Stem cell microcapsule for myocardial repair and preparation method thereof

Abstract

The invention provides a stem cell microcapsule for myocardial repair. The stem cell microcapsule comprises the following components: PLGA, dichloromethane, a DMEM culture medium containing 10% of fetal calf serum, stem cells, VEGF, IGF-1, polyvinyl alcohol and myocardial targeting peptide. The invention also provides a preparation method of the stem cell microcapsule for myocardial repair, which comprises the following steps: by taking a dichloromethane solution of PLGA as an oil phase and a stem cell suspension containing growth factors as a water phase, dropwise adding the water phase into the oil phase to obtain a W / O emulsion, mixing the W / O emulsion with an external water phase to obtain a W / O / W multiple emulsion, stirring, curing PLGA to form the microcapsule, and modifying the myocardial targeting peptide on the surface of the microcapsule, thereby obtaining the stem cell microcapsule for myocardial repair. The stem cell micro-capsule can be obtained. According to the stem cell microcapsule provided by the invention, growth factors are added, so that the survival rate of stem cells is remarkably improved, and proliferation and differentiation of the stem cells are promoted; the surface of the microcapsule is modified with specific myocardial targeting peptide, so that the damaged myocardial part can be specifically recognized, the stem cell microcapsule is guided to accurately reach a target area, and better homing ability is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a stem cell microcapsule for myocardial repair and a preparation method thereof. Background Art

[0002] Heart diseases such as myocardial infarction lead to the death of a large number of cardiomyocytes, and the heart's ability to repair itself is limited, which seriously affects heart function and the patient's normal life. Stem cells have the potential for self-renewal and multidirectional differentiation, and can differentiate into cardiomyocytes, vascular endothelial cells, smooth muscle cells, etc., thereby repairing damaged myocardium. As an emerging treatment strategy, stem cell therapy holds hope for myocardial repair. However, the clinical application of stem cell therapy faces many challenges. For example, after direct injection of stem cells, the efficiency of stem cell delivery to the target site is low. A large number of stem cells cannot accurately reach the damaged myocardium and are lost in other tissues and organs, resulting in poor treatment effect. At the same time, stem cells lack a suitable microenvironment in the body, and cell survival rate is low, making it difficult for them to effectively proliferate and differentiate into cardiomyocytes and play a role in repairing the myocardium. Traditional delivery methods cannot simultaneously solve the problems of low stem cell survival rate and low delivery efficiency to the target site, which limits the clinical application of stem cell therapy in the field of myocardial repair. Summary of the Invention

[0003] To address these issues, the present invention provides stem cell microcapsules for myocardial repair and a method for their preparation. These stem cell microcapsules are made from biodegradable polymers and are non-toxic and non-side effect. They also contain cell growth factors and are surface-modified with specific myocardial targeting peptides, enhancing stem cell survival while ensuring precise delivery to the target area, significantly increasing stem cell accumulation in damaged myocardium.

[0004] To achieve the above object, the technical solution adopted by the present invention is: A stem cell microcapsule for myocardial repair, characterized in that the microcapsule comprises the following components: PLGA, dichloromethane, DMEM culture medium containing 10% fetal bovine serum, stem cells, VEGF, IGF-1, polyvinyl alcohol, and a myocardial targeting peptide.

[0005] The core of the stem cell microcapsules of the present invention lies in the use of the polymer material PLGA as a carrier, which exhibits unprecedented advantages in biocompatibility and degradability. The carrier can gradually decompose into small molecules in the body, and these small molecules can be completely metabolized and excreted by the body naturally, fundamentally eliminating the occurrence of toxic side effects. Of particular importance is that the stem cell microcapsules of the present invention are exquisitely encapsulated with VEGF and IGF-1 growth factors. As the PLGA carrier gradually degrades, these growth factors continue to supply nutrients to the stem cells in a slow-release mode, while also providing key differentiation signals. This unique design significantly improves the survival rate of stem cells and effectively promotes the proliferation and differentiation of stem cells into cardiomyocytes. In addition, the present invention has achieved a major breakthrough in microcapsule surface modification technology. By modifying a specific myocardial targeting peptide (NGR peptide), the microcapsules are endowed with the remarkable ability to accurately identify damaged myocardial areas. The NGR peptide can specifically bind to the characteristic molecules of the damaged myocardium, guiding the microcapsules to the damaged myocardial area accurately and accurately, greatly improving the enrichment of stem cells in the damaged myocardium. This targeted delivery efficiency is far superior to the traditional direct injection of stem cells. In summary, the stem cell microcapsules of the present invention, with their innovative carrier design, growth factor sustained-release mechanism, and targeted modification technology, have successfully overcome the difficulties of low stem cell survival rate and low delivery efficiency to the target site in the traditional direct stem cell injection method, opening up a new path for clinical application in the field of myocardial repair, and have extremely high innovation and application value.

