Chondroitin sulfate proteoglycan analog microspheres and preparation method thereof

Abstract

The present invention relates to chondroitin sulfate proteoglycan analog microspheres and a preparation method thereof, belonging to the field of biomedicine technology. The preparation method comprises the following steps: S1, enzymatically hydrolyzing chondroitin sulfate using chondroitinase to obtain chondroitin sulfate oligosaccharides; S2, coupling reaction of the activated chondroitin sulfate oligosaccharides with methacryloylated gelatin with a low grafting rate to obtain chondroitin sulfate proteoglycan analogs; S3, dissolving the chondroitin sulfate proteoglycan analogs, methacryloylated gelatin with a high grafting rate, and a photoinitiator in water to obtain a dispersed phase; the oil phase material is the continuous phase; S4, using a microfluidic device to form the dispersed phase and the continuous phase into water-in-oil droplets, which are then cured by ultraviolet light and washed to obtain microspheres. The microspheres of the present invention can regulate the microenvironment of the lesion after spinal cord injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a chondroitin sulfate proteoglycan analog microsphere and a preparation method thereof. Background Art

[0002] The spinal cord is the place where the human body's nerves are most densely concentrated, and is responsible for transmitting nerve signals between the brain and various parts of the body. When the spinal cord is injured, the nerves may be compressed to varying degrees or even broken, leading to loss of nerve function. This complex physiological structure makes the treatment of spinal cord injury particularly difficult. Spinal cord injury, as a serious disabling injury, often causes varying degrees of paraplegia or quadriplegia. The somatic dysfunction and autonomic dysfunction it causes have seriously affected the patient's ability to take care of themselves and participate in social activities. Due to the fact that nerve cells cannot regenerate, improving the microenvironment of the injured area is the key to accelerating axon regeneration and synapse formation. Therefore, tissue engineering is an effective means to improve the microenvironment of the spinal cord lesion area.

[0003] Unlike other tissues, the central nervous system is specifically rich in chondroitin sulfate proteoglycans (CSPGs). These proteoglycans have a highly similar core protein structure, with glycosaminoglycan side chains of varying structure and density linked to the core protein peptide chain via serine residues. Their content and distribution exhibit remarkable spatiotemporal specificity. Under normal physiological conditions, CSPGs wrap around the soma or proximal dendrites of certain neurons in the form of a neural grid, participating in stabilizing existing synapses, inhibiting the morphology of abnormally linked synapses, and regulating interactions between cells and within the extracellular matrix. In pathological conditions, however, glial scars form at the site of injury, where CSPG content rapidly increases, inhibiting neural repair.

[0004] However, due to the complexity of CSPG structure and the difficulty of obtaining it, it is difficult to conduct in-depth research on the structure-activity mechanism of proteoglycans and nerve regeneration. At present, proteoglycans are mainly mixtures extracted from animal connective tissue, which makes quality control difficult, and there is a risk of viral contamination in biological products. There are no related commercial products. There are also few reports on the research of artificially synthesized proteoglycans. At the same time, spinal cord tissue engineering usually uses collagen, hyaluronic acid and other materials as hydrogel matrices. Due to their non-injectability, cutting the vertebrae will cause greater trauma. Therefore, it is necessary to artificially synthesize proteoglycan analogs to further study their structure-activity mechanism in various physiological and pathological conditions. Summary of the Invention

[0005] To address the above-mentioned technical problems, the present invention provides a chondroitin sulfate proteoglycan analog microsphere and its preparation method. These microspheres mimic the comb-shaped chain structure and sugar chain structure of natural proteoglycans, using methacryloylated gelatin as the core protein backbone. Sugar chains of varying sizes and subtypes are grafted onto the surface via an amidation reaction, thereby achieving controllable sugar chain subtypes and sizes of chondroitin sulfate proteoglycan analogs. To avoid the secondary trauma caused by direct implantation requiring vertebral incision, the microspheres are cross-linked under ultraviolet light using a microfluidic device to create injectable hydrogel microspheres, meeting the needs of minimally invasive treatment. Furthermore, the microspheres can adsorb growth factors from stem cell supernatant and encapsulate nanoparticles such as liposomes for controlled drug release, thus providing a new strategy for combined treatment of spinal cord injury. Sustained-release methylprednisolone replaces acute methylprednisolone pulse therapy, reducing the side effects of systemic administration. Successful liposome encapsulation can improve liposome stability, offering promising applications in the graded release of different drugs. The surface area and volume ratio of the hydrogel is improved, the contact area between cells and materials is increased, and it is more suitable to provide growth space for spinal cord regeneration.

