Gel composition and application thereof in preparation of medicine for treating limb ischemia

Abstract

The invention discloses a gel composition and application thereof in preparation of medicines for treating limb ischemia, the gel composition comprises mesenchymal stem cells and a gel carrier containing selenium, the gel carrier comprises a copolymer of PLGA and PEG, and in the gel carrier, the concentration of selenium is greater than 0 [mu] M and not greater than 2 [mu] M. The gel composition has a good prospect in application to preparation of medicines for treating limb ischemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine industry, and in particular to a gel composition and application thereof in preparing a medicine for treating limb ischemia. Background Art

[0002] Critical limb ischemia (CLI) is a stubborn disease accompanied by long-term inflammation, insufficient angiogenesis and tissue necrosis, which seriously affects the quality of life of patients and may even lead to amputation and death. Vascular reconstruction is the most effective treatment, but it is not suitable for all patients.

[0003] Mesenchymal stem cells (MSCs) are a type of multipotent stem cells with the potential for self-renewal and multidirectional differentiation. They are widely present in various tissues throughout the body, such as bone marrow, fat, umbilical cord and placenta. Due to their unique biological characteristics, they are considered to be a potential cell source for the treatment of limb ischemia. Existing studies have shown that MSCs have unique application value in the treatment of limb ischemia.

[0004] However, when MSCs are directly injected into the target area to treat limb ischemia, decreased MSC activity and low retention rate at the injection site after transplantation may occur, limiting the therapeutic effect of MSCs. Summary of the invention

[0005] The present disclosure is made in view of the above-mentioned prior art conditions, and its purpose is to provide a gel composition and its application in preparing a drug for treating limb ischemia.

[0006] To this end, the first aspect of the present disclosure provides a gel composition, including mesenchymal stem cells and a gel carrier containing selenium, wherein the gel carrier includes a copolymer of PLGA and PEG, and in the gel carrier, the concentration of selenium is greater than 0 μM and not greater than 2 μM. In the first aspect of the present disclosure, the use of a carrier gel to carry mesenchymal stem cells can control the release of MSCs, and improve the retention rate of MSCs after the gel composition is applied to the target area; and the selenium-containing gel carrier can improve the survival and immunoregulatory ability of MSCs under oxidative stress, protect the activity of MSCs, and help enhance the therapeutic effect of MSCs, especially the therapeutic effect on limb ischemia.

[0007] In the gel composition involved in the first aspect of the present disclosure, optionally, the mesenchymal stem cells are selected from any one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, human placenta mesenchymal stem cells and amniotic fluid mesenchymal stem cells.

[0008] In the gel composition involved in the first aspect of the present disclosure, optionally, the gel composition is liquid within a first temperature range and gelatinous within a second temperature range, the first temperature range is less than 33° C., and the second temperature range is not less than 33° C. In this case, the gel composition is a thermosensitive hydrogel, which can be easily injected at room temperature, gels after entering the body, and is suitable for clinical application.

[0009] In the gel composition involved in the first aspect of the present disclosure, optionally, the gel carrier encapsulates the mesenchymal stem cells. In this case, the gel carrier can be used to protect the activity of MSCs.

[0010] In the gel composition involved in the first aspect of the present disclosure, optionally, the gel carrier has a three-dimensional porous network structure, and the selenium is encapsulated in the three-dimensional porous network structure. In this case, selenium can be effectively maintained in the application environment, and selenium can be inhibited from being lost or migrated during the application process, thereby helping the gel composition to stably exert its efficacy.

[0011] In the gel composition involved in the first aspect of the present disclosure, optionally, the copolymer of PLGA and PEG is a PLGA-PEG-PLGA triblock copolymer, thereby facilitating the construction of a thermosensitive hydrogel and making the gel carrier have good biocompatibility and biodegradability.

[0012] In the gel composition involved in the first aspect of the present disclosure, optionally, in the gel carrier, the mass fraction of the copolymer of PLGA and PEG is 15%-25%, thereby making it easy to obtain a gel carrier with expected performance.

[0013] The second aspect of the present disclosure provides an application of a gel composition in preparing a drug for treating limb ischemia, wherein the gel composition is the gel composition described in any one of the first aspect of the present disclosure. In the present disclosure, the effect of the gel composition in treating limb ischemia is verified, and the gel composition can be used to prepare a drug for treating limb ischemia.

[0014] In the application involved in the second aspect of the present disclosure, optionally, the selenium in the gel composition induces GPX4 expression by regulating the transcription factor SP1 to protect the mesenchymal stem cells from oxidative damage.

[0015] In the application involved in the second aspect of the present disclosure, optionally, the gel composition inhibits macrophage M1 polarization and promotes macrophage M2 polarization, thereby accelerating angiogenesis and blood perfusion recovery in ischemic limbs.

[0016] According to the present disclosure, a gel composition and its application in preparing a medicine for treating limb ischemia can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a test result diagram about selenium involved in the present invention.

[0018] Figure 2 This is a test result diagram related to the regulatory factors of selenium on GPX4 transcription involved in the present invention.

