A kind of cuttlefish juice hydrogel microsphere and its preparation method and application

Abstract

The present invention relates to the field of biomaterial technology, and more specifically, to a cuttlefish juice hydrogel microsphere and a preparation method and application thereof, wherein the preparation method comprises uniformly mixing cuttlefish juice nanoparticles with methacrylated gelatin and then preparing the microspheres by microfluidic technology. The present invention constructs a cuttlefish juice hydrogel microsphere, which exhibits good biocompatibility and cell adhesion, and can effectively enhance the antioxidant capacity of nucleus pulposus cells, and ultimately promote the regeneration of nucleus pulposus tissue. The present invention not only provides a new strategy for the treatment of intervertebral disc degeneration, but also opens up new possibilities for the application of traditional Chinese medicine in modern medicine.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, in particular to cuttlefish juice hydrogel microspheres and a preparation method and application thereof. Background Art

[0002] Low back pain is a global public health problem that causes huge labor loss and medical burden to society, and intervertebral disc degeneration is one of the main causes of low back pain.

[0003] The intervertebral disc is composed of the central nucleus pulposus, the surrounding annulus fibrosus and the upper and lower cartilage endplates. It is an important load-bearing structure of the spine. In the degenerated intervertebral disc, a large number of nucleus pulposus cells are lost, the metabolism of the extracellular matrix is ​​unbalanced, and there is oxidative stress and chronic inflammatory response, which ultimately leads to the destruction of the intervertebral disc structure and the loss of functional integrity.

[0004] For disc degeneration, the existing treatment methods are mainly to relieve symptoms, such as bed rest and neurotrophic drug therapy. For patients with severe symptoms or ineffective conservative treatment, surgery may be required to remove the damaged disc, but surgery has the risk of reducing spinal mobility and accelerating the degeneration of adjacent segments. Therefore, there is still a lack of treatment options that can safely and effectively delay disc degeneration.

[0005] In addition, there is a vicious cycle in the development of disc degeneration: the diffusion and oxygenation of nutrients in the disc are impaired, leading to excessive accumulation of matrix decomposition products and reactive oxygen species, which further damage cell function, thereby accelerating the production of reactive oxygen species and worsening the microenvironment of the disc. In this context, the application of antioxidants and the supplementation of healthy nucleus pulposus cells to improve the microenvironment of disc degeneration is particularly important, which is considered to be a feasible research direction for delaying disc degeneration.

[0006] Modern scientific research shows that nanoparticles extracted from cuttlefish juice are rich in antioxidants. Its main ingredient, melanin, can effectively neutralize reactive oxygen through hydrogen atom transfer and single electron transfer mechanisms, and promote heme decomposition through the heme oxygenase-1 pathway to produce by-products with antioxidant effects, thereby enhancing the ability of cells to resist oxidative stress.

[0007] However, since the intervertebral disc is an avascular tissue and has a first-pass effect in the gastrointestinal tract and liver, the bioavailability of oral nanoparticles is limited, which may lead to poor therapeutic effects on intervertebral disc degeneration. Therefore, there is no precedent in the prior art for applying cuttlefish juice nanoparticles to treat intervertebral discs, and there is also a lack of an effective strategy to delay intervertebral disc degeneration. Summary of the invention

[0008] The invention provides a cuttlefish juice hydrogel microsphere and a preparation method and application thereof, which can overcome the defect that the bioavailability of cuttlefish juice nanoparticles is limited and cannot be used to delay intervertebral disc degeneration.

[0009] A method for preparing cuttlefish juice hydrogel microspheres comprises: uniformly mixing cuttlefish juice nanoparticles and methacrylated gelatin at a mass ratio of 1:1000-1:500, and then preparing the microspheres by using a microfluidic technology.

[0010] Methacryloyl gelatin, as a derivative of gelatin, a collagen degradation product, retains the key arginine-glycine-aspartic acid peptide sequence that promotes cell adhesion and proliferation. In addition, due to the introduction of methacrylate groups, it has photosensitivity and can quickly initiate polymerization under ultraviolet light of a specific wavelength, forming a stable hydrogel network structure with excellent mechanical properties. It can not only improve cell delivery, provide cells with a good adhesion and proliferation environment, and help cell survival and function; it can also be used as a drug carrier to achieve sustained and controlled release of growth factors, cytokines or other therapeutic agents, reducing the need for repeated administration.

[0011] Therefore, after mixing with cuttlefish juice nanoparticles, it can precisely control hydrogel formation and cell delivery. Its combination with cuttlefish juice nanoparticles can not only provide a highly biocompatible scaffold environment for nucleus pulposus cells, but also its excellent antioxidant properties help to reduce oxidative stress and protect cell viability and function in artificial nucleus pulposus, providing an effective strategy for the treatment of intervertebral disc degeneration in the field of tissue engineering and regenerative medicine.

