CN117757125A - Preparation method and application of methylpropionalized gelatin microsphere grafted with HAV polypeptide - Google Patents

Abstract

The invention discloses a preparation method and application of a methylpropionalized gelatin microsphere grafted with HAV polypeptide. The hydrogel microsphere prepared by the invention has dispersibility and good biocompatibility, and can carry rat tail vertebral nucleus pulposus cells to realize regeneration of the rat after the nuclear tissue is removed. The invention provides a new thought for the current research of using the polypeptide in cell and tissue engineering, and expands the application prospect of the polypeptide hydrogel material.

Description

Preparation method and application of methacrylated gelatin microspheres grafted with HAV polypeptide

Technical field

The invention belongs to the field of biological materials, and specifically relates to a preparation method and application of methacrylated gelatin microspheres grafted with HAV polypeptide.

Background technique

Lumbar disc herniation compresses the lumbar nerve roots and causes low back pain. In addition to conservative treatment such as bed rest, low back pain can be treated clinically by removing the protruding nucleus pulposus tissue through surgery to relieve the compression on the nerves. However, the removed nucleus pulposus tissue cannot regenerate, causing the intervertebral disc structure to be destroyed and lead to functional abnormalities, leading to recurrence of intervertebral disc herniation. Therefore, the use of tissue engineering to regenerate the resected nucleus pulposus tissue will become an effective means to completely cure intervertebral disc degeneration.

With the assistance of biological materials, transplanting nucleus pulposus cells into diseased intervertebral discs to achieve the regeneration of nucleus pulposus tissue is a common method for treating nucleus pulposus degeneration using tissue engineering. Usually, nucleus pulposus cells are combined with biomaterials that simulate the structure of their extracellular matrix (ECM), which not only helps the survival and proliferation of nucleus pulposus cells in vitro, but also promotes the coexistence of new tissue and native tissue. GelMA is prepared by the reaction of gelatin, a degradation product of collagen, and methacrylic anhydride (MA). In the presence of a photoinitiator, GelMA can form a hydrogel with good thermal stability under UV light irradiation. GelMA hydrogel retains the properties of gelatin, including the arginine-glycine-aspartic acid (RGD) peptide sequence that supports cell adhesion and proliferation, and has good biocompatibility and degradability. Therefore, GelMA can be used as an extracellular matrix-mimicking biomaterial in various studies of regenerative medicine and tissue engineering. However, GelMA is not injectable, and incision and implantation into the intervertebral disc will cause greater trauma. Therefore, GelMA hydrogel is made into injectable-sized GelMA hydrogel microspheres to meet the needs of minimally invasive treatment. In addition, microspheres increase the surface area and volume ratio of hydrogels, increase the contact area between cells and materials, and are more suitable for providing growth space for seed cells in tissue engineering. However, in addition to cell-ECM interactions, cell-cell interactions are also critical to the regulation of cell growth and development.

Contents of the invention

The purpose of this section is to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section, the abstract and the title of the invention to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the invention.

In view of the above and/or problems existing in the prior art, the present invention is proposed.

Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing methacrylated gelatin microspheres grafted with HAV polypeptides.

In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing methacrylated gelatin microspheres grafted with HAV polypeptide, including:

Dissolve methacrylic anhydride gelatin GelMA in a photoinitiator, use a microfluidic device to prepare hydrogel microspheres, and solidify;

Activate the carboxyl groups on the surface of GelMA hydrogel microspheres;

Soak the activated microspheres in a histidine-alanine-valine polypeptide solution, that is, a HAV polypeptide solution, to obtain GelMA microspheres grafted with HAV, which is methacrylated gelatin grafted with HAV polypeptides. Microspheres.

As a preferred embodiment of the preparation method of the present invention, GelMA is dissolved in a photoinitiator, wherein the photoinitiator is 0.1 to 0.2% LAP; the dosage ratio of GelMA to LAP is 100 to 150 mg: 1mL.