[0019] Figure 3 This is a test result diagram of the selenium-containing hydrogel involved in the present invention.

[0020] Figure 4 This is a graph showing test results related to the biocompatibility of the selenium-containing hydrogel involved in the present invention.

[0021] Figure 5 This is a test result diagram of the selenium-containing hydrogel involved in the present invention acting on MSCs.

[0022] Figure 6 This is a diagram showing the test results of the selenium-containing hydrogel-encapsulated MSCs on mice. DETAILED DESCRIPTION

[0023] Hereinafter, the preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same symbols are assigned to the same components, and repeated descriptions are omitted. In addition, the accompanying drawings are only schematic diagrams, and the ratio of the dimensions of the components or the shapes of the components may be different from the actual ones.

[0024] It should be noted that the terms "including" and "having" and any variations thereof in the present invention, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] In addition, the subheadings and the like involved in the following description of the present invention are not intended to limit the content or scope of the present invention, but are merely used as a reminder for reading. Such subheadings should neither be understood as being used to divide the content of the article, nor should the content under the subheading be limited to the scope of the subheading.

[0026] Abbreviations and their interpretations of the proper nouns involved in this disclosure:

[0027] MSC / MSCs: Mesenchymal stem cell / cells;

[0028] PBS: Phosphate-buffered saline;

[0029] PLGA: poly(lactic-co-glycolic acid);

[0030] PEG: polyethylene glycol.

[0031] The first aspect of the present disclosure relates to a gel composition. The gel composition can be used to prepare a drug for treating a disease. In the present disclosure, the gel composition may also be referred to as a "composite hydrogel", "hydrogel composite", "selenium-containing composition", etc.

[0032] Another aspect of the present disclosure relates to the use of a gel composition in the preparation of a drug for treating limb ischemia. The gel composition is the gel composition involved in the first aspect of the present disclosure. In the present disclosure, the effect of the gel composition in treating limb ischemia is verified, and the gel composition can be used to prepare a drug for treating limb ischemia.

[0033] Another aspect of the present disclosure relates to a preparation for treating limb ischemia, comprising the gel composition of the first aspect of the present disclosure. In the present disclosure, the effect of the gel composition in treating limb ischemia is verified, and the gel composition can be used to treat limb ischemia.

[0034] Hereinafter, the gel composition according to various aspects of the present disclosure will be described in detail.

[0035] In some examples, the gel composition may include repair cells and a gel carrier. The gel carrier can protect the repair cells and control the release of the repair cells, and improve the retention rate of the repair cells after the gel composition is applied to the target area. When the gel composition is used for disease treatment, a good therapeutic effect can be obtained, especially when it is used for the treatment of limb ischemia. In the present disclosure, the gel carrier may also be referred to as a hydrogel.

[0036] In some examples, the repair cells may include mesenchymal stem cells and / or functional cells differentiated from mesenchymal stem cells. For example, the functional cells may be osteoblasts, etc. In some examples, preferably, the repair cells may include mesenchymal stem cells. That is, in some examples, preferably, the gel composition may include mesenchymal stem cells. Below, taking the repair cells as mesenchymal stem cells as an example, the gel composition is described in detail.

[0037] In some examples, the gel carrier may contain selenium (Se). In this case, using a selenium-containing carrier gel to carry mesenchymal stem cells can improve the survival and immunomodulatory ability of MSCs under oxidative stress, protect the activity of MSCs, and help enhance the therapeutic effect of MSCs in the target area, especially the therapeutic effect on limb ischemia. In some examples, the gel carrier containing selenium can be called a selenium-containing hydrogel. The activity of MSCs can be protected by the selenium-containing hydrogel.

[0038] In some examples, selenium in the gel composition can protect mesenchymal stem cells from oxidative damage by increasing the transcription mechanism of GPX4. In some examples, selenium in the gel composition can protect MSCs from oxidative damage by regulating the transcription factor SP1 to induce GPX4 expression.

[0039] In some examples, the gel composition can inhibit macrophage M1 polarization and promote macrophage M2 polarization, thereby accelerating angiogenesis and blood perfusion recovery in ischemic limbs. In some examples, the gel composition including mesenchymal stem cells can inhibit macrophage M1 polarization and promote macrophage M2 polarization, thereby accelerating angiogenesis and blood perfusion recovery in ischemic limbs.

[0040] In some examples, mesenchymal stem cells can be selected from any one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, human placental mesenchymal stem cells and amniotic fluid mesenchymal stem cells. In other words, the repair cells can include one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, human placental mesenchymal stem cells and amniotic fluid mesenchymal stem cells. In some examples, preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells. Thus, it can help to improve the therapeutic effect of the gel composition on limb ischemia.

[0041] In some examples, the gel carrier can encapsulate mesenchymal stem cells. In this case, it is convenient to use the gel carrier to protect the activity of MSCs.

[0042] In some examples, the gel composition can be delivered to the target area by injection for treatment. Of course, it can be understood that the gel composition can also be delivered to the target area by other known methods.