[0006] The present invention also provides a method for preparing the above-mentioned stem cell microcapsules, characterized in that the method comprises the following steps: Step 1: Weigh PLGA powder and dissolve it in dichloromethane, and the resulting solution serves as the oil phase; Step 2: Take stem cells in the logarithmic growth phase and add DMEM medium containing 10% fetal bovine serum to the stem cells to adjust the stem cell concentration to 1×10 7 / mL, then add VEGF and IGF-1 and mix well as the aqueous phase; Step 3: adding the aqueous phase described in step 2 dropwise to the oil phase described in step 1, and stirring to obtain a W / O emulsion; Step 4: a solution containing 1% polyvinyl alcohol was measured as the external aqueous phase, and the W / O emulsion described in step 3 was added dropwise to the external aqueous phase to obtain a W / O / W emulsion. The mixture was stirred for 8 h to allow the PLGA to solidify and form microcapsules. Step 5: Mix the microcapsules described in step 4 with HEPES buffer, react at room temperature for 1 hour, add myocardial targeting peptide, continue to react for 4 hours, collect the microcapsules after reaction and wash with PBS to obtain stem cell microcapsules for myocardial repair.

[0007] Preferably, the mass concentration of PLGA in the oil phase in step 1 is 0.03 to 0.05 g / mL. The present invention uses PLGA as a microcapsule carrier. This carrier has good biocompatibility and degradability, can be metabolized and excreted by the body, is non-toxic to human tissues and cells, and is suitable for various biomedical applications. Dichloromethane is selected as the oil phase solution, which can fully dissolve and evenly disperse PLGA, which is conducive to forming a stable microcapsule structure. In addition, dichloromethane has a low boiling point and can be easily removed by volatilization at room temperature during the microcapsule preparation process, which is conducive to the solidification and molding of microcapsules. Within the above mass concentration range, PLGA has suitable viscosity and solubility in dichloromethane, which helps to form oil droplets of uniform size and regular shape during the emulsification process, thereby forming high-quality microcapsules.

[0008] Preferably, the mass concentration of VEGF in the aqueous phase in step 2 is 10~50 ng / mL, and the mass concentration of IGF-1 is 10~100 ng / mL. The present invention adds VEGF and IGF-1 growth factors to the microcapsules. VEGF can activate signaling pathways such as PI3K-Akt in stem cells, inhibit the expression of apoptosis-related proteins, thereby reducing the apoptosis of stem cells in the myocardial injury microenvironment and significantly improving their survival rate; IGF-1 binds to the IGF-1 receptor on the surface of stem cells, activates downstream signaling pathways such as PI3K-mTOR, provides survival signals for stem cells, and stimulates the metabolic activity of stem cells, providing a material and energy basis for cell proliferation, thereby promoting the proliferation of stem cells; within the above-mentioned mass concentration range, the two growth factors can more effectively play their roles in promoting stem cell survival and proliferation, inducing stem cells to differentiate into cardiomyocytes, promoting angiogenesis, and improving the myocardial microenvironment, so as to achieve better myocardial repair effects.

[0009] Preferably, the volume ratio of the aqueous phase to the oil phase in step 3 is 1:3. At this ratio, the oil phase, as the continuous phase, can fully encapsulate the aqueous phase, forming smaller and more uniform water droplets dispersed in the oil phase, reducing collisions and coalescence between the water droplets, thereby improving the stability of the emulsion. Furthermore, high-speed stirring, with a stirring time of 10 minutes and a rotation speed of 10,000 r / min, can further evenly disperse the aqueous phase in the oil phase, further refining the water droplet size and increasing the stability of the emulsion.

[0010] Preferably, the volume ratio of the W / O emulsion to the external aqueous phase in step 4 is 1:3. At this volume ratio, the W / O emulsion can be dispersed relatively evenly in the external aqueous phase, and the formed microcapsule structure is relatively regular, which is conducive to the subsequent slow release of growth factors and can continuously act on myocardial repair. At the same time, the relatively mild stirring condition of 500 r / min can ensure uniform mixing of the emulsion while maximally protecting the structural integrity of the W / O emulsion and the activity of the growth factors, ensuring that the growth factors can function normally during the myocardial repair process.

