CN113262240B - Mesenchymal stem cell delivery vector and application thereof - Google Patents

Abstract

The invention provides a carrier for mesenchymal stem cell delivery, which comprises the following components: type I collagen, melanin nanoparticles, and water. The vector can be used for delivery of genetically engineered stem cells and treatment of limb ischemia. When the cell delivery system is administered locally, thermally triggered in situ gelation can maintain stem cells at the injection site, and embedded MeNPs in the gel network can scavenge high levels of reactive oxygen species in the ischemic tissue, thereby promoting cell survival. By improving cell retention and survival, Grem1-MSC can continuously secrete high-concentration Grem1, thereby promoting the proliferation of the cells and accelerating the angiogenesis of an ischemic part by activating a PI3K-AKT signal pathway in vascular endothelial cells, and finally promoting blood flow perfusion and tissue repair. The invention provides a new means for delivering the genetic engineering stem cells and provides a basis for improving the cell treatment effect of ischemic diseases.

Description

Mesenchymal stem cell delivery vector and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a delivery vector capable of presenting mesenchymal stem cells and application thereof.

Background

Peripheral Arterial Disease (PAD) is a chronic ischemic disease in which blood flow in the lower extremities is impaired. Critical Limb Ischemia (CLI) is the most severe stage of PAD, with a high risk of cardiovascular disease, which can lead to amputation and even death. Recent clinical data show that mortality rates in CLI patients reach about 20% within 6 months of diagnosis and over 50% within 5 years. Currently, standard CLI treatment is to restore blood flow to the injured limb through surgery or intravascular revascularization. However, up to 30% of patients with CLI are not suitable for standard therapy due to excessive surgical risk or poor vascular condition itself. Therefore, there is an urgent need to develop an effective alternative to CLI treatment. One of the most promising approaches at present is stem cell therapy. Stem cells (e.g., Mesenchymal Stem Cells (MSCs)) are a group of undifferentiated cells that have the ability to restore tissue/organ function by direct differentiation into tissue or paracrine secretion of a variety of functional biomolecules, such as pro-angiogenic factors and anti-inflammatory mediators. However, several key problems severely limit the therapeutic outcome of stem cell therapy, especially the problem of poor survival rate after cell implantation. Studies have shown that the retention rate of implanted stem cells is less than 10% within 1-7 days after implantation.

In recent years, biomaterial-mediated cell delivery has become a promising approach to improve cell retention and survival. In particular, injectable melanin has attracted considerable interest in cell delivery due to its broad range of advantages, such as high water content, similarity to the native extracellular matrix (ECM), porous structure that can encapsulate stem cells within its framework, and the like. In addition, in addition to encapsulating stem cells, various pro-angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor (EGF), may be simultaneously assembled in a melanin scaffold, so that the problem of paracrine insufficiency of pro-angiogenic factors may be solved and cell survival and proliferation may be further enhanced. Despite these advantages, melanin injection still has difficulty overcoming the extremely critical obstacles of induction of stem cell loss and dysfunction due to the production of highly Reactive Oxygen Species (ROS) in ischemic tissues. It is well known that impaired blood flow in ischemic tissues often causes hypoxic microenvironments, and therefore the production of ROS is significantly increased, which damages cell membranes and oxidizes lipids leading to apoptosis. Thus, the ideal cell delivery system sought in the art would need to have the ability to not only promote cell retention and survival in ischemic tissues, but also to modulate hypoxic microenvironments to enhance therapeutic efficacy. However, no CLI system treatment technology has been developed to meet these criteria.

Disclosure of Invention

In order to solve the technical problems, the invention develops a mesenchymal stem cell delivery vector which can be used for delivering genetically engineered stem cells (Grem1-MSC) and treating limb ischemia. The delivery carrier consists of natural type I collagen and biomimetic designed melanin nanoparticles (Menps), and has strong ROS eliminating and ischemia repairing capabilities.

The invention adopts the following technical scheme to show the purposes of the invention:

a mesenchymal stem cell delivery vehicle comprising the following components: the carrier comprises the following components: type I collagen, MeNPs and water. The carrier can effectively remove ROS in an ischemic tissue, protect cell membranes and reduce stem cell apoptosis.