As a preferred embodiment of the preparation method of the present invention, the curing is curing the microsphere shape under ultraviolet light.

As a preferred embodiment of the preparation method of the present invention, the carboxyl groups on the surface of GelMA hydrogel microspheres are activated, and the activation method is to soak the GelMA hydrogel microspheres in a DMTMM solution.

As a preferred embodiment of the preparation method of the present invention, the concentration of the DMTMM solution is 10 to 15 mg/ml.

As a preferred embodiment of the preparation method of the present invention, the activated microspheres are soaked in a HAV polypeptide solution, wherein the concentration of the HAV polypeptide solution is 10 to 15 mg/ml.

Another object of the present invention is to overcome the deficiencies in the prior art and provide a methacrylated gelatin microsphere grafted with HAV polypeptide.

Another object of the present invention is to overcome the deficiencies in the prior art and provide a methacrylated gelatin microsphere grafted with HAV polypeptide for use in biomedicine.

Beneficial effects of the present invention:

(1) The GelMA-HAV hydrogel microspheres obtained by the present invention have good dispersibility.

(2) The hydrogel microspheres obtained by the present invention have good biocompatibility.

(3) The hydrogel microspheres obtained by the present invention can enhance the matrix synthesis ability of nucleus pulposus cells.

(4) The regeneration of the nucleus pulposus tissue can be achieved by implanting the GelMA-HAV hydrogel microspheres loaded with nucleus pulposus cells into the intervertebral disc after the nucleus pulposus has been removed.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings needed to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. Those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting any creative effort. in:

Figure 1 shows the preparation process of GelMA-HAV hydrogel microspheres prepared by the present invention.

Figure 2 shows the microstructure of GelMA-HAV hydrogel microspheres prepared by the present invention.

Figure 3 shows the effect of GelMA-HAV hydrogel microspheres prepared in the present invention on the viability of nucleus pulposus cells.

Figure 4 shows the effect of GelMA-HAV hydrogel microspheres prepared in the present invention on the proliferation of nucleus pulposus cells.

Figure 5 shows the effect of GelMA-HAV hydrogel microspheres prepared in the present invention on nucleus pulposus cell matrix synthesis.

Figure 6 shows the surgical method of nucleus pulposus resection.

Figure 7 shows the therapeutic effect detected by MRI.

Detailed ways

In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention will be described in detail below in conjunction with the examples in the description.

Many specific details are set forth in the following description to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described here. Those skilled in the art can do so without departing from the connotation of the present invention. Similar generalizations are made, and therefore the present invention is not limited to the specific embodiments disclosed below.

Second, reference herein to "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

Sources of raw materials and instruments used in the examples of the present invention:

Example 1

The preparation of GelMA-HAV hydrogel microspheres includes the following steps:

(1) Dissolve 100 mg GelMA in 1 mL of 0.1-0.2% photoinitiator LAP, use a microfluidic device to prepare the mixed hydrogel into hydrogel microspheres, and solidify the microsphere shape under ultraviolet light.

(2) Use 75% ethanol to clean the oil on the surface of the microspheres 2 to 3 times, then place it in PBS and observe the size and shape of the microspheres under an optical microscope.

(3) Soak the prepared GelMA microspheres in 10 mg/ml DMTMM (4-(4,6-dimethyl-1,3,5-triazinyl)-2,6-dimethylpyridine) solution , activate the carboxyl groups on the surface of GelMA microspheres.

(4) The activated GelMA microspheres are soaked in 10 mg/ml HAV peptide solution, HAV condenses with the activated carboxyl groups on the GelMA surface, and HAV is grafted to the surface of the microspheres.

(5) Freeze the HAV-grafted GelMA microspheres at -80°C for 8 to 12 hours, freeze-dry for 48 to 72 hours, and store at -20°C for subsequent use.

The preparation process of GelMA-HAV hydrogel microspheres is shown in Figure 1.