[0006] The first object of the present invention is to provide a method for preparing chondroitin sulfate proteoglycan analog microspheres, comprising the following steps:

[0007] S1, enzymatically hydrolyzing chondroitin sulfate with chondroitinase, and obtaining chondroitin sulfate oligosaccharides by ultrafiltration;

[0008] S2. activating the chondroitin sulfate oligosaccharide described in S1 with a cross-linking agent, and then subjecting it to a coupling reaction with a first methacryloylated gelatin, followed by dialysis and freeze-drying to obtain a chondroitin sulfate proteoglycan analog (GMCS); wherein the first methacryloylated gelatin (GelMA(L)) has a double bond grafting rate of 30%-40%, which facilitates covalent cross-linking between the remaining amino groups on the gelatin and the chondroitin sulfate;

[0009] S3, dissolving the chondroitin sulfate proteoglycan analog described in S2, a second methacryloylated gelatin, and a photoinitiator in water to obtain a dispersed phase; the second methacryloylated gelatin (GelMA(H)) has a double bond grafting rate of 70%-80%, and the microspheres have stronger mechanical properties after photocrosslinking;

[0010] The oil phase material is the continuous phase;

[0011] S4. The dispersed phase and the continuous phase described in S3 are made into water-in-oil droplets through a microfluidic device, and the chondroitin sulfate proteoglycan analog microspheres are obtained by UV curing and washing.

[0012] In one embodiment of the present invention, in S1, the chondroitinase is selected from chondroitin sulfate ABC enzyme (ChABC) or chondroitin sulfate AC enzyme (ChAC);

[0013] And / or, the chondroitin sulfate is selected from one or more of chondroitin sulfate A (CSA), chondroitin sulfate C (CSC) and chondroitin sulfate E (CSE).

[0014] In one embodiment of the present invention, in S1, the enzymatic hydrolysis conditions are: temperature of 20°C-37°C, time of 20h-30h;

[0015] And / or, during the enzymatic hydrolysis process, the concentration of chondroitinase is 0.2 mg / mL-0.5 mg / mL, and the concentration of chondroitin sulfate is 1 mg / mL-5 mg / mL.

[0016] In one embodiment of the present invention, in S1, chondroitin sulfate oligosaccharides of different molecular weights can be obtained by ultrafiltration.

[0017] In one embodiment of the present invention, in S2, the cross-linking agent includes EDC and NHS; the mass ratio of EDC to NHS is 1:(1-3);

[0018] And / or, the mass ratio of the chondroitin sulfate oligosaccharide, the first methacryloylated gelatin and the cross-linking agent is (40-60):250:(6-11).

[0019] In one embodiment of the present invention, in S3, the photoinitiator is selected from photoinitiator LAP;

[0020] And / or, the oil phase material is selected from one or more of Span 80, Span 60, paraffin oil, castor oil and soybean oil.

[0021] In one embodiment of the present invention, in S3, the mass ratio of the chondroitin sulfate proteoglycan analog to the second methacryloylated gelatin is 1:(2-4); the mass ratio of the second methacryloylated gelatin to the photoinitiator is (10-40):1.

[0022] In one embodiment of the present invention, in S4, the specifications of the microfluidic device are: channel depth of 30μm-60μm, necking width of 60μm-120μm, inner diameter of the pipeline of 0.3mm-0.6mm, and outer diameter of the pipeline of 1mm-2mm.

[0023] In one embodiment of the present invention, in S4, the microfluidic device uses a dual-channel injection pump, the chip uses a T-channel microfluidic chip, and the pipeline uses a tygon hose.

[0024] In one embodiment of the present invention, in S4, the process parameters of the microfluidic device are: the flow rate of the dispersed phase is 0.3 mL / h-0.8 mL / h, and the flow rate of the continuous phase is 5 mL / h-10 mL / h.

[0025] In one embodiment of the present invention, in S4, the UV curing conditions are: wavelength of 365nm-405nm, irradiation power of 50mW·cm -2 -200mW·cm -2 , the irradiation time is 30s-180s.

[0026] And / or, the washing is first performed with petroleum ether and then with water.