Preferably, the mass percentages of the type I collagen and the MeNPs in the carrier are 0.3-0.4% of the type I collagen, 0.02-0.05% of the MeNPs and 99.55-99.68% of water. Through a great deal of experimental research by the inventor, the components of the carrier of the invention can form good in-situ hydrogel within the mass percentage range, and the gel of the invention can not be formed or the technical effect of the invention can not be realized beyond the numerical value range.

Preferably, the mass percentages of type I collagen, MeNPs and water in the carrier are 0.4% type I collagen, 0.05% MeNPs and 99.55% water. Through a large number of experimental researches of the inventor, the in-situ hydrogel is formed when the components of the carrier are in the mass percentage, and the oxidation free radicals can be eliminated to the maximum extent.

The invention also provides a preparation method of the mesenchymal stem cell delivery vector, which comprises the following steps:

s1 preparing MeNPs;

s2 an aqueous solution of injectable type I collagen mixed with Menps was prepared.

Preferably, the method for preparing MeNPs according to step S1 includes the following steps: highly water-dispersible MeNPs are prepared by dissolving melanin granules in sodium hydroxide solution and then neutralizing by adding dilute aqueous hydrochloric acid under sonication.

Preferably, the method for preparing the mixed aqueous solution of injectable type I collagen and MeNPs of step S2 includes the following steps: type I collagen is dissolved in 2% by volume of acetic acid aqueous solution at 4 ℃, then sodium hydroxide aqueous solution is added to adjust the pH of the solution to be neutral, and finally, MeNPs dispersed in the aqueous solution are added to obtain injectable aqueous mixed solution of type I collagen and the MeNPs.

Further, the reactive oxygen species scavenging performance of the aqueous solution of injectable type I collagen mixed with the MeNPs was examined.

The invention also provides a hydrogel for treating limb ischemia, which is characterized by comprising 0.4 mass percent of type I collagen, 0.05 mass percent of MeNPs, 1.2 mass percent of GREM1-MSCs mesenchymal stem cells and 98.35 mass percent of water.

The invention also provides a preparation method of the hydrogel for treating limb ischemia, which is characterized by comprising the following steps:

s1, preparing a genetic engineering GREM1-MSCs mesenchymal stem cell, and constructing a stem cell over-expressing GREM1 gene;

s2 preparing MeNPs;

s3 preparing a mixed aqueous solution of injectable type I collagen and Menps;

s4 Grem1-MSC was mixed with the aqueous solution of step two at 4 deg.C, then the mixture was injected into ischemic lower limbs, and finally hydrogel containing Grem1-MSC was formed by heat-triggered gelation in situ.

Specifically, the invention prepares genetically engineered stem cells (Grem1-MSCs) and constructs stem cells over-expressing GREM1 genes. The method specifically comprises the following steps:

1. amplifying a promoter sequence attB4-promoter-attB1R (EF1 alpha promoter) with attB4 and attB1R sites by a PCR reaction, and then carrying out a recombination reaction between BP sites with an entry vector pDONTMP 4-P1R under the action of BP Clonase TM II Enzyme Mix to generate an entry clone pENTR-5' -EF1 alpha;

2. amplifying a target gene sequence attB1-gene-attB2 with attB1 and attB2 sites by PCR reaction, and then carrying out recombination reaction between BP sites with attP1 and attP2 sites of an entry vector pDONRTM221 under the action of BP Clonase II Enzyme Mix to generate an entry clone pENTR-GREM 1;

3. replacing attR1 and attR2 sites in pLenti6/BLOCK-iT-DEST with attR4 and attR2 by enzyme digestion and ligation reaction, replacing PGK with a promoter SV40 of a resistance gene, and replacing a Blasticidin sequence of the resistance gene with a Puromycin sequence to obtain target vectors pDESTR4-Puromycin-R2 and pDESTR 4-Blasticidin-R2;

4. under the action of LR Clonase TM II Enzyme Mix, the entry clone is connected with the target vector through site-specific connection reaction, and the vector pLV/final-PGK-Puromycin-EF1 alpha-GREM 1(EF1 alpha-GREM 1) is obtained.