[0012] As a preference, the method specifically comprises the following steps:

[0013] adding cuttlefish ink nanoparticles and methacrylated gelatin into a photoinitiator and uniformly mixing them to obtain a composite;

[0014] The composite was prepared into cuttlefish juice hydrogel microspheres by microfluidics technology, and then cured by ultraviolet light to obtain cured microspheres;

[0015] The solidified microspheres are washed and freeze-dried to obtain cuttlefish juice hydrogel microspheres.

[0016] Preferably, the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphite, which is prepared from deionized water or phosphate buffer.

[0017] Preferably, the concentration of the photoinitiator is 0.05%-0.25%.

[0018] Preferably, the concentration of the cuttlefish ink nanoparticles is 0.05-0.5 mg / ml, and the concentration of the methacrylated gelatin is 50-200 mg / ml.

[0019] Preferably, the cuttlefish juice nanoparticles are extracted from cuttlefish juice by differential centrifugation.

[0020] Preferably, extracting cuttlefish juice nanoparticles from cuttlefish juice by differential centrifugation comprises the following steps:

[0021] Centrifuge the squid ink at 1000-3000 rpm / min for 5 min;

[0022] Adjust the speed to 10000-15000 rpm / min and centrifuge again for 10 min;

[0023] The precipitate was recovered and freeze-dried to obtain dried cuttlefish juice nanoparticles.

[0024] Preferably, a mixed solution of ethanol and isopropanol is used to clean the solidified microspheres, wherein the volume ratio of ethanol to isopropanol is 1:1-5:1.

[0025] A cuttlefish juice hydrogel microsphere is prepared by the aforementioned method for preparing cuttlefish juice hydrogel microspheres. The prepared cuttlefish juice hydrogel microspheres not only have good biocompatibility, cell adhesion and antioxidant properties, but also can effectively load rat nucleus pulposus cells to achieve regeneration of nucleus pulposus tissue after removal in rats.

[0026] A use of the aforementioned cuttlefish juice hydrogel microspheres in intervertebral disc degeneration products.

[0027] The present invention has significant technical effects due to the adoption of the above technical solution:

[0028] The present invention constructs a cuttlefish juice hydrogel microsphere, which exhibits good biocompatibility and cell adhesion, and can effectively enhance the antioxidant capacity of nucleus pulposus cells, ultimately promoting the regeneration of nucleus pulposus tissue. The present invention not only provides a new strategy for the treatment of intervertebral disc degeneration, but also opens up new possibilities for the application of traditional Chinese medicine in modern medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The preparation flow chart of cuttlefish juice hydrogel microspheres GelMA@CINPs;

[0030] Figure 2 The amino acid and monosaccharide analysis results of squid juice nanoparticles CNIPs are shown in Figure 2.

[0031] Figure 3 The microstructure of squid juice hydrogel microspheres GelMA@CINPs;

[0032] Figure 4This is a comparison of the effects of squid juice hydrogel microspheres GelMA@CINPs and ordinary GelMA microspheres on the survival of nucleus pulposus cells;

[0033] Figure 5 This is a comparison of the effects of squid juice hydrogel microspheres GelMA@CINPs and ordinary GelMA microspheres on the proliferation of nucleus pulposus cells;

[0034] Figure 6 This is a comparison of the effects of squid juice hydrogel microspheres GelMA@CINPs and ordinary GelMA microspheres on the antioxidant capacity of nucleus pulposus cells;

[0035] Figure 7 This is a staining image of the pathological section of the nucleus pulposus tissue regenerated by cuttlefish juice hydrogel microspheres loaded with nucleus pulposus cells. DETAILED DESCRIPTION

[0036] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the embodiments are only for explaining the present invention and are not intended to limit it.

[0037] like Figure 1 As shown, a method for preparing cuttlefish juice hydrogel microspheres comprises the following steps:

[0038] Step S1, first centrifuge the cuttlefish juice at a speed of 2000 rpm / min for 5 min;

[0039] Step S2, adjusting the speed to 12000 rpm / min, and centrifuging again for 10 min;

[0040] Step S3, recovering the precipitate and freeze-drying it to obtain cuttlefish juice nanoparticles;

[0041] Step S4, preparing 0.1% of the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite LAP with PBS, and adding 100 μg of cuttlefish ink nanoparticles CNIPs and 100 mg of methacrylated gelatin GelMA thereto, respectively, and mixing them thoroughly;

[0042] Step S5, preparing the composite in step S4 into cuttlefish juice hydrogel microspheres by microfluidics technology, and curing them into a stable shape by ultraviolet light;

[0043] Step S6, mixing ethanol and isopropanol in a volume ratio of 5:1, and soaking and cleaning the microspheres with the mixed solution;

[0044] Step S5, recovering the microspheres and freeze-drying them to obtain dried cuttlefish juice hydrogel microspheres GelMA@CINPs.