[0027] The second object of the present invention is to provide chondroitin sulfate proteoglycan analog microspheres prepared by the method described above, wherein the diameter of the chondroitin sulfate proteoglycan analog microspheres is 50 μm-200 μm.

[0028] The technical solution of the present invention has the following advantages over the prior art:

[0029] (1) The chondroitin sulfate proteoglycan analogs described in the present invention are covalently linked through amide bonds, and their size and subtype are controllable, providing a technical solution for regulating the microenvironment after spinal cord injury and for in-depth research on the structure-activity mechanism of chondroitin sulfate proteoglycans of different subtypes and molecular weights in various physiological and pathological conditions.

[0030] (2) The chondroitin sulfate proteoglycan analogues of the present invention are photocrosslinkable, which expands their functions and applications and has good application prospects in the biomedical field.

[0031] (3) The diameter of the chondroitin sulfate proteoglycan analog microspheres described in the present invention is 50 μm-200 μm, and they have good stability and swelling properties. At the same time, the pore structure of the microspheres allows nano-scale liposomes to be embedded therein, and the stability of the microspheres can protect the liposomes from being destroyed during transportation and release, giving the liposomes certain controlled release properties, which helps to achieve directional delivery and precise release of the liposomes.

[0032] (4) The chondroitin sulfate proteoglycan analog microspheres described in the present invention, due to their negative charge, can adsorb growth factors through electrostatic interactions, mimicking the extracellular matrix composition of the human spinal cord and regulating the microenvironment of the lesion after spinal cord injury. This lays a technical foundation for subsequent research on the mechanism of action of different subtypes of chondroitin sulfate proteoglycans on neural repair.

[0033] (5) The chondroitin sulfate proteoglycan analog microspheres prepared by the preparation method of the present invention are uniform in size, so that methylprednisolone is evenly distributed in the chondroitin sulfate proteoglycan microspheres, thereby being continuously released at the site of spinal cord injury, reducing the nonspecific distribution of the drug in the body, reducing the fluctuation of blood drug concentration, and thus reducing its toxic side effects on the lungs and gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0035] Figure 1 This is the UV absorption graph of chondroitin sulfate A oligosaccharide in Test Example 1 of the present invention;

[0036] Figure 2 1H NMR spectra of GelMA (L) and GMCS in Test Example 2 of the present invention;

[0037] Figure 3 1H NMR spectra of chondroitin sulfate A oligosaccharide and GMCS in Test Example 2 of the present invention;

[0038] Figure 4 The contact angle diagrams of GelMA (L) and GMCS in Test Example 5 of the present invention are shown below. The left diagram is for GelMA (L) and the right diagram is for GMCS.

[0039] Figure 5 The swelling results of GelMA(H) microspheres and GMCS microspheres in Test Example 6 of the present invention are shown in the figure; the upper figure is a microscopic image of GelMA(H) microspheres, the middle figure is a microscopic image of GMCS microspheres, and the lower figure shows the change in microsphere diameter after analysis using Image J software;

[0040] Figure 6 This is the release curve of methylprednisolone from the GMCS microspheres in Test Example 7 of the present invention;

[0041] Figure 7 Characterization images of GelMA(H) microspheres, GMCS microspheres, and liposome-encapsulated GMCS microspheres in Test Example 8 of the present invention; the upper image is a SEM image of GelMA(H) microspheres, the middle image is a SEM image of GMCS microspheres, and the lower image is a SEM image of liposome-encapsulated GMCS microspheres;

[0042] Figure 8 This is an SDS-PAGE image of the growth factors adsorbed by the GMCS microspheres in the supernatant of stem cells in Test Example 9 of the present invention;

[0043] Figure 9 The changes in the storage modulus (G') and loss modulus (G") of the chondroitin sulfate proteoglycan analog GMCS microspheres in Test Example 10 of the present invention. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. It should be understood that the specific embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0045] In the present invention, unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art to which the present invention belongs.

[0046] In the present invention, unless stated otherwise, the term "and / or" used in the present invention includes any and all combinations of one or more of the associated listed items.

[0047] In the present invention, unless otherwise stated, the experimental methods used in the examples of the present invention are conventional methods unless otherwise stated, and the materials, reagents, etc. used are all commercially available unless otherwise stated.

[0048] In the present invention, unless otherwise specified, the microfluidic device used in the embodiments of the present invention uses a dual-channel syringe pump, the chip uses a T-channel microfluidic chip, and the pipeline uses a tygon hose; the specifications of the microfluidic device are: channel depth of 50 μm, neck width of 100 μm, pipeline inner diameter of 0.51 mm, and pipeline outer diameter of 1.52 mm.