Example 2

The GelMA-HAV hydrogel microspheres prepared in Example 1 were characterized, and SEM was used to examine the micromorphology of the GelMA-HAV hydrogel microspheres.

The specific method is to dehydrate the prepared hydrogel microspheres, dry them at the critical point, and then freeze and brittle them. Place the cross section on the stage, use a plasma sputtering instrument to spray gold for 45 seconds, and place it under SEM for detection. As shown in Figure 2, it can be seen that grafting HAV polypeptide does not affect the porous structure of GelMA.

Example 3

Testing the in vitro biocompatibility of the GelMA-HAV hydrogel microspheres prepared in Example 1 includes the following steps:

(1) Use dead and live cell staining to detect the effect of hydrogel microspheres on cell viability:

Rat nucleus pulposus cells were inoculated into culture bottles and cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% double antibody.

When amplification reaches the P1 generation, use 0.25% trypsin to digest, centrifuge at 1500 rpm for 5 minutes and count the cell volume. Place the nucleus pulposus cells and hydrogel microspheres in a 15 mL centrifuge tube at a ratio of 10 ⁵ /4 mg microspheres and co-culture for 3 days. After three days, the microspheres loaded with nucleus pulposus cells were transferred to a low-adhesion 24-well plate.

On days 1, 3, and 5, add serum-free DMEM/F12 solution containing dead and live cell staining reagents, and incubate in a 37°C, 5% _CO2 incubator in the dark for 20 minutes. After the incubation, use a laser confocal microscope (scale bar = 200 μm) to take pictures and count the number of dead and living cells. As shown in Figure 3, it can be seen that the cell survival of the two groups of microspheres is more than 90%, indicating that GelMA-HAV microspheres do not affect Cell viability.

(2) Nucleus pulposus cells and hydrogel microspheres were co-cultured in a 15mL centrifuge tube at a ratio of 10 ⁵ /4 mg microspheres for 3 days. GelMA-HAV hydrogel microspheres loaded with nucleus pulposus cells were inoculated into a 96-well plate. , incubate with CCK-8 working solution at 37°C in the dark for 1 hour on days 1, 4, and 7.

A spectrophotometer was used to measure the absorbance (OD) value at 450 nm. The measurement results are shown in Figure 4. It can be seen that HAV has no obvious effect on cell proliferation.

(3) Nucleus pulposus cells and hydrogel microspheres were co-cultured in a 15mL centrifuge tube at a ratio of 10 ⁵ /4 mg microspheres for 3 days. GelMA-HAV hydrogel microspheres loaded with nucleus pulposus cells were inoculated into a 24-well plate. , add 0.3% Triton X-100 permeability for 10 min, and then block in 5% bovine serum albumin (BSA). After 20 minutes, the blocking solution was recovered, and ACAN primary antibody was added and incubated overnight at 4°C. The next day, recover the primary antibody and add the secondary antibody working solution coupled with Cy5 for incubation. The secondary antibody was incubated for 1 hour, and the cytoskeleton was stained with Phalloidin iFluor 594 reagent for 20 minutes, and then DAPI was added to stain the nucleus.

An inverted fluorescence microscope was used to capture images, as shown in Figure 5. It can be seen that ACAN is excited to emit red light, the cytoskeleton is excited to emit green light, and the cell nucleus is excited to emit blue light, indicating that GelMA-HAV hydrogel microspheres can enhance the nucleus pulposus. Cell matrix synthesis ability.

Example 4

Applying the GelMA-HAV hydrogel microspheres prepared in Example 1 to carry nucleus pulposus cells to regenerate nucleus pulposus tissue in vivo includes the following steps:

(1) Discectomy:

Male SD rats were ordered from the Animal Center of Soochow University. After being completely anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1.5 mL/kg), the Co7-8 and Co8-9 segments of the caudal intervertebral discs were disinfected with iodophor. A posterior midline incision is used to separate the soft tissue layer by layer and expose the intervertebral disc. Use a 23G needle to pierce the annulus fibrosus, and use a microspoon to dig out the NP tissue, as shown in Figure 6.