[0045] Example 2 Amino Acid and Monosaccharide Analysis of Cuttlefish Ink Nanoparticles

[0046] The amino acid composition of CINPs was analyzed in detail by an amino acid analyzer, and the composition of monosaccharides in CINPs was determined using high-performance liquid chromatography (HPLC).

[0047] Comparative Example 1

[0048] The same as Example 1, except that in step S4 of this example, no cuttlefish juice nanoparticles were added, and finally ordinary GelMA microspheres were obtained.

[0049] Comparative Example 2 Cell activity and proliferation test

[0050] The GelMA@CINPs microspheres and GelMA microspheres obtained in Example 1 and Comparative Example 1 were immersed in 75% ethanol solution for disinfection for 30 minutes, then rinsed three times with PBS solution, and placed in a six-well plate to dry naturally for subsequent use.

[0051] Rat nucleus pulposus cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. Fresh medium was replaced every two days in the incubator at 37°C and 5% CO2. When the cells grew to 70%-80%, they were passaged, and P1-P3 cells were selected for subsequent experiments. In a six-well plate, 100 μL of medium containing 5×105 rat nucleus pulposus cells was added to each well. After 30 minutes of culture in the incubator, 2.5 mL of medium was added to each well. After 3 days of co-culture, the two hydrogel microspheres loaded with nucleus pulposus cells were transferred.

[0052] Live / dead staining reagent was used to evaluate the activity of cells on GelMA@CINPs microspheres and GelMA microspheres. The specific operation was to plant two microspheres containing nucleus pulposus cells in a 24-well plate, and after 1, 3 and 5 days of culture, incubate them at 37°C in the dark for 30 minutes using live / dead staining reagent, and then observe the cell status using a fluorescence microscope.

[0053] The proliferation ability of nucleus pulposus cells on GelMA@CINPs microspheres and GelMA microspheres was evaluated using a CCK-8 kit. Microspheres loaded with nucleus pulposus cells were seeded in a 96-well plate and cultured in a medium containing 10% CCK-8 solution for 1 hour after 1, 3, and 5 days of culture. Finally, the absorbance was measured at a wavelength of 450nm using a spectrophotometer to evaluate cell proliferation.

[0054] Comparative Example 3 Antioxidant Capacity Test

[0055] The GelMA@CINPs microspheres and GelMA microspheres obtained in Example 1 and Comparative Example 1 were co-cultured with nucleus pulposus cells in complete culture medium at 37°C and 5% CO2 for 48 hours. After that, the culture medium was replaced with a culture medium containing 1mM hydrogen peroxide (H2O2) and cultured for 24 hours, maintaining the same other culture conditions. After oxidative stress intervention, the cells were washed with PBS solution, and then incubated with 10 μM DCFH-DA solution at 37°C for 20 minutes. After washing again with PBS solution, the samples were observed using a fluorescence microscope.

[0056] Comparative Example 4: In vivo experiment to reconstruct the nucleus pulposus tissue of rat intervertebral disc

[0057] Six-week-old male SD rats were purchased and housed in an environment with controlled humidity (50-60%), temperature (22-24°C), and light cycle (6 am to 6 pm). Before surgery, SD rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1.5 mL / kg). A longitudinal incision was made 2 cm above the coccyx. After separating the ligaments, the annulus fibrosus was penetrated with a 22-gauge needle, and the nucleus pulposus tissue was extracted with a 5-mL syringe. The remaining nucleus pulposus tissue was removed with a curette. The rats were randomly divided into seven experimental groups: sham group, discectomy group, nucleus pulposus cell (NPCs) group, GelMA microsphere group, GelMA microsphere + NPCs group, GelMA@CINPs microsphere group, and GelMA@CINPs microsphere + NPCs group. After removing the nucleus pulposus, the cell suspension or microsphere solution (10 μL) was injected into the intervertebral disc by syringe, and then the annulus fibrosus was sutured. For three consecutive days after surgery, penicillin (80,000 units per rat) was injected into the thigh muscle of the rats to prevent infection.