[0049] In the present invention, unless otherwise specified, the preparation of the liposome solution used in the examples of the present invention comprises the following steps: 60 mg of lecithin and 15 mg of cholesterol are weighed and dissolved in 30 mL of chloroform at 50° C., poured into an eggplant-shaped bottle, and subjected to rotary evaporation from slow to fast speed in a 50° C. water bath to form a phospholipid membrane, which is then vacuum-dried for 12 h and incubated with a phosphate buffer solution at 50° C. for 30 min to form a liposome solution, which is then ultrasonicated in an ice bath and passed through a 0.45 μm and 0.22 μm membrane three times, and placed at 4° C. until next use.

[0050] Example 1

[0051] The chondroitin sulfate proteoglycan analog microspheres and the preparation method thereof of this embodiment specifically include the following steps:

[0052] S1. Preparation of chondroitin sulfate oligosaccharides: 10 mg of chondroitin sulfate A was dissolved in 50 mM Tris / 60 mM sodium acetate (pH = 8.0) enzymatic hydrolysis buffer, 110 μL of 1 mg / mL ChABC was added, and the mixture was shaken at 37°C and 100 rpm for 24 h. After shaking, the mixture was boiled and filtered to remove protein. The mixture was then passed through 10 kDa, 5 kDa, 3 kDa, and 1 kDa ultrafiltration centrifuge tubes in sequence, and centrifuged at 4°C and 4000 rpm for 50 min to obtain chondroitin sulfate A oligosaccharides of 1 kDa-3 kDa, 3 kDa-5 kDa, and 5 kDa-10 kDa.

[0053] S2. Preparation of methacryloylated gelatin: Two portions (10 g) of type A gelatin were heated in a water bath at 50°C for 1 h to dissolve, and then 15 mL and 2 mL of methyl methacrylate (MA) were slowly added dropwise, respectively. The mixture was reacted at 50°C in the dark for 24 h. The mixture was dialyzed using a 3500 Da dialysis bag at 37°C in the dark for five days and freeze-dried to obtain methacryloylated gelatin GelMA (H) with a high double bond grafting rate and methacryloylated gelatin GelMA (L) with a low double bond grafting rate, respectively.

[0054] S3. Preparation of chondroitin sulfate proteoglycan analogs: 50 mg of chondroitin sulfate A oligosaccharide was dissolved in 10 mL of MES (pH = 5.5), and activated for 1 h by adding 3 mg of EDC and 4 mg of NHS. The oligosaccharide was then mixed with 250 mg of GelMA (L) dissolved in 125 mL of PBS (pH = 7.4) and reacted in the dark for 24 h to obtain a coupling product. Finally, the chondroitin sulfate proteoglycan analog GMCS was obtained after dialysis and lyophilization.

[0055] S4. Preparation of microspheres: 100 mg of GelMA (H) and 5 mg of photoinitiator LAP were mixed in 1 mL of deionized water to obtain a first pregel solution; 40 mg of chondroitin sulfate proteoglycan analog GMCS, 100 mg of GelMA (H) and 5 mg of photoinitiator LAP were mixed in 1 mL of deionized water to obtain a second pregel solution; the two pregel solutions were used as dispersed phases; 2.5 mL of Span 80 was added to 50 mL of paraffin oil as the continuous phase; the dispersed phase (flow rate of 0.5 mL / h) and the continuous phase (flow rate of 8 mL / h) were made into oil-in-water droplets using a microfluidic device and exposed to 405 nm, 100 mW·cm in the subsequent pipeline. -2 Two types of hydrogel microspheres were formed under ultraviolet light for 1 minute, and then washed four times with petroleum ether and water in sequence, and dried to obtain chondroitin sulfate proteoglycan analog GMCS microspheres.

[0056] Example 2

[0057] The chondroitin sulfate proteoglycan analog microspheres and the preparation method thereof of this embodiment specifically include the following steps:

[0058] S1. Preparation of chondroitin sulfate oligosaccharides: 10 mg of chondroitin sulfate A was dissolved in 50 mM Tris / 60 mM sodium acetate (pH = 8.0) enzymatic hydrolysis buffer, 110 μL of 1 mg / mL ChABC was added, and the mixture was shaken at 37°C and 100 rpm for 24 h. After shaking, the mixture was boiled and filtered to remove protein. The mixture was then passed through 10 kDa, 5 kDa, 3 kDa, and 1 kDa ultrafiltration centrifuge tubes in sequence, and centrifuged at 4°C and 4000 rpm for 50 min to obtain chondroitin sulfate A oligosaccharides of 1 kDa-3 kDa, 3 kDa-5 kDa, and 5 kDa-10 kDa.