[0043] In some examples, the gel carrier may be a thermosensitive hydrogel. That is, in some examples, the gel composition may be a thermosensitive hydrogel. In some examples, the gel composition may be liquid in a first temperature range and gel in a second temperature range. Thus, it is convenient to inject the liquid gel composition, which is in a gel state after injection into the target area, and can limit the diffusion of internal substances (such as selenium and mesenchymal stem cells), and play a long-term protective role on repair cells.

[0044] In some examples, the first temperature range may be less than 33° C. In this case, the gel composition is in a liquid state at room temperature and can be easily injected and used at room temperature. In other words, the gel composition is injectable within the first temperature range. Therefore, at room temperature, the gel composition is injectable.

[0045] In some examples, the second temperature range may be no less than 33° C. The human body temperature is usually around 37° C. In this case, the gel composition can gel after entering the body, limiting the diffusion of internal substances (such as selenium and mesenchymal stem cells) and exerting a long-term protective effect on repair cells.

[0046] In some examples, preferably, the gel composition can be liquid in a first temperature range and gel in a second temperature range, the first temperature range can be less than 33° C., and the second temperature range can be not less than 33° C. In this case, the gel composition is a thermosensitive hydrogel, which can be easily injected at room temperature, gels after entering the body, and is suitable for clinical applications.

[0047] In some examples, the gel carrier may have good biocompatibility and biodegradability, thereby facilitating the gel composition to be placed in a living body for effect.

[0048] In some examples, selenium can be included in the gel composition by adding a selenium-containing substance to the gel carrier. For example, selenium can be included in the gel composition by adding selenite, nano-selenium, or other selenium precursor compounds to the gel carrier. In other words, in some examples, the gel composition can include selenite, nano-selenium, or other selenium precursor compounds.

[0049] In some examples, the selenite may be any one or more of sodium selenite, potassium selenite and ammonium selenite. In some examples, preferably, the selenite may be sodium selenite. In some examples, preferably, the gel composition may include sodium selenite.

[0050] In some examples, in the gel carrier, the concentration of selenium can range from 0 μM to 2 μM. In this case, selecting a suitable concentration can facilitate the protection of repair cells. In some examples, in the gel carrier, the concentration of selenium can be greater than 0 μM and not greater than 2 μM. For example, the concentration of selenium can be 0.1 μM, 0.2 μM, 0.3 μM, 0.5 μM, 0.8 μM, 1 μM, 1.2 μM, 1.5 μM, 1.6 μM, 1.8 μM or 2 μM.

[0051] In some examples, within the range of 0 μM to 2 μM selenium concentration, selenium supplementation has a dose-dependent protective effect on repair cells. That is, within the range of 0 μM to 2 μM, the protective effect of selenium on repair cells increases with increasing concentration.

[0052] In some examples, the gel carrier may have a three-dimensional porous network structure. Thus, the overall stability of the gel composition can be improved. In some examples, selenium may be located in the three-dimensional porous network structure. In this case, selenium can be effectively maintained in the application environment, and selenium can be inhibited from being lost or migrated during the application process, thereby helping the gel composition to stably exert its efficacy. In some examples, the repair cells may be located in the three-dimensional porous network structure. Thus, it is convenient to limit the diffusion of the repair cells by the skeleton polymer, and it is convenient to protect the repair cells.

[0053] In some examples, the gel carrier may include a backbone polymer. In some examples, the backbone polymer may include a copolymer of PLGA and PEG. In other words, in some examples, the gel carrier may include a copolymer of PLGA and PEG. In this case, it is possible to facilitate obtaining a gel carrier with desired performance.

[0054] In some examples, the copolymer of PLGA and PEG can be a PLGA-PEG-PLGA triblock copolymer. In other words, in some examples, the backbone polymer can include a PLGA-PEG-PLGA triblock copolymer. Thus, it is easy to construct a thermosensitive hydrogel while making the gel carrier have good biocompatibility and biodegradability.

[0055] In some examples, the mass fraction of the copolymer of PLGA and PEG can be 15%-25%. For example, the mass fraction of the copolymer of PLGA and PEG can be 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%. Thus, it is easy to obtain a gel carrier with expected performance.

[0056] In some examples, preferably, the gel composition may include mesenchymal stem cells and a gel carrier containing selenium, the gel carrier including a copolymer of PLGA and PEG, and in the gel carrier, the concentration of selenium is greater than 0 μM and not greater than 2 μM. In this case, the use of a carrier gel to carry mesenchymal stem cells can control the release of MSCs, and improve the retention rate of MSCs after the gel composition is applied to the target area; and the selenium-containing gel carrier can improve the survival and immunoregulatory ability of MSCs under oxidative stress, protect the activity of MSCs, and help enhance the therapeutic effect of MSCs, especially the therapeutic effect on limb ischemia.

[0057] As mentioned above, the present disclosure also provides a preparation for treating limb ischemia, including a gel composition. In some examples, the preparation may further include pharmaceutically acceptable excipients.