The experimental groups are as follows: the Sham group only incises the skin without puncturing the annulus fibrosus; the Discetomy group involves simple nucleus pulposus resection without the injection of microspheres or cells; the GelMA group and GelMA-HAV group simply inject GelMA microspheres and GelMA-HAV after nucleus pulposus resection. The HAV microspheres and NPCs group were simply injected with nucleus pulposus cells after nucleus pulposus resection. The GelMA+NPCs and GelMA-HAV+NPCs groups were injected with GelMA microspheres and GelMA-HAV microspheres loaded with nucleus pulposus cells after nucleus pulposus resection.

(2) Imaging observation:

MRI examination was performed 4 weeks after surgery. After being anesthetized with 4% sodium pentobarbital, the rats were placed on the MRI examination table. An MRI sagittal SE sequence T2WI scan was performed centered on the Co7-8 and Co8-9 intervertebral discs.

The scanning parameters are set as: TR: 3000ms, TE: 80ms, FOV: 200mm×200mm, Thick: 1.4mm.

Observe the signal intensity changes of the intervertebral disc on T2WI and score according to the modified Thompson criteria: Grade I: normal; Grade II: extremely slight signal intensity change but visible reduction in high signal area area; Grade III: moderate signal intensity reduction; IV: Significant signal strength loss.

The scan results are shown in Figure 7. It can be seen that the water content of the intervertebral discs in the Discectomy, GelMA, and GelMA-HAV groups has not recovered, while the NPCs group has slightly recovered. The GelMA+NPCs group has a slightly improved repair effect compared with the NPCs group. The GelMA-HAV+ The NPCs group has the most obvious recovery effect.

Example 5

The preparation of GelMA-HAV hydrogel microspheres includes the following steps:

(1) Dissolve 130 mg GelMA in 1 mL of 0.1 to 0.2% photoinitiator LAP, use a microfluidic device to prepare the mixed hydrogel into hydrogel microspheres, and solidify the microsphere shape under ultraviolet light.

(2) Use 75% ethanol to clean the oil on the surface of the microspheres 2 to 3 times, then place it in PBS and observe the size and shape of the microspheres under an optical microscope.

(3) Soak the prepared GelMA microspheres in 13 mg/ml DMTMM (4-(4,6-dimethyl-1,3,5-triazinyl)-2,6-dimethylpyridine) solution , activate the carboxyl groups on the surface of GelMA microspheres.

(4) The activated GelMA microspheres are soaked in 10 mg/ml HAV peptide solution, HAV condenses with the activated carboxyl groups on the GelMA surface, and HAV is grafted to the surface of the microspheres.

(5) Freeze the HAV-grafted GelMA microspheres at -80°C for 8 to 12 hours, freeze-dry for 48 to 72 hours, and store at -20°C for subsequent use.

Example 6

The preparation of GelMA-HAV hydrogel microspheres includes the following steps:

(1) Dissolve 150 mg GelMA in 1 mL of 0.1 to 0.2% photoinitiator LAP, use a microfluidic device to prepare the mixed hydrogel into hydrogel microspheres, and solidify the microsphere shape under UV light.

(2) Use 75% ethanol to clean the oil on the surface of the microspheres 2 to 3 times, then place it in PBS and observe the size and shape of the microspheres under an optical microscope.

(3) Soak the prepared GelMA microspheres in 15 mg/ml DMTMM (4-(4,6-dimethyl-1,3,5-triazinyl)-2,6-dimethylpyridine) solution , activate the carboxyl groups on the surface of GelMA microspheres.

(4) The activated GelMA microspheres are soaked in 10 mg/ml HAV peptide solution, HAV condenses with the activated carboxyl groups on the GelMA surface, and HAV is grafted to the surface of the microspheres.