[0059] S2. Preparation of methacryloylated gelatin: Two portions (10 g) of type A gelatin were heated in a water bath at 50°C for 1 h to dissolve, and then 15 mL and 2 mL of methyl methacrylate (MA) were slowly added dropwise, respectively. The mixture was reacted at 50°C in the dark for 24 h. The mixture was dialyzed using a 3500 Da dialysis bag at 37°C in the dark for five days and freeze-dried to obtain methacryloylated gelatin GelMA (H) with a high double bond grafting rate and methacryloylated gelatin GelMA (L) with a low double bond grafting rate, respectively.

[0060] S3. Preparation of chondroitin sulfate proteoglycan analogs: 50 mg of chondroitin sulfate A oligosaccharide was dissolved in 10 mL of MES (pH = 5.5), and activated for 1 h by adding 3 mg of EDC and 4 mg of NHS. The oligosaccharide was then mixed with 250 mg of GelMA (L) dissolved in 125 mL of PBS (pH = 7.4) and reacted in the dark for 24 h to obtain a coupling product. Finally, the chondroitin sulfate proteoglycan analog GMCS was obtained after dialysis and lyophilization.

[0061] S4. Preparation of microspheres: 40 mg of GMCS, 100 mg of GelMA (H), 40 mg of methylprednisolone (MPSS), and 5 mg of LAP were mixed in 1 mL of deionized water to obtain a pregel solution as the dispersed phase. 2.5 mL of Span 80 was added to 50 mL of paraffin oil as the continuous phase. The dispersed phase (flow rate of 0.5 mL / h) and the continuous phase (flow rate of 8 mL / h) were prepared into water-in-oil droplets using a microfluidic device and exposed to 405 nm, 100 mW·cm in the subsequent pipeline. -2 Hydrogel microspheres were formed under ultraviolet light for 1 minute, and then washed four times with petroleum ether and water in sequence, and dried to obtain drug chondroitin sulfate proteoglycan analog GMCS microspheres.

[0062] Example 3

[0063] The chondroitin sulfate proteoglycan analog microspheres and the preparation method thereof of this embodiment specifically include the following steps:

[0064] S1. Preparation of chondroitin sulfate oligosaccharides: 10 mg of chondroitin sulfate A was dissolved in 50 mM Tris / 60 mM sodium acetate (pH = 8.0) enzymatic hydrolysis buffer, 110 μL of 1 mg / mL ChABC was added, and the mixture was shaken at 37°C and 100 rpm for 24 h. After shaking, the mixture was boiled and filtered to remove protein. The mixture was then passed through 10 kDa, 5 kDa, 3 kDa, and 1 kDa ultrafiltration centrifuge tubes in sequence, and centrifuged at 4°C and 4000 rpm for 50 min to obtain chondroitin sulfate A oligosaccharides of 1 kDa-3 kDa, 3 kDa-5 kDa, and 5 kDa-10 kDa.

[0065] S2. Preparation of methacryloylated gelatin: Two portions (10 g) of type A gelatin were heated in a water bath at 50°C for 1 h to dissolve, and then 15 mL and 2 mL of methyl methacrylate (MA) were slowly added dropwise, respectively. The mixture was reacted at 50°C in the dark for 24 h. The mixture was dialyzed using a 3500 Da dialysis bag at 37°C in the dark for five days and freeze-dried to obtain methacryloylated gelatin GelMA (H) with a high double bond grafting rate and methacryloylated gelatin GelMA (L) with a low double bond grafting rate, respectively.

[0066] S3. Preparation of chondroitin sulfate proteoglycan analogs: 50 mg of chondroitin sulfate A oligosaccharide was dissolved in 10 mL of MES (pH = 5.5), and activated for 1 h by adding 3 mg of EDC and 4 mg of NHS. The oligosaccharide was then mixed with 250 mg of GelMA (L) dissolved in 125 mL of PBS (pH = 7.4) and reacted in the dark for 24 h to obtain a coupling product. Finally, the chondroitin sulfate proteoglycan analog GMCS was obtained after dialysis and lyophilization.