[0058] In some examples, in addition to preparations for treating limb ischemia, the gel composition can also be used in the preparation of therapeutic drugs for other diseases that need to be delivered to the target area. In some examples, the gel composition can be used as a protective agent for repair cells, and the gel composition can improve the vitality and retention rate of repair cells, thereby improving the treatment effect.

[0059] In summary, according to the present disclosure, a gel composition and its use in preparing a drug for treating limb ischemia can be provided.

[0060] To facilitate understanding of the present invention, the present invention is further explained below with reference to specific embodiments in conjunction with the accompanying drawings, and the specific embodiments do not constitute a limitation on the embodiments of the present invention.

[0061] Those skilled in the art should understand that the drawings are merely schematic diagrams of embodiments, and the components in the drawings are not necessarily necessary for implementing the present invention.

[0062] Experimental methods and procedures

[0063] In the examples disclosed herein, the cell protection ability of selenium on mesenchymal stem cells was evaluated, and a thermosensitive injectable selenium-containing hydrogel was prepared using PLGA-PEG-PLGA triblock copolymer as a cell carrier, and the biocompatibility, biodegradability and cell protection ability of the selenium-containing hydrogel were determined. The beneficial application value of thermosensitive selenium-containing hydrogel encapsulating MSCs for limb ischemia was verified at the cell and animal experimental levels.

[0064] In this embodiment, the test materials used are all conventional test materials in the art and can be purchased through commercial channels.

[0065] method

[0066] Mesenchymal stem cell isolation

[0067] Mesenchymal stem cells were isolated from human umbilical cord. Fresh umbilical cords were washed with PBS on a laminar flow clean bench. The umbilical cord outer membrane and umbilical cord blood vessels were removed to obtain Wharton's jelly. Wharton's jelly was cut into small pieces and cultured in α-MEM medium (Gibco, NY, USA) containing 10% FBS (Gibco) at 37°C, 5% CO2. Cell adhesion and growth were observed, and the medium was changed every 3-4 days. All study participants provided informed consent. The above protocol was approved by the Ethics Committee of Qilu Hospital of Shandong University (Jinan, China).

[0068] Cell viability

[0069] Cell viability was determined using the Cell Counting Kit-8 (CCK-8) (Solarbio, Beijing, China) according to the manufacturer's instructions. 1 × 10 4 The cells were cultured for 30 min and the optical density (OD) at 450 nm was measured.

[0070] Calcein / PI detection kit (Beyotime, Shanghai, China) was used to stain live and dead cells simultaneously. Calcein AM / PI detection solution was prepared according to the manufacturer's protocol. Cells were washed twice with PBS and incubated with Calcein AM / PI detection solution at 37°C for 15 min. Fluorescence was detected using an ECLIPSE Ni fluorescence microscope (Nikon, Tokyo, Japan). Cell counting was performed using ImageJ software.

[0071] Antioxidant capacity assay

[0072] The antioxidant capacity was determined using the ABTS method (Beyotime). 6 The cells were centrifuged at 10000 g for 10 min to obtain the supernatant. 20 μL of the supernatant was incubated with 200 μL of ABTS reagent for 15 min, and the OD value was measured at 414 nm to evaluate its antioxidant capacity.

[0073] RNA extraction and qRT-PCR

[0074] Total RNA was extracted using Trizol reagent (Takara, Shiga, Japan). Reverse transcription was performed using PrimeScript RT reagent Kit (Takara), and qRT-PCR was performed using SYBR Green PCR Master Mix (Takara) according to the manufacturer's instructions. β-actin was used for mRNA normalization.

[0075] Transient transfection

[0076] Small interfering RNA (siRNA) was purchased from Thermo Fisher (Waltham, USA): siGPX4 (10848), siSP1 (116546), siGTF2I (289315), siSRF (115344), siMYB (107687) and siNRF1 (107597). Lipofectamine 3000 (Invitrogen, Carlsbad, USA) was used for siRNA transient transfection according to the manufacturer's instructions. qRT-PCR was used to evaluate the siRNA transfection efficiency.

[0077] Exosome extraction and protein concentration determination

[0078] Exosomes were isolated by differential centrifugation. Exosome-free medium was used 48 h before exosome isolation. Cell culture medium was centrifuged at 10,000 × g for 30 min at 4 °C to remove cell debris, and then the supernatant was centrifuged at 100,000 × g for 70 min at 4 °C to obtain exosomes. The supernatant was discarded, and the exosomes were resuspended in PBS and stored at -80 °C. An equal volume of RIPA reagent (Beyotime) was added to the exosomes, and the protein concentration was determined using a BCA protein assay kit (Beyotime).

[0079] Lipid peroxidation and ROS assay

[0080] The cells were stained with 5 μM BODIPY 581 / 591C11 (Thermo Fisher) for 20 min and washed twice with PBS. The lipid-reactive oxygen species (lipid-ros) content was detected by flow cytometry.