(5) Freeze the HAV-grafted GelMA microspheres at -80°C for 8 to 12 hours, freeze-dry for 48 to 72 hours, and store at -20°C for subsequent use.

Example 7

The preparation of GelMA-HAV hydrogel microspheres includes the following steps:

(1) Dissolve 100 mg GelMA in 1 mL of 0.1-0.2% photoinitiator LAP, use a microfluidic device to prepare the mixed hydrogel into hydrogel microspheres, and solidify the microsphere shape under ultraviolet light.

(2) Use 75% ethanol to clean the oil on the surface of the microspheres 2 to 3 times, then place it in PBS and observe the size and shape of the microspheres under an optical microscope.

(3) Soak the prepared GelMA microspheres in 10 mg/ml DMTMM (4-(4,6-dimethyl-1,3,5-triazinyl)-2,6-dimethylpyridine) solution , activate the carboxyl groups on the surface of GelMA microspheres.

(4) The activated GelMA microspheres are soaked in 13 mg/ml HAV peptide solution, HAV condenses with the activated carboxyl groups on the GelMA surface, and HAV is grafted to the surface of the microspheres.

(5) Freeze the HAV-grafted GelMA microspheres at -80°C for 8 to 12 hours, freeze-dry for 48 to 72 hours, and store at -20°C for subsequent use.

Example 8

The preparation of GelMA-HAV hydrogel microspheres includes the following steps:

(1) Dissolve 100 mg GelMA in 1 mL of 0.1-0.2% photoinitiator LAP, use a microfluidic device to prepare the mixed hydrogel into hydrogel microspheres, and solidify the microsphere shape under ultraviolet light.

(2) Use 75% ethanol to clean the oil on the surface of the microspheres 2 to 3 times, then place it in PBS and observe the size and shape of the microspheres under an optical microscope.

(3) Soak the prepared GelMA microspheres in 10 mg/ml DMTMM (4-(4,6-dimethyl-1,3,5-triazinyl)-2,6-dimethylpyridine) solution , activate the carboxyl groups on the surface of GelMA microspheres.

(4) The activated GelMA microspheres are soaked in 15 mg/ml HAV peptide solution, HAV condenses with the activated carboxyl groups on the GelMA surface, and HAV is grafted to the surface of the microspheres.

(5) Freeze the HAV-grafted GelMA microspheres at -80°C for 8 to 12 hours, freeze-dry for 48 to 72 hours, and store at -20°C for subsequent use.

Comparative example 1

The preparation of GelMA-HAV hydrogel microspheres includes the following steps:

(1) Dissolve 100 mg GelMA in 1 mL of 0.1-0.2% photoinitiator LAP, use a microfluidic device to prepare the mixed hydrogel into hydrogel microspheres, and solidify the microsphere shape under ultraviolet light.

(2) Use 75% ethanol to clean the oil on the surface of the microspheres 2 to 3 times, then place it in PBS and observe the size and shape of the microspheres under an optical microscope.

(3) Soak the prepared GelMA microspheres in 10 mg/ml DMTMM (4-(4,6-dimethyl-1,3,5-triazinyl)-2,6-dimethylpyridine) solution , activate the carboxyl groups on the surface of GelMA microspheres.

(4) The activated GelMA microspheres are soaked in 9 mg/ml HAV peptide solution, HAV condenses with the activated carboxyl groups on the GelMA surface, and HAV is grafted to the surface of the microspheres.

(5) Freeze the HAV-grafted GelMA microspheres at -80°C for 8 to 12 hours, freeze-dry for 48 to 72 hours, and store at -20°C for subsequent use.