5. The expression vector and the Packaging plasmid ViraPower Lentiviral Packaging Mix were co-transfected into 293FT cells at 4.5ug each using Lipofectamine 2000 for amplification. Collecting virus-containing supernatant culture solution after 48-72h, centrifuging at 4 deg.C and 3000rpm for 15min, and filtering to remove cell debris. Centrifuging at 50,000g for 2 hr at 4 deg.C, concentrating the supernatant, and storing in a refrigerator at-80 deg.C. MSC cells were taken and added to the supernatant. 5 days after virus infection, Puromycin is added for screening for 4-6 days, and the successfully infected MSC over-expressing GREM1 can be obtained.

Further, Grem1-MSCs performance was examined, including: digesting Grem1-MSCs from the culture flask, extracting mRNA, and detecting the expression condition of Grem1 by qPCR; extracting protein, and detecting Grem1 protein expression by WB; flow cytometry detected CD29, CD44, CD73, and CD90 expression.

The invention also provides the application of the melanin nano-particles in any one of the following applications:

(1) the application of the melanin nano-particles in preparing a preparation for eliminating the active oxygen of tissues or cells;

(2) the application of the melanin nano-particles in preparing preparations or medicines for treating limb ischemia or limb repair;

(3) the application of the melanin nano-particles in preparing angiogenesis promoting preparations or medicines.

The invention has the beneficial effects that: the injectable collagen/Menps mixed aqueous solution is used for delivery of genetically engineered stem cells (Grem1-MSCs) and treatment of limb ischemia. When the cell delivery system is administered locally, heat-triggered in situ gelation can maintain Grem1-MSC at the injection site, and embedded MeNPs in the gel network can scavenge high levels of ROS in ischemic tissues, thereby promoting cell survival. By improving cell retention and survival, Grem1-MSC can continuously secrete high-concentration Grem1, thereby promoting the proliferation of the cells and accelerating the angiogenesis of an ischemic part by activating a PI3K-AKT signal pathway in vascular endothelial cells, and finally promoting blood flow perfusion and tissue repair. The methods developed herein provide new treatment options for various ischemic diseases based on cell therapy, such as limb ischemia and myocardial infarction.

Drawings

FIG. 1 is a schematic diagram showing the results of the measurement of the reactive oxygen species scavenging reaction of the mixed aqueous solution of type I collagen and Menps: a microscopic morphology (a) and size distribution (B) of melanin nanoparticles; (C) is prepared by mixing melanin nanoparticle solution with 100 μ M H₂O₂After incubating for 10 minutes, adding benzidine (TMB) and horseradish peroxidase (HRP) to develop a color chart; (D) melanin nanoparticle solutions of different concentrations and 100 μ M H₂O₂After incubation for 10 minutes, adding fluorescence emission spectra of HRP and a reactive oxygen species (DFCH); a photograph (E) of an injectable collagen hydrogel doped with melanin nanoparticles and a microscopic topography (F) thereof; (G) injectable collagen hydrogel doped with melanin nanoparticles of different concentrations and 100 mu M H₂O₂After incubating for 10 minutes, adding TMB and HRP for developing; (H) injectable collagen hydrogel doped with melanin nanoparticles of different concentrations and 100 mu M H₂O₂After 10 minutes of incubation, fluorescence emission spectra of HRP and reactive oxygen indicator DFCH were added.

FIG. 2 is a schematic diagram of the performance detection result of the constructed GREM 1-MSCs: (A) expression of intracellular GREM1 mRNA from GREM 1-MSCs; (B) expression of GREM 1-intracellular GREM1 protein in MSCs; (C) expression of GREM1 protein in supernatant of GREM1-MSCs culture medium; (D) GREM1-MSCs cell surface marker expression; (E) expression of VEGF, EGF, HGF and PLGF growth factors in supernatants of GREM1-MSCs culture medium.

FIG. 3 is a schematic representation of the results of experiments on live and dead cells on collagen or hybrid hydrogels: (A) experiments on live and dead cells of MSCs or Grem1-MSCs on collagen or hybrid hydrogels (100. mu. M H)₂O₂Cultured for different times) live cells stained green fluorescence with calcein AM and dead cells stained red fluorescence with EthD-1. (B) Flow cytometry of MSCs or Grem1-MSCs for detection of collagen or Mixed hydrogels, followed by detection in a medium containing 100. mu. M H₂O₂For different periods of time.