[0081] The cells were lysed by ultrasonication, centrifuged at 10000×g for 10 min, and the supernatant was collected. The malondialdehyde (MDA) concentration was detected by measuring the OD value at 532 nm using a malondialdehyde (MDA) assay kit (Beyotime).

[0082] Intracellular reactive oxygen species (ROS) were measured using a reactive oxygen species assay kit (Beyotime). Briefly, cells were incubated with 10 μM fluorescent probe DCFH-DA at 4°C for 30 min and washed twice in PBS. Fluorescence distribution was observed using a fluorescence microscope (Nikon). Fluorescence intensity was quantitatively measured using ImageJ software.

[0083] Immunofluorescence staining

[0084] Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.25% Triton X-100 for 10 min, then blocked with 1% BSA for 30 min and incubated with GPX4 antibody (Servicebio, Wuhan, China) at a dilution of 1:500 overnight at 4°C. Cells were washed twice in PBS and incubated with secondary antibodies for 1 h at room temperature. DAPI stained the nuclei. Visualization was performed using an ECLIPSE nickel fluorescence microscope (Nikon). Fluorescence intensity was quantified using ImageJ software.

[0085] Preparation of hydrogel

[0086] PLGA-PEG-PLGA triblock copolymer (1600-1500-1600, No.: 15376-372) was purchased from Daigang Biomaterial (Jinan, China) and dissolved in PBS overnight to obtain PLGA-PEG-PLGA precursor solution. Sodium selenite (cat. 10102-18-8) was purchased from Sigma-Aldrich (St. Louis, USA), vortexed and added to the PLGA-PEG-PLGA solution, and incubated at room temperature for 2 hours to obtain selenium-containing PLGA-PEG-PLGA solution.

[0087] Cell lines and animals

[0088] MH-S cells (mouse alveolar macrophage cell line) and L929 cells (mouse fibroblast cell line) purchased from Servicebio were cultured in 1640 medium (Gibco) containing 10% FBS (Gibco) under standard conditions (37°C, 5% CO2). C57BL / 6 mice were purchased from Huafukang Company, Beijing, China. All animal experiments were performed in accordance with current ethical guidelines and approved by the Animal Ethics Committee of Qilu Hospital of Shandong University (Jinan, China).

[0089] Characterization of Selenium-containing PLGA-PEG-PLGA Hydrogels

[0090] Gel temperature: Gel temperature is determined by the test tube inversion test. 1 mL of sample is placed in a glass tube and heated from 20°C to 60°C at a rate of 0.5°C / min. The glass tube is flipped once per minute, and the gel temperature is determined when the gel does not flow downward under gravity.

[0091] Gel time: Gel time is determined by the test tube inversion test. A 1 ml sample at room temperature in a glass tube is placed in a 37°C water bath and the tube is inverted every 5 seconds. Gel time is defined as the time when the gel does not flow downward under the action of gravity.

[0092] Syringability: Samples were injected through a 25 gauge needle to test syringeability.

[0093] Rheological analysis: The rheological behavior was measured using a rheometer (TA Instruments, New Castle, USA) and heated from 20°C to 60°C at a rate of 0.5°C / min.

[0094] Morphology observation: The samples were transferred to a scanning electron microscope (Helios G4 UC, Thermo Fisher) at −180 °C using a low-temperature transfer system (PP3010T, Quorum, Knutsford, UK) to observe the surface morphology.

[0095] Selenium release analysis: 1 g of selenium-containing hydrogel was added to 10 mL of PBS at pH 7.4 and incubated for 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. PBS was collected at different time points and centrifuged at 1000 g for 5 min. Se concentration was analyzed by inductively coupled plasma mass spectrometry (iCAP Q, Thermo Fisher). The selenium release rate was calculated by dividing the selenium content in PBS by the total selenium content in the hydrogel.

[0096] Cytotoxicity test

[0097] The in vitro biocompatibility of the hydrogel was evaluated using the CCK-8 method. L929 cells (STCC20025G, Servicebio) were cultured in MEM medium with or without hydrogel for 3 days. The medium was then replaced with fresh MEM medium and CCK-8 reagent was added. After incubation for 30 minutes, the OD value at 450 nm was measured using a microplate reader (Multiskan SkyHigh, Thermo Fisher).

[0098] Hemolysis test

[0099] Fresh mouse whole blood was centrifuged at 500×g for 10 min to obtain red blood cells, which were diluted to 5% in physiological saline. 300 μL of red blood cells were mixed with 700 μL of hydrogel, incubated at 37°C for 1 h, centrifuged at 500×g for 10 min, and the OD value of the supernatant at 540 nm was measured using an enzyme marker (Thermo Fisher). The hemolysis rate was calculated by dividing the OD value of the test group by the OD value of the positive control (double distilled water).

[0100] In vitro and in vivo degradation studies

[0101] 1 mL of 20 wt% PLGA-PEG-PLGA solution was inoculated in a 24-well plate to form a gel at 37°C. After gelation, the hydrogel was placed in a cell culture dish and incubated with 10 mL of pH 7.4 PBS containing collagenase IV (1 μg / mL) at 37°C for 21 days. At each set time point, the PBS was removed, the hydrogel was freeze-dried and weighed. The degradation rate was calculated by dividing the mass of the freeze-dried hydrogel by the mass of the PLGA-PEG-PLGA triblock copolymer used to prepare the hydrogel.

