stem cell membrane slightly reduce the secretion amount of the two inflammatory factors, and the bionic nanoparticles coated by the mesenchymal stem cell membrane overexpressed by PD-L1 obviously reduce the level of the two inflammatory factors, which indicates that the overexpression of PD-L1 greatly enhances the immunosuppressive and anti-inflammatory abilities of the mesenchymal stem cell membrane.

Example 5: inflammation targeting ability of PD-L1 overexpressed mesenchymal stem cell membrane coated biomimetic nanoparticles

(1) C57BL/6 mice were weighed and randomly divided into 2 groups, free cy5.5 and cy5.5 labeled biomimetic nanoparticles, respectively. A hepatitis model was established by intraperitoneal injection of LPS (100. mu.g/kg) and D-GalN (1.25g/kg) based on the body weight of the mice.

(2) Injecting bionic nanoparticles coated by the membrane of the free Cy5.5 and Cy5.5 marked PD-L1-overexpressed mesenchymal stem cell into the hepatitis mouse in the step (1) through veins, carrying out in-vivo tracing by using a small animal imager at 1, 2, 3, 4 and 5 hours after injection, killing the mouse after 5 hours, carrying out dissection, and detecting the distribution condition of the nanoparticles in each main organ. As shown in fig. 10, although the signals of nanoparticles are also present in other organs, the biomimetic nanoparticles are mainly enriched in the liver of hepatitis mice, and it can be seen that the nanoparticles have the inflammation targeting ability.

The liver harvested in step (2) was sectioned, in which the lymphocyte marker CD45 was immunofluorescent-stained, the nucleus was stained with DAPI, and the sections were subsequently observed using a zeiss confocal microscope. As can be seen from fig. 11, the PD-L1 overexpressed mesenchymal stem cell membrane-coated biomimetic nanoparticles were significantly enriched in liver and were spatially very close to CD45 labeled lymphocytes, suggesting that a close interaction between them may occur.

Example 6: effect analysis of PD-L1 over-expressed mesenchymal stem cell membrane coated bionic nanoparticles on hepatitis treatment

(1) C57BL/6 mice were weighed and randomly divided into 5 groups, namely a negative control group, a positive control group, a PLGA group, a common membrane group and a PD-L1 overexpression membrane group. In addition to the negative control group, other groups were made to model hepatitis by intraperitoneal injection of LPS (100. mu.g/kg) and D-GalN (D-galactosamine, 1.25g/kg) based on the body weight of mice.

(2) After the hepatitis model is successfully established, intravenous administration treatment is immediately carried out. Wherein the negative control group is not treated, the positive control group is given with PBS (the volume is about 200 mu L) with the same amount as the control, and the other three groups are respectively given with 20 mu g PLGA nanometer nucleus, 40 mu g total protein amount of bionic nanoparticles coated by common mesenchymal stem cell membranes and 40 mu g total protein amount of bionic nanoparticles coated by mesenchymal stem cell membranes and PD-L1 over-expressed by total protein amount.

(3) After 5 hours of dosing, mice were sacrificed and serum and liver samples were collected.

And (3) further processing the liver sample harvested in the step (3), taking a proper amount of liver tissue to crush the liver tissue into a single cell suspension, and then performing immunophenotyping on macrophages and T cells in the liver tissue by using flow cytometry, wherein the macrophage cells are stained by CD45, F4/80, CD80 and PD-1, and the T cells are stained by CD45, CD3, CD44, CD69 and PD-1. The results show that compared with the negative control group, the hepatitis mice of the positive control group have a large number of lymphocyte infiltrates in the liver, macrophages are remarkably polarized to M1 proinflammatory phenotype, and T cells also present a high activation state; similar to the in vitro results, the PLGA nanophase core has little influence on the phenotypes of the two immune cells, and the bionic nanoparticles coated by the common mesenchymal stem cell membrane prevent macrophages from activating to a pro-inflammatory phenotype, namely M1 type, to a certain extent and suppress the activation state of T cells, but the bionic nanoparticles coated by the mesenchymal stem cell membrane overexpressed by PD-L1 remarkably inhibit the polarization of the macrophages to M1 type and strongly and effectively inhibit the activation of the T cells, which indicates that the immunosuppressive action of the mesenchymal stem cell membrane is greatly enhanced by the overexpression of PD-L1 (figure 12).

Taking a proper amount of liver tissues harvested in the step (3), grinding and cracking to obtain liver tissue lysate, and detecting the levels of inflammatory factors TNF-alpha, IL-6 and IL-1 beta by using an ELISA kit. As shown in FIG. 14, the levels of inflammatory factors such as TNF-. alpha., IL-6 and IL-1. beta. were sharply increased in liver tissues of hepatitis mice of the positive control group as compared with healthy mice of the negative control group; similarly, PLGA nanophase core has little influence on the level of the inflammatory factors, the bionic nanoparticles coated by common mesenchymal stem cell membrane slightly reduce the secretion amount of the inflammatory factors, and the bionic nanoparticles coated by the mesenchymal stem cell membrane overexpressed by PD-L1 very effectively reduce the level of the inflammatory factors, which shows that the overexpression of PD-L1 obviously enhances the immunosuppressive and anti-inflammatory capability of the mesenchymal stem cell membrane and inhibits the outbreak of cytokine storm.