FIG. 4 is a schematic diagram of the angiogenic effects of Grem1-MSCs and MSCs: (A) flow cytometry was used to examine the survival of human umbilical vein endothelial cells HUVECs on collagen or hybrid hydrogels. (B) HUVECs survival and Caspase 3 expression were detected by immunofluorescence. (C) HUVECs proliferation rate after supernatant concentration treatment. (D) The case of clone formation. (E) Capillary vessel formation. (F) And (3) analyzing the expression of AKT and p-AKT in HUVECs by Western blot.

Fig. 5. Results of different hydrogel treatment of hind limb ischemic mice are shown schematically: (A) laser doppler perfusion images of hindlimb ischemic mice treated with collagen hydrogel, hybrid hydrogel, collagen hydrogel or hybrid hydrogel (hybrid Gel3/Grem1-MSCs), and hindlimb recovery on day 21. (B) And quantitatively analyzing the blood perfusion recovery condition. (C) Tissue damage score. (D) Quantitatively analyzing the recovery condition of the ischemic injury tissues of the hind limbs. (E) HE staining showed tissue repair. (F) CD31 staining showed capillary repair after injury.

Detailed Description

In order to show technical solutions, purposes and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments.

Example 1

The mesenchymal stem cell delivery carrier comprises collagen type I, MeNPs and water, wherein the mass percentage of the MeNPs is 0.4% of the collagen type I, 0.05% of the MeNPs and 99.55% of the water.

Example 2

The embodiment provides a preparation method of a mesenchymal stem cell delivery vector, which comprises the following specific steps:

the method comprises the following steps: melanin nanoparticles (melanon nanoparticles, MeNPs) were prepared by the following steps:

highly water-dispersible MenPs were prepared by dissolving 20mg of melanin granules in 10ml of sodium hydroxide solution (NaOH, 0.1M) and then neutralizing by adding 0.1M aqueous hydrochloric acid under sonication. The resulting MeNPs showed a regular spherical morphology (fig. 1A). The MeNPs size distribution was found to be 8-40 nm by Dynamic Light Scattering (DLS) analysis, with an average size of about 20nm (FIG. 1B).

Step two: the preparation method comprises the following steps of preparing a mixed aqueous solution of injectable type I collagen and Menps: 20mg of type I collagen was dissolved in 5mL of a 2% by volume aqueous acetic acid solution at 4 ℃ to prepare a collagen solution having a concentration of 4mg/mL, followed by addition of 1M sodium hydroxide to adjust the pH of the solution to neutrality, and addition of 2.5mg of MeNPs to the solution to prepare a mixed aqueous solution having a type I collagen content of 0.4% by mass, a MeNPs content of 0.05% by mass, and a water content of 99.55% by mass. The mixed aqueous solution of type I collagen and MeNPs is placed at 37 ℃ for 3-5 minutes to form the thermally triggered in-situ gel, namely the collagen-MeNPs hybrid hydrogel (figure 1E). The observation of the scanning electron microscope shows that the formed collagen-Menps hybrid hydrogel has a porous network-like structure inside, and the microstructure is similar to that of the traditional collagen hydrogel (FIG. 1F). The collagen-Menps hybrid hydrogel is the mesenchymal stem cell delivery vector.

Test example 1

The MeNPs prepared in example 2 were evaluated for their ability to scavenge Reactive Oxygen Species (ROS) as follows:

the experimental principle is as follows: (1) horse Red Peroxidase (HRP) can catalyze H₂O₂Generating oxygenGas, then oxidizing colorless 3,3',5,5' -Tetramethylbenzidine (TMB) to make it display dark blue, when H₂O₂Is purged without oxygen generation, the TMB cannot be oxidized and continues to be colorless. (2) In HRP catalysis of H₂O₂In the case of oxygen generation, ROS probe 2', 7' -Dichlorodihydrofluorescein (DFCH) was oxidized and strong fluorescence was observed at 525nm (FIG. 1D)