[0102] The in vivo degradation test was performed by injecting 200 μL of 20 wt% hydrogel subcutaneously into the inguinal region of mice. The mice were observed and histologically examined at each set time point.

[0103] In vivo tracking of hydrogel-encapsulated mesenchymal stem cells

[0104] The cells were stained with 1 μM CM-Dil dye (Invitrogen) for 30 min, washed with PBS, and centrifuged at 300×g for 5 min. CM-Dil-stained MSCs were coated with hydrogels and injected subcutaneously into the inguinal region of mice. Mesenchymal stem cells were detected using the Maestro in vivo imaging system (CRi, Woburn, USA).

[0105] Establishment of limb ischemia mouse model

[0106] A mouse limb ischemia model was established. Briefly, limb ischemia was induced by femoral artery ligation and transection, while the contralateral limb served as a control. All animal experiments were performed in accordance with current ethical guidelines and approved by the Animal Ethics Committee of Qilu Hospital of Shandong University.

[0107] Histology

[0108] The muscle tissue was fixed with 4% formalin and embedded in paraffin. 4 μm sections were taken and stained with hematoxylin and eosin (H&E). Masson staining was performed using the Masson staining kit (Solarbio) according to the manufacturer's instructions.

[0109] For immunofluorescence staining, sections were incubated with the following antibodies after de-affinity, antigen retrieval and blocking: CD31 (Servicebio), CD163 (Servicebio) and iNOS (Servicebio) overnight at 4°C, followed by incubation with secondary antibodies at 1:500 dilution for 1 hour at room temperature. Visualization was performed using an ECLIPSE nickel fluorescence microscope (Nikon).

[0110] ELISA

[0111] 100 μL of cell culture supernatant was added to an ELISA plate (Abcam, Cambridge, UK) pre-coated with PEG2, TGF-β, IDO, TSG6, TNF-α, IL-1β, and IL-6 antibodies and incubated at room temperature for 2 h. After washing, biotin-labeled detection antibodies were added and incubated at 37°C for 1 h. Color was developed with avidin-horseradish peroxidase. OD values ​​were measured at 450 nm using a microplate reader (Thermo Fisher).

[0112] Experimental results analysis

[0113] 1. Selenium protects mesenchymal stem cells from oxidative damage

[0114] We isolated MSCs from human umbilical cord and investigated whether selenium supplementation could protect MSCs from oxidative damage.

[0115] Figure 1 This is a test result diagram about selenium involved in the present invention.

[0116] Our analysis showed that H2O2 induced cell death in the concentration range of 100-1000 μM, whereas Se supplementation had a dose-dependent protective effect on MSCs in the concentration range of 0-2 μM ( Figure 1 (Part A). Since selenium treatment above 2 μM is cytotoxic, we chose 2 μM as the selenium concentration for subsequent experiments. Notably, the addition of 2 μM Se significantly improved the decrease in antioxidant capacity and ROS generation induced by H2O2 ( Figure 1 Next, the mRNA levels of some major antioxidant enzymes were measured, and the results showed that Se increased the expression of Se-containing antioxidant enzymes, especially GPX4, but not Se-free antioxidant enzymes, suggesting that GPX4 may be the main executor of antioxidant activity after Se treatment ( Figure 1 Part D in the middle). Se induced a significant increase in GPX4 protein level ( Figure 1 (E). Erastin is a ferroptosis inducer that causes cell death through antioxidant consumption and GPX4 degradation. We found that Se also reduced erastin-induced mortality and ferroptosis biomarkers such as lipid ROS and MDA in MSCs ( Figure 1 (see FH section in the figure).

[0117] To further confirm the critical role of selenium-induced GPX4 expression in cell protection, we knocked down GPX4 using specific small interfering RNA (siRNA) and evaluated the knockdown efficiency using qRT-PCR. Knockdown of GPX4 abolished the protective effect of selenium against H2O2-induced cell death and decreased antioxidant capacity ( Figure 1 (Parts I and J in the figure).

[0118] In summary, selenium can be used as a new protective agent with powerful antioxidant effects, providing new possibilities for improving MSC vitality.

[0119] 2. Selenium protects MSCs from oxidative damage by regulating GPX4 transcription

[0120] Figure 2 This is a test result diagram related to the regulatory factors of selenium on GPX4 transcription involved in the present invention.

[0121] To further investigate the role of Se in GPX4 transcription, we first treated MSCs under oxidative stress with Se and the transcription inhibitor actinomycin D (Act D). The results showed that Se increased the expression level of mRNA, but this upregulation was offset by ActD ( Figure 2 (Part A in the middle), indicating that Se protects against oxidative damage by regulating GPX4 transcription.