[0067] S4. Preparation of microspheres: 40 mg of GMCS, 100 mg of GelMA (H), 5 mg of LAP, and 200 μL of liposome solution were mixed in 1 mL of deionized water to obtain a pregel solution as the dispersed phase. 2.5 mL of Span 80 was added to 50 mL of paraffin oil as the continuous phase. The dispersed phase (flow rate of 0.5 mL / h) and the continuous phase (flow rate of 8 mL / h) were prepared into water-in-oil droplets using a microfluidic device and exposed to 405 nm, 100 mW·cm in the subsequent pipeline. -2 The hydrogel microspheres were formed under ultraviolet light for 1 minute, and then washed with petroleum ether and water for 4 times, and dried to obtain liposome-encapsulated chondroitin sulfate proteoglycan analog GMCS microspheres.

[0068] Comparative Example 1 is basically the same as Example 1, except that in S3, the amount of GelMA (L) used is 150 mg (the mass ratio of chondroitin sulfate A oligosaccharide to GelMA (L) is 1:3).

[0069] Comparative Example 2 is basically the same as Example 1, except that in S3, the amount of GelMA (L) used is 350 mg (the mass ratio of chondroitin sulfate A oligosaccharide to GelMA (L) is 1:7).

[0070] Comparative Example 3 is basically the same as Example 1, except that in S4, the amount of GMCS, a chondroitin sulfate proteoglycan analog, is 20 mg (the mass ratio of GMCS to GelMA (H) is 1:5).

[0071] Comparative Example 4 is basically the same as Example 1, except that in S4, the amount of chondroitin sulfate proteoglycan analog GMCS is 60 mg (the mass ratio of chondroitin sulfate proteoglycan analog GMCS to GelMA (H) is 3:5).

[0072] Test Example 1

[0073] The chondroitin sulfate A oligosaccharide of Example 1 was subjected to UV analysis at a wavelength of 200-500 nm. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that chondroitin sulfate A oligosaccharide has obvious ultraviolet absorption at 232nm, which is the characteristic absorption peak of the unsaturated double bond produced at the non-reducing end of the sugar chain when chondroitin sulfate restriction endonuclease degrades chondroitin sulfate A, indicating its successful enzymatic hydrolysis.

[0074] Test Example 2

[0075] 15 mg of each of the chondroitin sulfate A oligosaccharide, methacrylated gelatin GelMA (L), GelMA (H) and chondroitin sulfate proteoglycan analog GMCS of Example 1 and the chondroitin sulfate proteoglycan analog GMCS of Comparative Examples 1-2 were dissolved in 0.5 mL of D2O and then placed in a nuclear magnetic resonance tube for testing using a nuclear magnetic resonance spectrometer. The results are as follows: Figure 2-Figure 3 As shown. Figure 2-Figure 3It can be seen that GelMA (L) and GelMA (H) show peaks at 5.3ppm and 5.6ppm, which are the chemical shifts of the propyleneamine double bond. After integration, the double bond grafting rates of GelMA (L) and GelMA (H) are 34% and 74%, respectively. The GMCS peak at 2.0ppm-2.5ppm shows the methyl hydrogen related to the acetylamino group, the peak at 3.0ppm-4.0ppm shows the hydrogen related to the sugar ring, and the peak at 4.5ppm-5.5ppm shows the hydrogen related to the carboxyl group and the sulfate group, indicating that the chondroitin sulfate A oligosaccharide is successfully modified onto the protein core GelMA (L). With the increase of the feed ratio of chondroitin sulfate A oligosaccharide to GelMA in GMCS, the peak intensities at 2.0ppm-2.5ppm, 3.0ppm-4.0ppm and 4.5ppm-5.5ppm gradually increased, and the oligosaccharide grafting degree increased. When the feed ratio of chondroitin sulfate A oligosaccharide to GelMA (L) was 1:5, the oligosaccharide grafting degree of GMCS was the highest.

[0076] Test Example 3

[0077] 1 mg / mL of each of the chondroitin sulfate A oligosaccharide, methacrylated gelatin GelMA (L), and chondroitin sulfate proteoglycan analog GMCS in Example 1 were subjected to potential analysis using a Zeta potential analyzer. The results are shown in Table 1:

[0078] Table 1

[0079] Sample GelMA(L) Chondroitin sulfate A oligosaccharide GMCS Potential (mV) -4.566 -15.731 -9.820

[0080] As can be seen from Table 1 , due to the covalent binding of chondroitin sulfate A oligosaccharide, the electronegativity of GMCS is enhanced compared with GelMA (L), which improves its binding ability to growth factors.