MeNPs at concentrations of 0.1mg/mL, 0.25mg/mL, 0.5mg/mL were mixed with hydrogen peroxide (H) at a concentration of 100. mu.M, respectively₂O₂) Incubation of this H₂O₂The concentration approaches the ROS level in the identified ischemic tissue. As a result, as shown in FIG. 1C, when MeNPs were added at a concentration of 0.5mg/mL, H was₂O₂Effectively cleared, oxidation of TMB was inhibited and no blue color was observed; when the MeNPs were added at a concentration of 0.5mg/mL, the DFCH fluorescence intensity was significantly reduced, and only a weak signal was observed. The above experimental results show that Menps can effectively remove H₂O₂。

Test example 2

The collagen-MeNPs hybrid hydrogel prepared in example 2 was evaluated for its ability to scavenge Reactive Oxygen Species (ROS) as follows:

with the evaluation method in step one, as shown in FIGS. 1G and 1H, oxidation of both TMB and DFCH was suppressed, and blue light was reduced (FIG. 1G), and fluorescence intensity was reduced at 525nm (FIG. 1H). In particular Hybrid Gel3 (type I collagen, MeNPs in mass% of the vehicle 0.4% type I collagen and 0.05% MeNPs and 99.55% water), blue light of oxidized TMB was not visible, no blue color was observed, and very weak fluorescence of oxidized DFCH was detected at 525 nm. These results indicate that the hybrid hydrogels prepared by the present invention can effectively scavenge H₂O₂。

The mass percentages of the type I collagen and the MeNPs in the Hybrid Gel 1 (comparative example) are as follows: collagen type I0.2%, MeNPs 0.05%, water 99.75%.

The mass percentages of the type I collagen and the MeNPs in Hybrid Gel 2 (experimental example 1) are as follows: collagen type I0.3%, MeNPs 0.02%, water 99.68%.

The mass percentages of the type I collagen and the MeNPs in Hybrid Gel3 (Experimental example 2) are as follows: collagen type I0.4%, MeNPs 0.05%, water 99.55%.

Example 3

This example provides a hydrogel for treating limb ischemia, comprising type I collagen, MeNPs, water, and GREM1-MSCs mesenchymal stem cells, wherein the type I collagen is 0.4% by mass, the MeNPs is 0.05% by mass, the GREM1-MSCs mesenchymal stem cells are 1.2% by mass, and the water is 98.35% by mass.

Example 4

This example provides a hydrogel for treating limb ischemia, which is prepared by the following steps:

the method comprises the following steps: constructing over-expressed genetically engineered stem cells (Grem 1-MSCs):

constructing an over-expression Grem1 plasmid, transferring the over-expression Grem1 plasmid into MSCs by using lentivirus, and stably expressing Grem1, wherein the specific steps are as follows:

firstly, an entry clone pENTR-GREM1 is generated through PCR reaction amplification and a recombination reaction among BP sites, and then the entry clone is connected with a target vector through enzyme digestion and connection reaction, so that a lentivirus expression vector pLV/final-PGK-Puromycin-EF1 alpha-GREM 1(EF1 alpha-GREM 1) is obtained. The expression vector and the lentiviral Packaging plasmid ViraPowerLentiviral Packaging Mix were co-transfected into 293FT cells at 4.5ug each using Lipofectamine 2000 for lentiviral amplification. Collecting virus-containing upper layer culture solution after 48-72h, and storing concentrated virus solution for later use. Adding virus solution into MSCs cells. 5 days after virus infection, Puromycin is added for screening for 4-6 days, and MSCs (GREM1-MSCs) which successfully infect and over-express ISL1 can be obtained.

As shown in fig. 2A and 2B, high levels of Grem1 mRNA, determined experimentally by reverse transcription quantitative polymerase chain reaction (qRT-PCR, fig. 2A), and high Grem1 protein expression, determined by western blot analysis (fig. 2B), all showed stable expression of Grem1 in genetically engineered cells (Grem 1-MSCs). Furthermore, more than 3-fold secretion of Grem1 could also be induced from Grem1-MSC to the culture medium using gene manipulation techniques compared to parental MSCs (fig. 2C). Notably, transduction of Grem1 plasmid did not affect the sternness of MSCs, as evidenced by similar expression of MSCs and the typical sternness markers for Grem1-MSCs (including CD29, CD44, CD73 and CD90) (fig. 2D). Furthermore, this Grem1 transfection also did not show a significant effect on the secretion of other pro-angiogenic factors, such as VEGF, EGF, Hepatocyte Growth Factor (HGF) and placental growth factor (PLGF) (fig. 2E).