[0122] We then collected transcription factors (TFs) that Se might regulate based on previously published studies. To identify the regulatory factors of GPX4 transcription by Se, we screened the hTFtarget database (http: / / bioinfo.life.hust.edu.cn / hTFtarget) and performed Venn diagram analysis, identifying five TFs (SP1, GTF2I, SRF, MYB, and NRF1) ( Figure 2 Part B). Using siRNA to knock out these five TFs, it was found that selenium-induced GPX4 transcription was only inhibited by SP1 knockout ( Figure 2 SP1 knockout reduced GPX4 protein levels and abolished the cytoprotective effect of selenium supplementation ( Figure 2 D and E parts).

[0123] Therefore, we demonstrated that selenium induced GPX4 expression to protect MSCs from oxidative damage via regulating the transcription factor SP1.

[0124] 3. Preparation and characterization of selenium-containing PLGA-PEG-PLGA hydrogels

[0125] Figure 3 This is a test result diagram of the selenium-containing hydrogel involved in the present invention.

[0126] In this study, we prepared PLGA-PEG-PLGA hydrogels (hereinafter referred to as Gel) with mass fractions of 15wt%, 20wt% and 25wt% and PLGA-PEG-PLGA hydrogels containing Se (2μM) (hereinafter referred to as Se / Gel). The liquid-gel transition temperature windows of Gel and Se / Gel were measured. Figure 3Part A). When the temperature rises from 20°C to 60°C, Gel and Se / Gel exist in three states: solution, gel and suspension. The addition of Se (2 μM) has no significant effect on the gelation properties of Gel. Gel and Se / Gel with a mass fraction of 20 wt% were selected for further experiments. Their solution-gel transition temperature is about 33°C. Therefore, they are easy to be incorporated into MSCs at room temperature and gel after injection into the body, which is suitable for clinical application.

[0127] 20wt% Gel and Se / Gel have high injectability at room temperature (25℃) Figure 3 The gel time of the two samples at body temperature (37°C) was 66.00±5.56s and 67.33±5.69s, respectively. Figure 3 As the temperature rises from room temperature (25°C) to body temperature (37°C), 20wt% Gel and Se / Gel both transform from aqueous solution to gel. The rheological properties of Gel and Se / Gel were analyzed by rheometer ( Figure 3 The storage modulus (G′) was observed to be higher than the loss modulus (G′), indicating that the solution was transformed into a hydrogel. The surface morphology of Gel and Se / Gel showed a three-dimensional porous network structure ( Figure 3 (Part F), the Se release rate of Se / Gel within 24 h was maintained at 27.67%±2.52%, indicating that Se / Gel has good Se in situ retention performance.

[0128] 4. Biocompatibility of Se-containing Hydrogels in Vitro and in vivo

[0129] Figure 4 This is a graph showing test results related to the biocompatibility of the selenium-containing hydrogel involved in the present invention.

[0130] To evaluate the cytotoxicity of the novel Se / Gel, we cultured L929 cells in 96-well plates and measured cell viability with or without Se / Gel seeding on the surface ( Figure 4 The results showed that the addition of Se / Gel had no significant effect on the proliferation activity of cells ( Figure 4 We performed an in vitro hemolysis test to further evaluate the biocompatibility of Se / Gel. Compared with the positive control (double distilled water), the hemolysis rates of Gel and Se / Gel were both less than 5% ( Figure 4 In addition, in vitro and in vivo degradation studies were performed to evaluate the use of Se / Gel as a local implant material ( Figure 4 The in vitro degradation rates of Gel and Se / Gel were not significantly different, reaching 82.33±3.05% and 81.67±6.80% after 21 days, respectively. Figure 4In vivo degradation studies showed that Gel and Se / Gel gradually degraded over time during the 21-day observation period ( Figure 4 (G section). There was no significant difference in the number of inflammatory cells infiltrating the injection site of the hydrogel and Se / Gel. The results showed that there was obvious inflammatory cell infiltration in the surrounding subcutaneous tissue on the 7th and 14th days after injection of Gel and Se / Gel. However, on the 21st day, the inflammatory response was significantly weakened as the hydrogel degraded. In summary, these results demonstrate that Se / Gel has good tissue compatibility.

[0131] 5. Selenium-containing hydrogels can improve the survival and immunoregulatory ability of MSCs under oxidative stress.

[0132] Figure 5 This is a test result diagram of the selenium-containing hydrogel involved in the present invention acting on MSCs.

[0133] MSCs were encapsulated in PLGA-PEG-PLGA hydrogels (to obtain Gel / MSC) or selenium-containing PLGA-PEG-PLGA hydrogels (to obtain Se / Gel / MSC) at a density of 1×10 6 cells / mL, such as Figure 5 As shown in Part A. We found that under H2O2 or Erastin-induced oxidative stress, the activity of Se / Gel / MSC reached 72.01±6.03% and 78.33±7.57%, respectively, while the activity of Gel / MSC was 35.67±9.79% and 47.33±11.06%, respectively. Figure 5 (Part B), indicating that Se / Gel has a strong cytoprotective effect against oxidative damage.