[0011] Preferably, the volume ratio of the microcapsules to the HEPES buffer in step five is 1:5, and the volume proportion of the myocardial targeting peptide is 1% to 5%. The HEPES buffer used in the present invention contains EDC and NHS. EDC can convert the carboxyl groups on the surface of the microcapsules into more active intermediates, which are convenient for reacting with NHS to form active esters, thereby enhancing the binding ability of the microcapsules and the myocardial targeting peptide (NGR peptide). The modified microcapsules can specifically identify damaged myocardial sites and accurately reach the target area. At the above volume ratio, the HEPES buffer can provide a good dispersion environment for the microcapsules, increase the contact area between the microcapsules and the activator components in the buffer, and facilitate the subsequent modification reaction of the microcapsule surface by the activator. The above volume proportion of the NGR peptide solution can ensure that there is sufficient NGR peptide to specifically bind to the surface of the activated microcapsules, so that the microcapsules have good myocardial targeting ability.

[0012] The beneficial effects of the present invention are: (1) This invention innovatively constructs a stem cell microcapsule. The core of the invention is the use of a polymer material, PLGA, as a carrier. It exhibits unprecedented advantages in terms of biocompatibility and degradability. The carrier can gradually decompose into small molecules in the body, and these small molecules can be completely metabolized and excreted by the body, fundamentally eliminating the occurrence of toxic side effects. In the process of preparing microcapsules, dichloromethane is selected as the oil phase solution, which can fully dissolve and evenly disperse PLGA, which is conducive to the formation of a stable microcapsule structure. In addition, dichloromethane has a low boiling point and can be easily removed by volatilization at room temperature, which is conducive to the solidification and molding of microcapsules.

[0013] (2) The stem cell microcapsules of the present invention are exquisitely encapsulated with VEGF and IGF-1 growth factors. As the PLGA carrier gradually degrades, these growth factors continue to supply nutrients to the stem cells in a slow release mode, while also providing key differentiation signals. VEGF can activate signaling pathways such as PI3K-Akt in stem cells, inhibit the expression of apoptosis-related proteins, thereby reducing stem cell apoptosis in the myocardial injury microenvironment and significantly improving their survival rate; IGF-1 binds to the IGF-1 receptor on the surface of stem cells, activates downstream signaling pathways such as PI3K-mTOR, provides survival signals for stem cells, and stimulates the metabolic activity of stem cells, providing a continuous material and energy basis for cell proliferation, thereby promoting the proliferation and differentiation of stem cells. This unique design significantly improves the survival rate of stem cells and efficiently promotes the proliferation and differentiation of stem cells into cardiomyocytes. This effect has not been reported in the prior art.

[0014] (3) The present invention has achieved a major breakthrough in microcapsule surface modification technology. By modifying a specific myocardial targeting peptide (NGR peptide), the microcapsule is endowed with the excellent ability to accurately identify damaged myocardial sites. The NGR peptide can specifically bind to the characteristic molecules of the damaged myocardial site, guiding the microcapsule to accurately and accurately reach the damaged myocardial area, greatly improving the enrichment of stem cells in the damaged myocardium. This targeted delivery efficiency is much higher than the traditional method of direct injection of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of detecting the number of living cells using the CCK-8 kit.

[0016] Figure 2 This is a graph showing the concentration changes of growth factor VEGF over time.

[0017] Figure 3 This is a graph showing the concentration changes of growth factor IGF-1 over time.

[0018] Figure 4 This is the result of Transwell chamber cell migration detection. DETAILED DESCRIPTION

[0019] Example 1: Preparation of stem cell microcapsules The stem cell microcapsules for myocardial repair of this embodiment include the following components: PLGA, dichloromethane, DMEM culture medium containing 10% fetal bovine serum, stem cells, VEGF, IGF-1, polyvinyl alcohol, and myocardial targeting peptide (NGR peptide).