Step two, preparing collagen-Menps hybrid hydrogel

The collagen type I-MenPs mixed aqueous solution was prepared according to the method of example 2, and the mixed aqueous solution was added to a 12-well cell culture plate containing the above-mentioned GREM1-MSCs in a volume of 500. mu.L per well, and then the cell culture plate was placed in a cell culture chamber (at 37 ℃) for 10 minutes to form a collagen-Menps hybrid hydrogel containing GREM1-MSCs in the cell culture plate by heat-triggered in situ gelation, i.e., the hydrogel for treating ischemia in the limb.

Test example 3

1. Evaluation of cell survival promoting Capacity of collagen-Menps hybrid hydrogels

Following successful construction of Grem1-MSCs, collagen-MeNPs hybrid hydrogels were evaluated for their ability to promote stem cell survival in an in vitro simulated ischemic microenvironment. The results of the dead and live cell assay showed that both MSCs and Grem1-MSCs could adhere and survive on their surface, whether collagen hydrogel (without melanin nanoparticles) or collagen-MeNPs hybrid hydrogel (fig. 3Ai and Av). However, 100. mu.M of H was added₂O₂MSC cultured on the surface of collagen hydrogel could be significantly inhibited from surviving (fig. 3 Aii). By H₂O₂After 8 hours of treatment, many apoptotic cells (red fluorescence) were observed, and the morphology of most residual MSCs collapsed (fig. 3 Aiii). Although, genetic engineering could enhance ROS tolerance of Grem1-MSC to some extent (FIG. 3 Avi). However, long-term exposure to ROS still clearly caused apoptosis of Grem1-MSCs cultured on the surface of collagen hydrogel, such as bright red fluorescence and cell morphology collapse (fig. 3 Avii). In contrast, even with the addition of 100. mu. M H, the MenPs can scavenge ROS₂O₂The survival rates of MSCs (FIG. 3Aiv) and Grem1-MSCs (FIG. 3Aviii) cultured on the surface of collagen-Menps hybrid hydrogels remained high. Similar results were also shown in flow cytometry analysis, as shown in FIG. 3B, using 100 μ MH₂O₂Apoptosis rate of Grem1-MSC cultured on collagen hydrogel surface reached about 30% or 70% after 3 or 8 hours of treatment; further apoptosis analysis showed that this H₂O₂Treatment can activate caspase 3 expression, thereby inducing apoptosis of Grem 1-MSC. However, since MenPs can scavenge ROS, H is used₂O₂After 8h of treatment, survival of Grem1-MSC cultured on collagen-MeNPs hybrid hydrogel surface was higher than 80% (fig. 3B).

2. Evaluation of in vitro pro-angiogenic effects

High levels of ROS in ischemic tissue not only affect the survival of implanted stem cells, but can also induce apoptosis of vascular endothelial cells, a cell type that is of utmost importance in angiogenesis and tissue regeneration. In the present invention, since collagen-MeNPs hybrid hydrogels show good ROS scavenging effect, we speculate that this cell delivery system can also protect vascular endothelial cells from ROS-induced damage. As expected, the flow cytometry analysis results indicated that the hybrid hydrogel did significantly attenuate ROS-induced damage to Human Umbilical Vein Endothelial Cells (HUVEC), and that the hybrid hydrogel and 100. mu. M H₂O₂After a duration of 8h, approximately 85% of the cells remained viable (FIG. 4A). In contrast, the lack of Menps in scavenging ROS contained 100. mu. M H₂O₂More than 50% of the cells were apoptotic after 8h of culture on the collagen hydrogel. Further apoptosis analysis showed that this ROS-induced cell damage was mainly due to activation of caspase 3 expression to induce apoptosis (fig. 4B).