And (4) taking a proper amount of liver tissues harvested in the step (3) for H & E staining, and performing pathological observation. According to the slicing result, compared with the negative control group, the hepatitis mice in the positive control group have a large amount of inflammatory cell infiltration in the liver, and a large amount of liver cells are in a broken state, so that the liver is seriously damaged; PLGA nanoparticies had little effect on inflammatory cell infiltration in liver and liver injury, normal mesenchymal stem cell membrane-coated biomimetic nanoparticles slightly reduced inflammatory cells infiltrating liver, while PD-L1-overexpressed mesenchymal stem cell membrane-coated biomimetic nanoparticles significantly inhibited inflammatory cell infiltration in liver, but did not appear to have significant rescue effect on liver cell disruption (fig. 15).

Determining four indexes of liver function on the serum sample obtained in the step (3), wherein the result shows that compared with a negative control group, the ALT, AST and TBA levels in the serum of the hepatitis mouse of the positive control group are increased sharply, the ALB level is obviously reduced, and the liver function is seriously damaged; the PLGA nano-core and the bionic nano-particles coated by the common mesenchymal stem cell membrane have little influence on the four liver function indexes, while the bionic nano-particles coated by the mesenchymal stem cell membrane overexpressed by the PD-L1 obviously recover the liver function, which suggests the rescue effect of the PD-L1 overexpression of the invention for inhibiting the burst of the cytokine on the liver function (figure 16).

Example 7: effect of PD-L1-overexpressed mesenchymal stem cell membrane-coated biomimetic nanoparticles on survival rate of hepatitis mice

(1) C57BL/6 mice were weighed and randomly divided into 4 groups, positive control group, PLGA group, common membrane group and PD-L1 overexpression membrane group. A hepatitis model was established by intraperitoneal injection of LPS (100. mu.g/kg) and D-GalN (1.25g/kg) based on the body weight of the mice.

(2) After the hepatitis model is successfully established, intravenous administration treatment is immediately carried out. Wherein, the positive control group is given with equal amount of PBS (volume about 200 μ L) as control, and the other three groups are respectively given with corresponding 20 μ g PLGA nano-core, 40 μ g total protein amount of bionic nano-particles coated by common mesenchymal stem cell membrane, and 40 μ g total protein amount of bionic nano-particles coated by PD-L1 over-expressed mesenchymal stem cell membrane.

The treatment effect is detected, and the survival time and survival rate of the mice are recorded. As shown in figure 17, except for the PD-L1 overexpression membrane group, all the hepatitis mice in other groups died within 8 hours, and the survival time and survival rate of the mice with the PD-L1 overexpression membrane group are improved, which indicates that the bionic nanoparticles coated by the PD-L1 overexpression mesenchymal stem cell membranes have a good treatment effect on acute hepatitis induced by LPS and D-GalN.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. Use of a mesenchymal stem cell membrane-coated biomimetic nanoparticle with surface over-expressed by PD-L1 molecule in preparation of a therapeutic preparation for inflammation, wherein the inflammation comprises inflammation of heart, liver, spleen, and lung; the bionic nanoparticle comprises a nano-core and a mesenchymal stem cell membrane wrapped outside the nano-core; the nano-core comprises a polymer with biocompatibility, and the membrane surface of the mesenchymal stem cell membrane overexpresses a PD-L1 molecule; the mesenchymal stem cell membrane is derived from mesenchymal stem cells which are treated by gamma interferon so that the surface of the membrane excessively expresses PD-L1 molecules.

2. The use according to claim 1, wherein the inflammation therapeutic agent is administered by intravenous injection.

3. Use according to claim 1, characterized in that: the polymer with biocompatibility is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, polyglycolic acid, polycaprolactone, polylysine, polyglutamic acid and glucan.

4. Use according to claim 1, characterized in that: the biocompatible polymer has a molecular weight of 10-1000 kDa.

5. Use according to claim 1, characterized in that: the particle size of the bionic nano-particles is 50-1000 nm.

6. Use according to claim 1, characterized in that: the preparation method of the bionic nanoparticle coated by the mesenchymal stem cell membrane with the surface PD-L1 molecule over-expressed comprises the following steps:

mixing the nano-core and the vesicle of the mesenchymal stem cell membrane, and repeatedly extruding by a homogenizer to obtain the bionic nano-particle, wherein the nano-core comprises a polymer with biocompatibility, and the membrane surface of the mesenchymal stem cell membrane excessively expresses a PD-L1 molecule.

7. Use according to claim 6, characterized in that: the preparation method of the vesicle of the mesenchymal stem cell membrane comprises the following steps:

(1) resuspending and crushing the washed mesenchymal stem cells with the membrane surface over-expressing PD-L1 molecules in a cell crushing solution, and separating out precipitates in a cell crushing mixture to obtain cell membranes;

(2) and extruding the cell membrane through a membrane by using a homogenizer to obtain the vesicle of the mesenchymal stem cell membrane.

8. The use according to claim 6, characterized in that the preparation method of the nanocore comprises the following steps:

dissolving a biocompatible polymer in an organic solvent, slowly dripping the obtained solution into a PVA aqueous solution, carrying out ultrasonic treatment, and then fully mixing to ensure that the biocompatible polymer is solidified into nano particles, thus obtaining the nano core.