[0081] Test Example 4

[0082] 4 mg of each of the chondroitin sulfate A oligosaccharide, methacrylated gelatin GelMA (L), and chondroitin sulfate proteoglycan analog GMCS of Example 1 were analyzed by combustion method. The results are as follows: Figure 1 As shown:

[0083] Table 2

[0084] Sample N[％] C[％] S[％] H[％] GelMA(L) 13.24 35.53 3.820 5.730 Chondroitin sulfate A oligosaccharide 25.69 47.20 0.721 5.399 GMCS 18.62 43.63 1.895 5.903

[0085] As can be seen from Table 2, the N, C, and S element contents of GMCS are closer to the average contents of the raw materials GelMA (L) and chondroitin sulfate A oligosaccharide, which proves the successful synthesis of chondroitin sulfate proteoglycan analogs.

[0086] Test Example 5

[0087] The contact angle test was performed on GelMA (L) of Example 1 and GMCS, a chondroitin sulfate proteoglycan analog. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that the contact angle of GelMA (L) in the left figure is 80.3°, and the contact angle of the chondroitin sulfate proteoglycan analog GMCS in the right figure is 66.3°, indicating that the introduction of a large number of hydroxyl groups on the chondroitin sulfate A sugar chain can form hydrogen bonds with water, thereby increasing the hydrophilicity of the protein core skeleton.

[0088] Test Example 6

[0089] Based on Example 1, the swelling of GelMA (H) microspheres and GMCS microspheres, a chondroitin sulfate proteoglycan analog, was compared by immersing them in PBS at 37°C. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that both microspheres reached swelling equilibrium within 5 minutes, and the volume of the microspheres after swelling was between 100 μm and 200 μm. Among them, the volume of the chondroitin sulfate proteoglycan analog GMCS microspheres was larger, which verified that the introduction of hydroxyl groups on the CSA sugar chain can form hydrogen bonds with water, thereby improving the hydrophilicity of the protein core skeleton.

[0090] Test Example 7

[0091] 120 mg of the drug-containing chondroitin sulfate proteoglycan analog GMCS microspheres of Example 2 were subjected to a drug release test in PBS (pH = 7.4) at 37°C and 100 rpm. 1 mL of samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 9 days to measure their UV absorption at 238 nm, and 1 mL of PBS (pH = 7.4) was added at the same time to maintain the total volume unchanged. The results are shown in FIG. Figure 6 As shown. Figure 6 It can be seen that the drug-containing chondroitin sulfate proteoglycan analog GMCS microspheres can release methylprednisolone in a sustained manner within 7 days.

[0092] Test Example 8

[0093] Based on Example 1, GelMA (H) microspheres and GMCS microspheres, as well as GMCS microspheres loaded with liposomes of Example 3, were swollen in water, and after being freeze-dried in liquid nitrogen, they were subjected to gold spraying and scanning electron microscopy (SEM) analysis. The results are as follows: Figure 7 As shown. Figure 7It can be seen that the GelMA (H) microspheres in the upper figure have a smooth surface and small and dense pores; the diameter of the swelled chondroitin sulfate proteoglycan analog GMCS microspheres in the middle figure is still around 200 μm. Due to the grafting of sugar chains, the pores of the microspheres become larger and the surface becomes rough; the lower figure shows that the liposomes are successfully encapsulated by the chondroitin sulfate proteoglycan analog GMCS microspheres. Subsequently, biological macromolecules such as proteins and nucleic acids can be encapsulated in the liposomes to improve their in vivo stability for targeted delivery into cells.

[0094] Test Example 9

[0095] The GMCS microspheres of Example 1 were incubated in the stem cell supernatant at 4°C for 24 hours to fully absorb the growth factors therein. The supernatant was dried on the surface with filter paper and then rinsed twice with deionized water. The microspheres were then soaked in 1M NaCl at 4°C for 12 hours and then shaken for 1 hour for elution. The eluted growth factors were analyzed by SDS-PAGE. The results are as follows: Figure 8 As shown, 1 is Maker, 2 is stem cell supernatant containing growth factors, 3 is stem cell supernatant after soaking GMCS microspheres, 4 is deionized water after the first rinse, 5 is deionized water after the second rinse, and 6 is 1M NaCl after elution. Figure 8 It can be seen that the GMCS microspheres, a chondroitin sulfate proteoglycan analog, successfully adsorbed growth factors from the stem cell supernatant containing growth factors into their interior.