[0058] After the MRI assessments at weeks 4 and 8, samples were collected from the caudal vertebrae of the rats. The collected samples were first fixed in 4% formaldehyde solution for one week and then immersed in 10% ethylenediaminetetraacetic acid (EDTA) solution for 6 weeks. After processing, the samples were embedded in paraffin and 5-mm-thick sections were cut from them using a microtome. After deparaffinization, the sections were stained with hematoxylin-eosin (HE) and Safranin O-fast green (SO) to observe the structural characteristics of the intervertebral disc and the deposition of proteoglycans.

[0059] According to Example 2, the cuttlefish juice nanoparticles were analyzed for amino acids and monosaccharides. Figure 2 As shown, the results showed that proline accounted for 21% of the total amino acids and fucose accounted for 58% of the monosaccharide composition.

[0060] The GelMA@CINPs microspheres and GelMA microspheres obtained in Example 1 and Comparative Example 1 were photographed under a scanning electron microscope and found that the pore size of the GelMA@CINPs hydrogel microspheres was reduced compared to that of GelMA. Figure 3 shown.

[0061] Through the experiments of Comparative Examples 2-3, it was found that the cell survival rate of the two groups of microspheres was above 90%, indicating that the squid juice hydrogel microspheres would not affect the cell viability. Figure 4 shown.

[0062] In addition, if Figure 5 , Figure 6 As shown, cuttlefish juice hydrogel microspheres not only have a better effect in promoting cell proliferation, but also can significantly improve the antioxidant capacity of nucleus pulposus cells.

[0063] The in vivo experiment of Comparative Example 4 was used to reconstruct the nucleus pulposus tissue of rat intervertebral discs. The results of the case sections indicated that implanting squid juice hydrogel microspheres loaded with nucleus pulposus cells into the intervertebral discs from which the nucleus pulposus had been removed could promote the regeneration of the nucleus pulposus tissue. Figure 7 shown.

[0064] It is easy to understand that those skilled in the art can combine, split, reorganize, etc. the embodiments of the present application to obtain other embodiments based on one or several embodiments provided in the present application, and these embodiments do not exceed the protection scope of the present application.

[0065] The present invention and its implementation methods are described schematically above, and the description is not restrictive. The embodiments shown in the embodiments are only part of the implementation methods of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by the embodiments and designs a structure and an implementation method similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing cuttlefish juice hydrogel microspheres, characterized in that: include: The cuttlefish juice nanoparticles and methacrylated gelatin were uniformly mixed in a mass ratio of 1:1000-1:500 and then prepared by microfluidic technology; adding cuttlefish ink nanoparticles and methacrylated gelatin into a photoinitiator and uniformly mixing them to obtain a composite; The composite is prepared into cuttlefish juice hydrogel microspheres by microfluidics technology, and then cured by ultraviolet light to obtain cured microspheres; The solidified microspheres are washed and freeze-dried to obtain cuttlefish juice hydrogel microspheres; The cuttlefish juice nanoparticles are extracted from the cuttlefish juice by differential centrifugation.

2. The method for preparing cuttlefish juice hydrogel microspheres according to claim 1, characterized in that: The photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphite, which is prepared by deionized water or phosphate buffer.

3. The method for preparing cuttlefish juice hydrogel microspheres according to claim 1 or 2, characterized in that: The concentration of the photoinitiator is 0.05%-0.25%.

4. The method for preparing cuttlefish juice hydrogel microspheres according to claim 1, characterized in that: The concentration of the cuttlefish ink nanoparticles was 0.05-0.5 mg / ml, and the concentration of the methacrylated gelatin was 50-200 mg / ml.

5. The method for preparing cuttlefish juice hydrogel microspheres according to claim 1, characterized in that: Extracting cuttlefish juice nanoparticles from cuttlefish juice by differential centrifugation specifically comprises the following steps: Centrifuge the squid ink at 1000-3000 rpm / min for 5 min; Adjust the speed to 10000-15000 rpm / min and centrifuge again for 10 min; The precipitate was recovered and freeze-dried to obtain dried cuttlefish juice nanoparticles.

6. The method for preparing cuttlefish juice hydrogel microspheres according to claim 1, characterized in that: The solidified microspheres are cleaned with a mixed solution of ethanol and isopropanol, wherein the volume ratio of ethanol to isopropanol is 1:1-5:

1.

7. A cuttlefish juice hydrogel microsphere, characterized in that: The cuttlefish juice hydrogel microspheres are prepared by the method for preparing the cuttlefish juice hydrogel microspheres according to any one of claims 1 to 6.

8. Use of the cuttlefish juice hydrogel microspheres according to claim 7 in intervertebral disc degeneration products.

Images