3. Evaluation of the angiogenesis promoting Effect of the hydrogel for the treatment of ischemia in limbs of example 3

After verifying the protective ability of the above hybrid hydrogels to prevent ROS-induced HUVEC injury, we next evaluated whether the hydrogels of example 3 for treating limb ischemia could exert a pro-angiogenic effect to promote HUVEC proliferation and angiogenesis. We collected Grem1-MSCs medium cultured on hybrid hydrogels (called conditioned Medium Grem1-MSCs-CM) and then examined the proliferation of HUVECs incubated in this conditioned medium. As shown in FIG. 4C, Grem1-MSCs-CM treatment promoted the proliferation of HUVEC and increased the number of cells by more than 10-fold within 6 days, much higher than HUVEC cells incubated in MSCs-CM. The results of the clone formation analysis (FIG. 4D) showed a similar trend, and more clones could be formed for HUVECs treated with Grem 1-MSCs-CM. In addition to improving the proliferation of HUVECs, Grem1-MSCs-CM treatment also enhanced their ability to form more capillary-like structures (fig. 4E), clearly demonstrating better pro-angiogenic effects of Grem1-MSCs compared to control MSCs.

Since transduction of the Grem1 plasmid did not affect secretion of other pro-angiogenic factors (including VEGF, EGF, HGF and PLGF), we predicted that the better pro-angiogenic effect of Grem1-MSCs was largely dependent on their ability to secrete more Grem 1. Grem1 has been shown to promote vascular endothelial cell proliferation and angiogenesis by specifically recognizing VEGFR2 expressed on endothelial cells and subsequently activating the downstream PI3K-AKT signaling pathway. Inspired by these findings, we analyzed the activity of the PI3K-AKT signaling pathway in conditioned medium-treated HUVECs using immunoblotting. As shown in FIG. 4F, although there was no significant difference in total AKT expression, the expression of phosphorylated AKT (p-AKT) was upregulated in cells treated with Grem1-MSCs-CM, indicating activation of the PI3K-AKT signaling pathway. If HUVEC were treated with AKT inhibitors (LY294002), phosphorylation of 59AKT was significantly down-regulated, resulting in limited cell proliferation (FIG. 4C), fewer colonies formed (FIG. 4D) and capillary-like structures (FIG. 4E). All these results strongly demonstrate that increased secretion of Grem1 by transduction of Grem1 plasmid can promote the pro-angiogenic effect of Grem1-MSC by specific recognition between Grem1 and VEGFR2, followed by activation of downstream PI3K-AKT signaling pathway in HUVECs.