The GelMA-HAV hydrogel microspheres prepared in Examples 5 to 8 and Comparative Examples 1 and 2 were used to carry nucleus pulposus cells to regenerate nucleus pulposus tissue in vivo. Male SD rats were ordered from the Animal Center of Suzhou University and injected intraperitoneally for 3 After complete anesthesia with % sodium pentobarbital (1.5 mL/kg), the coccygeal intervertebral disc Co7-8 and Co8-9 segments were disinfected with iodophor. A posterior midline incision is used to separate the soft tissue layer by layer and expose the intervertebral disc. A 23G needle was used to pierce the annulus fibrosus, and a microspoon was used to dig out the NP tissue. After the nucleus pulposus was removed, GelMA-HAV microspheres loaded with nucleus pulposus cells were injected.

MRI examination was performed 4 weeks after surgery. After being anesthetized with 4% sodium pentobarbital, the rats were placed on the MRI examination table. An MRI sagittal SE sequence T2WI scan was performed centered on the Co7-8 and Co8-9 intervertebral discs. The scanning parameters were the same as those in Example 4. The order of excellence in the repair effect was Example 8, Example 7, Example 6, and Example 5, Comparative Example 1, but the repair effects are far inferior to the GelMA-HAV+NPCs group of Example 4.

The present invention proposes a method for preparing GelMA-HAV hydrogel microspheres and applies it in the field of nucleus pulposus tissue regeneration. The GelMA-HAV hydrogel microspheres prepared by this method have good dispersibility and biocompatibility. Implanting the GelMA-HAV hydrogel microspheres loaded with nucleus pulposus cells into the intervertebral disc after the nucleus pulposus tissue has been removed can enhance the matrix synthesis ability of the nucleus pulposus cells and achieve the regeneration of the nucleus pulposus tissue. This is a new step for the current use of polypeptides in cell and tissue engineering. The research provides new ideas and expands the application prospects of hydrogel microspheres.

The present invention chooses to graft HAV polypeptides onto GelMA hydrogel microspheres to artificially construct a surface similar to the aggregation state of nucleus pulposus cells for the culture of nucleus pulposus cells. While satisfying the cell-ECM interaction, it can also pass through the cell-cell Interact, regulate intracellular signaling pathways, and enhance the ECM synthesis ability of nucleus pulposus cells on the surface of microspheres.

It should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solution of the present invention can be carried out. Modifications or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of the present invention.

Claims (8)

1. A preparation method of methyl-propylene gelatin microspheres grafted with HAV polypeptide is characterized in that: comprising the steps of (a) a step of,

dissolving methacrylic anhydride gelatin GelMA in a photoinitiator, preparing hydrogel microspheres by using a microfluidic device, and curing;

activating carboxyl groups on the surface of the GelMA hydrogel microsphere;

soaking the activated microsphere in histidine-alanine-valine polypeptide solution, namely HAV polypeptide solution, to obtain a GelMA microsphere grafted with HAV, namely a methylpropionalized gelatin microsphere grafted with HAV polypeptide.

2. The method of manufacturing according to claim 1, wherein: the GelMA is dissolved in a photoinitiator, wherein the photoinitiator is LAP with the concentration of 0.1-0.2%; the dosage ratio of GelMA to LAP is 100-150 mg:1mL.

3. The method of manufacturing according to claim 1, wherein: the curing is to cure the microsphere shape under ultraviolet irradiation.

4. The method of manufacturing according to claim 1, wherein: the carboxyl on the surface of the GelMA hydrogel microsphere is activated, wherein the activation method is to soak the GelMA hydrogel microsphere in DMTMM solution.

5. The method of manufacturing according to claim 4, wherein: the concentration of the DMTMM solution is 10-15 mg/ml.

6. The method of manufacturing according to claim 1, wherein: the activated microsphere is soaked in HAV polypeptide solution, wherein the concentration of the HAV polypeptide solution is 10-15 mg/ml.

7. A methylpropionalized gelatin microsphere grafted with HAV polypeptide produced by the process of any of claims 1 to 6.

8. Use of the methylpropionalized gelatin microsphere grafted with HAV polypeptide according to claim 7 in biological medicine.