[0020] This embodiment also provides a method for preparing stem cell microcapsules for myocardial repair, the specific steps of which are as follows: Step 1: Accurately weigh 3 g of PLGA powder and slowly add it to 100 mL of dichloromethane under magnetic stirring until no obvious powder particles remain in the solution and a homogeneous and transparent solution is obtained as the oil phase; Step 2: Collect the logarithmic growth phase stem cells, add fresh DMEM medium containing 10% fetal bovine serum, and gently pipette to fully disperse the cells. Then, use a cell counting plate to count the cells. By adding or reducing DMEM medium containing 10% fetal bovine serum, repeatedly adjust the cell suspension volume until the cell concentration reaches exactly 1×10 7 Take 100 mL of the cell suspension, add VEGF and IGF-1 growth factors with a final concentration of 10 ng / mL, and mix thoroughly to use as the aqueous phase; Step 3: Measure 60 mL of the oil phase solution described in step 1, and use a sterile rubber-tipped dropper to draw 20 mL of the aqueous phase solution described in step 2. Stir the oil phase solution while slowly adding the aqueous phase. After the aqueous phase is completely added, stir at 10,000 rpm for 10 minutes to allow the aqueous phase to be fully dispersed in the oil phase and form a stable W / O emulsion. Step 4: Measure 60 mL of a solution containing 1% polyvinyl alcohol as the external aqueous phase, and slowly add the W / O emulsion described in step 3 in a volume ratio of 1:3 between the W / O emulsion and the external aqueous phase, stir at 500 r / min for 30 min to obtain a W / O / W emulsion, and continue stirring for 8 h. During the stirring process, the dichloromethane gradually evaporates, and the PLGA solidifies to form microcapsules. The microcapsules are collected by centrifugation at 6000 r / min for 10 min; Step 5. The microcapsules obtained in step 4 were washed repeatedly with PBS three times. The washed microcapsules were evenly mixed with HEPES buffer at a volume ratio of 1:5, and allowed to react at room temperature for 1 hour. Then, a 1% by volume solution of myocardial targeting peptide (NGR peptide) was added. After continuing the reaction for 4 hours, the microcapsules were collected by centrifugation at 6000 r / min for 10 minutes, and then washed with PBS to obtain stem cell microcapsules for myocardial repair.

[0021] Example 2: In vitro performance testing of stem cell microcapsules (1) Detection of viable cell number The above stem cell microcapsules and unencapsulated stem cells in the logarithmic growth phase were prepared into a concentration of 1×10 5100 μL of cell suspension was added to each well of a 96-well plate. Eight replicate wells were set up for each group, and 100 μL of the corresponding cell suspension was added to each well. The 96-well plate was gently shaken to evenly distribute the cells at the bottom of the wells. The seeded 96-well plate was then placed in an incubator and incubated at 37°C. At each assay time point (24, 48, 72, and 96 hours), the 96-well plate was removed from the incubator and 10 μL of CCK-8 solution was added to two wells in the microcapsule and stem cell groups, respectively. The cells were incubated in a 37°C incubator for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. The absorbance value is proportional to the number of viable cells; that is, a higher OD value indicates a greater number of viable cells.

[0022] Test results such as Figure 1 As shown, at each time point, the absorbance values ​​of the stem cell microcapsule group were higher than those of the stem cell group, indicating that under identical culture conditions, the number of viable cells in the stem cell microcapsule group was higher than that in the stem cell group at the same time. Once the nutrients in the cell suspension were depleted, the number of viable cells in the stem cell group stopped increasing and began to decrease over time. However, in the stem cell microcapsule group, the microcapsules contained the growth factors VEGF and IGF-1, which activated corresponding signaling pathways and stimulated stem cell metabolic activity. This not only maintained a relatively high stem cell survival rate by inhibiting the expression of apoptosis-related proteins, but also provided continuous nutritional support and differentiation signals to stem cells, promoting their proliferation and differentiation, ultimately resulting in a higher number of viable cells. This suggests that these two growth factors can effectively promote stem cell survival and proliferation, induce stem cell differentiation, and contribute to better myocardial repair outcomes.

[0023] (2) Sustained-release performance test Dilute the stem cell microcapsules with PBS buffer to a concentration of 1×10 5 50 mL of cell suspension was prepared and cultured at 37°C with constant shaking. Buffer was taken at different time points and the concentrations of VEGF and IGF-1 were detected using ELISA kits. Figure 2 is the concentration change diagram of VEGF, Figure 3 This is a graph showing changes in IGF-1 concentration.

[0024] The results showed that as the microcapsules degraded, the growth factors were slowly released over the 14 days tested, and the concentration of growth factors in the buffer solution gradually increased, indicating that the resulting stem cell microcapsules exhibited sustained-release properties. PLGA, a microcapsule carrier with excellent biocompatibility and degradability, degrades gradually. This property allows for sustained and slow growth factor release, helping to maintain effective growth factor concentrations and achieving long-term, stable release. This sustained-release property of the microcapsules ensures a continuous supply of material and energy for stem cells, ensuring their survival and proliferation over an extended period.