[0096] Test Example 10

[0097] 1g of each of the GMCS microspheres of Example 1 and Comparative Examples 3-4 was placed on a strain-controlled rheometer at a constant temperature of 25°C. The frequency was swept in the shear rate range of 0.1rad / s-100rad / s. The results are shown in FIG. Figure 9 As shown. Figure 9 It can be seen that when the feed ratio of GMCS and GelMA in the composition of the chondroitin sulfate proteoglycan analog GMCS microspheres is 2:5, the storage modulus (G') and loss modulus (G") of the hydrogel microspheres both reach the maximum, indicating that the chondroitin sulfate proteoglycan analog GMCS microspheres under this ratio have the optimal viscoelasticity.

[0098] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing chondroitin sulfate proteoglycan analog microspheres, characterized in that: The following steps are involved: S1, enzymatically hydrolyzing chondroitin sulfate with chondroitinase, and obtaining chondroitin sulfate oligosaccharides by ultrafiltration; S2. activating the chondroitin sulfate oligosaccharide described in S1 with a cross-linking agent, and then subjecting it to a coupling reaction with a first methacryloylated gelatin, dialyzing, and freeze-drying to obtain a chondroitin sulfate proteoglycan analog; the double bond grafting rate of the first methacryloylated gelatin is 30%-40%; and the mass ratio of the chondroitin sulfate oligosaccharide, the first methacryloylated gelatin, and the cross-linking agent is (40-60):250:(6-11); S3. Dissolving the chondroitin sulfate proteoglycan analog, the second methacrylated gelatin, and a photoinitiator described in S2 in water to obtain a dispersed phase; the double bond grafting rate of the second methacrylated gelatin is 70%-80%; and the mass ratio of the chondroitin sulfate proteoglycan analog to the second methacrylated gelatin is 1:(2-4); The oil phase material is the continuous phase; S4. The dispersed phase and the continuous phase described in S3 are made into water-in-oil droplets through a microfluidic device, and the chondroitin sulfate proteoglycan analog microspheres are obtained by UV curing and washing.

2. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S1, the chondroitinase is selected from chondroitin sulfate ABC enzyme or chondroitin sulfate AC enzyme; And / or, the chondroitin sulfate is selected from one or more of chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate E.

3. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S1, the enzymatic hydrolysis conditions are: temperature of 20°C-37°C, time of 20h-30h; And / or, during the enzymatic hydrolysis process, the concentration of chondroitinase is 0.2 mg / mL-0.5 mg / mL, and the concentration of chondroitin sulfate is 1 mg / mL-5 mg / mL.

4. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S2, the cross-linking agent includes EDC and NHS; the mass ratio of EDC to NHS is 1:(1-3).

5. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S3, the photoinitiator is selected from photoinitiator LAP; And / or, the oil phase material is selected from one or more of Span 80, Span 60, paraffin oil, castor oil and soybean oil.

6. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S3, the mass ratio of the second methacrylated gelatin to the photoinitiator is (10-40):

1.

7. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S4, the specifications of the microfluidic device are: channel depth of 30 μm-60 μm, constriction width of 60 μm-120 μm, inner diameter of the pipeline of 0.3 mm-0.6 mm, and outer diameter of the pipeline of 1 mm-2 mm.

8. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S4, the process parameters of the microfluidic device are: the flow rate of the dispersed phase is 0.3 mL / h-0.8 mL / h, and the flow rate of the continuous phase is 5 mL / h-10 mL / h.

9. The method for preparing chondroitin sulfate proteoglycan analog microspheres according to claim 1, characterized in that: In S4, the UV curing conditions are: wavelength of 365nm-405nm, irradiation power of 50mW·cm -2 -200mW·cm -2 , irradiation time is 30s-180s; And / or, the washing is first performed with petroleum ether and then with water.

10. A chondroitin sulfate proteoglycan analog microsphere prepared by the method according to any one of claims 1 to 9, characterized in that: The diameter of the chondroitin sulfate proteoglycan analog microspheres is 50 μm-200 μm.

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