4. Evaluation of hydrogel for treating limb ischemia of example 3 improves blood perfusion and limb repair

The ability of the hybrid hydrogel to transport Grem1-MSC in vivo for CLI treatment was evaluated using a hindlimb ischemic mouse model. After C57/BL6 mice are anesthetized and fixed, femoral artery and vein are separated by using a glass needle under a dissecting microscope, femoral artery is exposed, the femoral artery is cut and ligated from the inguinal ligament to the knee part, the branch is stripped, the ligation and stripping are carried out together, and the mouse lower limb unilateral limb ischemia model is established. Blood perfusion of injured hind limbs was monitored by laser doppler perfusion imager to assess the therapeutic effect of Hybrid Gel3 delivered Grem 1-MSC. As shown in fig. 5A and 5B, neither collagen gel nor hybrid hydrogel achieved the desired therapeutic effect due to the absence of pro-angiogenic factors (fig. 5A), and blood perfusion of the injured limb was maintained at a low level (fig. 5B). With the existence of Grem1-MSC, the high concentration of the angiogenesis promoting factor Grem1 is continuously secreted, and the collagen gel-Grem 1-MSC locally injected Grem1-MSC can improve the blood flow to a certain extent. In contrast, using Hybrid hydrogels to scavenge ROS and promote stem cell survival, local injections of Hybrid Gel3 into Grem1-MSCs (called Hybrid Gel3/Grem1-MSCs) could achieve better therapeutic results and perfuse stronger blood into injured limbs than mice treated with collagen Gel/Grem 1-MSC. Similar trends could be found in the results of the tissue damage score analysis (fig. 5C). Compared with the treatment with collagen Gel/Grem 1-MSCs, the use of Hybrid Gel3/Grem1-MSCs can significantly repair ischemic tissues, thereby causing a significant decrease in tissue damage score. The results of the lesion severity analysis also showed that Hybrid Gel3/Grem1-MSC had superior therapeutic efficacy to the other formulations (FIG. 5D). Over a long evaluation period of 21 days, mice treated with Hybrid Gel3/Grem1-MSC had the lowest foot necrosis rate (10%) and highest limb retention rate (90%) compared to other formulation treated mice. To determine whether the better therapeutic results of Grem1-MSCs encapsulated in the hybrid hydrogel were due to their pro-angiogenic effect, mice were sacrificed for histological analysis. As shown in fig. 5E, the muscles of the hind limbs of mice treated with hybrid gel3/Grem1-MSC were more compact than the hind limb muscles of mice treated with other formulations, which means that hybrid gel3/Grem1-MSC were more effective in tissue repair. Further Immunohistochemical (IHC) staining analysis showed stronger CD31 expression in hindlimbs of mice treated with hybrid gel3/Grem1-MSCs (fig. 5F), indicating that the use of Grem1-MSCs encapsulated in hybrid hydrogel can significantly enhance the formation of more capillaries in injured limbs. In general, the use of hybrid hydrogels for the delivery of Grem1-MSC can take advantage of its ROS scavenging properties to promote the survival of stem cells in ischemic tissues, thereby significantly accelerating blood flow restoration and improving limb function through Grem 1-mediated angiogenesis.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A vector for mesenchymal stem cell delivery, comprising the following components: type I collagen, melanin nanoparticles, and water; the mass percentages of the type I collagen and the melanin nano particles in the carrier are 0.3-0.4% of type I collagen, 0.02-0.05% of melanin nano particles and 99.55-99.68% of water.

2. The carrier of claim 1, wherein the mass percentages of the type I collagen and the melanin nanoparticles in the carrier are 0.4% of type I collagen, 0.05% of melanin nanoparticles and 99.55% of water.

3. A method of preparing the mesenchymal stem cell delivery vehicle of claim 1, comprising the steps of:

s1 preparing melanin nano particles;

s2 an injectable aqueous mixture of type I collagen and melanin nanoparticles is prepared.

4. The method of claim 3, wherein the step S1 of preparing the melanin nanoparticles comprises the steps of: the melanin particles are dissolved in an aqueous solution of sodium hydroxide, and then neutralized by adding a dilute aqueous solution of hydrochloric acid under ultrasonic treatment to prepare highly water-dispersed melanin nanoparticles.

5. The method of claim 3, wherein the step S2 is a method for preparing an injectable aqueous mixture of type I collagen and melanin nanoparticles, comprising the steps of: dissolving type I collagen in acetic acid water solution at 4 ℃, then adding sodium hydroxide water solution to adjust the pH of the solution to be neutral, and finally adding melanin nano particles dispersed in the water solution to prepare the water solution capable of injecting type I collagen and melanin nano particles.

6. The method according to claim 3, wherein the aqueous solution contains 0.3 to 0.4% by mass of type I collagen, 0.02 to 0.05% by mass of melanin nanoparticles, and 99.55 to 99.68% by mass of water.

7. The hydrogel for treating limb ischemia is characterized by comprising 0.4 mass percent of type I collagen, 0.05 mass percent of melanin nanoparticles, 1.2 mass percent of GREM1-MSCs mesenchymal stem cells and 98.35 mass percent of water.

8. A method of preparing a hydrogel for the treatment of limb ischemia as claimed in claim 7, comprising the steps of:

s1, preparing a genetic engineering GREM1-MSCs mesenchymal stem cell, and constructing a stem cell over-expressing GREM1 gene;

s2 preparing melanin nano particles;

s3 preparing a mixed aqueous solution of injectable type I collagen and melanin nanoparticles;

s4 Grem1-MSC was mixed with an aqueous solution of S3 at 4 deg.C and hydrogel containing Grem1-MSC was formed by thermally triggered in situ gelation.

Images