[0134] Extract exosomes stripped from Gel / MSC or Se / Gel / MSC, measure exosome protein concentration, and evaluate the paracrine activity of MSCs ( Figure 5 In the Gel / MSC group, the concentration of exosome proteins decreased by nearly three quarters under oxidative stress, while the paracrine activity of MSCs in the Se / Gel encapsulation group was significantly increased by more than 3 times compared with the Gel encapsulation group ( Figure 5 In addition, we measured the secretion of immunomodulatory factors by MSCs. The results showed that H2O2 or Erastin exposure significantly reduced the secretion of immunomodulatory factors by Gel / MSCs, but the addition of selenium offset the inhibitory effect ( Figure 5 Finally, in vivo tracking of hydrogel-encapsulated MSCs revealed that Se / Gel increased cell retention at the injection site compared with Gel ( Figure 5 H section).

[0135] These observations suggest that Se / Gel has the key advantage of promoting MSC survival and enhancing immunomodulatory capacity.

[0136] 6. Se / Gel administration of MSCs improved blood perfusion in the limb ischemia mouse model.

[0137] Figure 6 This is a diagram showing the test results of the selenium-containing hydrogel-encapsulated MSCs on mice.

[0138] To evaluate its therapeutic potential in vivo, we established a mouse limb ischemia model and injected Se / Gel-encapsulated MSCs subcutaneously into the injury site ( Figure 6 Part A), blood perfusion was monitored during the 21-day observation period ( Figure 6 (Part B).

[0139] Laser speckle imaging showed that almost no blood flow signals were detected in the injured limb immediately after femoral artery transection. On days 7 and 21, blood perfusion of the ischemic limb was significantly restored after Se / Gel / MSC treatment compared with that after Gel / MSC and Se / Gel treatment ( Figure 6 Histological examination showed focal necrosis and obvious inflammatory cell infiltration in muscle tissue after Gel / MSC or Se / Gel treatment. In contrast, there was less necrosis and inflammatory cell infiltration after Se / Gel / MSC treatment ( Figure 6 Masson staining was used to distinguish collagen and muscle tissue. The results showed that collagen formation was abundant in muscle samples of the Se / Gel group; collagen formation was reduced after Gel / MSC treatment, and collagen formation was further significantly reduced after Se / Gel / MSC treatment ( Figure 6 CD31 immunofluorescence staining showed that the capillary density increased the most after Se / Gel / MSC treatment compared with the other two groups ( Figure 6 (see Section G in the Appendix).

[0140] These results suggest that Se / Gel-delivered MSCs effectively alleviated ischemia-induced tissue damage and increased capillary formation, thereby promoting recovery from limb ischemia.

[0141] In summary, selenium protects MSCs from oxidative damage by increasing the transcription mechanism of GPX4. Selenium-containing hydrogels have good biocompatibility, biodegradability and antioxidant properties. They are easily injected into the target area in liquid form at room temperature and become gels at body temperature to limit the diffusion of selenium, thereby exerting a cytoprotective effect on mesenchymal stem cells. In addition, selenium-containing hydrogels containing MSCs can effectively inhibit macrophage M1 polarization and promote macrophage M2 polarization, thereby accelerating angiogenesis and blood perfusion recovery in ischemic limbs.

[0142] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A gel composition, characterized in that: The invention comprises mesenchymal stem cells and a gel carrier containing selenium, wherein the gel carrier comprises a copolymer of PLGA and PEG, and the concentration of selenium in the gel carrier is greater than 0 μM and not greater than 2 μM.

2. The gel composition according to claim 1, characterized in that The mesenchymal stem cells are selected from any one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, human placenta mesenchymal stem cells and amniotic fluid mesenchymal stem cells.

3. The gel composition according to claim 1, characterized in that The gel composition is in liquid form within a first temperature range and in gel form within a second temperature range, wherein the first temperature range is less than 33°C and the second temperature range is not less than 33°C.

4. The gel composition according to claim 1, characterized in that The gel carrier encapsulates the mesenchymal stem cells.

5. The gel composition according to claim 1 or 4, characterized in that: The gel carrier has a three-dimensional porous network structure, and the selenium is encapsulated in the three-dimensional porous network structure.

6. The gel composition according to claim 1, characterized in that The copolymer of PLGA and PEG is a PLGA-PEG-PLGA triblock copolymer.

7. The gel composition according to claim 1 or 6, characterized in that: In the gel carrier, the mass fraction of the copolymer of PLGA and PEG is 15%-25%.

8. Use of a gel composition in preparing a drug for treating limb ischemia, characterized in that: The gel composition is the gel composition according to any one of claims 1 to 7.

9. The use according to claim 8, characterized in that: Selenium in the gel composition induces GPX4 expression by regulating transcription factor SP1 to protect the mesenchymal stem cells from oxidative damage.

10. The use according to claim 8, characterized in that: The gel composition inhibits macrophage M1 polarization and promotes macrophage M2 polarization, thereby accelerating angiogenesis and blood perfusion recovery in ischemic limbs.

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