[0025] (3) Homing ability test A Transwell chamber was used to simulate the in vivo blood circulation and myocardial tissue environment. The supernatant of damaged myocardial cell culture was added to the lower chamber, with the liquid level close to but not exceeding the bottom of the upper chamber. Stem cell microcapsules and non-encapsulated stem cell suspensions were added to the upper chamber, respectively. After 24 hours of culture, the Transwell chamber was removed, and the non-migrated cells in the upper chamber were gently wiped off with a cotton swab. The cells in the lower chamber were stained with crystal violet and counted.

[0026] Figure 4 Results showed that the number of stem cell microcapsules migrating was significantly greater than that of non-encapsulated stem cells. This is because the supernatant of damaged cardiomyocytes contains overexpressed aminopeptidase N, and the myocardial targeting peptide (NGR peptide) on the microcapsule surface specifically recognizes this overexpressed aminopeptidase N. This specific recognition is not a simple binding process, but rather triggers a series of complex biological reactions, resulting in greater migration motivation. When the NGR peptide recognizes and binds to aminopeptidase N, it activates certain intracellular signaling pathways, causing changes in the extracellular matrix surrounding the stem cell microcapsules, creating a more favorable environment for migration. This binding may also affect the morphology and motility of the stem cell microcapsules themselves, enabling more efficient migration to the damaged myocardium. This synergistic effect ultimately demonstrates that stem cell microcapsules have superior homing ability compared to non-encapsulated stem cells, enabling more precise and efficient migration to damaged myocardial areas, laying a solid foundation for subsequent tissue repair and regeneration.

[0027] In summary, the stem cell microcapsules prepared by the present invention are beneficial to improving the survival rate of stem cells, promoting stem cell proliferation and differentiation, and can specifically identify damaged myocardial sites, significantly improving the enrichment of stem cells in damaged myocardial sites, and have better homing ability. The above embodiments are only partial implementation methods of the present invention. In actual applications, the materials, preparation processes, etc. can be adjusted and optimized according to specific needs. The scope of protection of the present invention is not limited to the above embodiments, but also includes various modifications and improvements made based on the technical solutions of the present invention.

Claims

1. A stem cell microcapsule for myocardial repair, characterized in that: The microcapsule includes the following components: PLGA, dichloromethane, DMEM culture medium containing 10% fetal bovine serum, stem cells, VEGF, IGF-1, polyvinyl alcohol, and myocardial targeting peptide.

2. A method for preparing the stem cell microcapsule according to claim 1, characterized in that: The method comprises the following steps: Step 1: Weigh PLGA powder and dissolve it in dichloromethane, and the resulting solution serves as the oil phase; Step 2: Take stem cells in the logarithmic growth phase and add DMEM medium containing 10% fetal bovine serum to the stem cells to adjust the stem cell concentration to 1×10 7 / mL, then add VEGF and IGF-1 and mix well as the aqueous phase; Step 3: adding the aqueous phase described in step 2 dropwise to the oil phase described in step 1, and stirring to obtain a W / O emulsion; Step 4: a solution containing 1% polyvinyl alcohol was measured as the external aqueous phase, and the W / O emulsion described in step 3 was added dropwise to the external aqueous phase to obtain a W / O / W emulsion. The mixture was stirred for 8 h to allow the PLGA to solidify and form microcapsules. Step 5: Mix the microcapsules described in step 4 with HEPES buffer, react at room temperature for 1 hour, add myocardial targeting peptide, continue to react for 4 hours, collect the microcapsules after reaction and wash with PBS to obtain stem cell microcapsules for myocardial repair.

3. The method according to claim 2, characterized in that The mass concentration of PLGA in the oil phase in step 1 is 0.03~0.05 g / mL.

4. The method according to claim 2, characterized in that The mass concentration of VEGF in the aqueous phase in step 2 is 10-50 ng / mL, and the mass concentration of IGF-1 is 10-100 ng / mL.

5. The method according to claim 2, characterized in that The volume ratio of the water phase to the oil phase in step 3 is 1:

3.

6. The method according to claim 2, characterized in that The volume ratio of the W / O emulsion and the external aqueous phase in step 4 is 1:

3.

7. The method according to claim 2, characterized in that In step 5, the volume ratio of the microcapsules to the HEPES buffer is 1:5, and the volume proportion of the myocardial targeting peptide is 1% to 5%.

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