Recombinant keratin microsphere injection and preparation method thereof

Abstract

The invention discloses a recombinant keratin microsphere injection and a preparation method thereof, and relates to the technical field of biological medicines. The recombinant keratin microsphere injection comprises hydrogel microspheres which are prepared from recombinant keratin RK37 and methacrylated hyaluronic acid through a photo-crosslinking reaction. In the hydrogel microspheres, the mass volume concentration of the methacrylated hyaluronic acid is 4%, the mass volume concentration of the recombinant keratin RK37 is 1%, and the mass volume concentration of the photoinitiator is 0.5%. The photoinitiator is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphate, and the photoinitiator is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphate. Compared with the existing sodium hyaluronate injection and medical chitosan injection, the recombinant keratin RK37 hydrogel microsphere injection disclosed by the invention not only can increase the lubrication degree of knee joints and buffer mechanical pressure stress suffered by the joints, but also has the effects of promoting cartilage matrix formation and inhibiting inflammatory response, and has a stronger effect of treating osteoarthritis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a recombinant keratin microsphere injection and a preparation method thereof. BACKGROUND

[0002] At present, the injection used for treating knee osteoarthritis mainly includes sodium hyaluronate injection and medical chitosan injection, which mainly plays a role in reducing pain and protecting articular cartilage by increasing the lubricity of the knee joint and buffering the mechanical pressure stress of the joint. At present, there are many patents for sodium hyaluronate injection, which cover preparation process, crosslinking agent addition, and drug efficacy improvement, and the patent numbers include CN118403006, CN101112381A, etc. Medical chitosan injection is another commonly used joint cavity injection, which is a high molecular polysaccharide and can play a role in lubricating the joint and reducing the friction of the joint, and can also protect the articular cartilage through a physical barrier. At present, the patents for medical chitosan injection mainly focus on the preparation method, such as a method for extracting and preparing medical chitosan injection from shrimp shells or crab shells (publication number: CN114748499A) and a preparation method of a compound crosslinked medical chitosan preparation (publication number: CN104491846B).

[0003] Sodium hyaluronate injection and medical chitosan injection can only play a role in relieving osteoarthritis by reducing friction, increasing the lubricity of the knee joint, and forming a physical barrier on the surface of the articular cartilage. These two injections do not have the effects of promoting cartilage repair and anti-inflammation, and the American Association of Orthopedic Surgeons (AAOS) guidelines do not recommend using sodium hyaluronate injection to treat knee osteoarthritis. AAOS believes that based on existing clinical research, there is little difference in short-term efficacy between sodium hyaluronate joint cavity injection and placebo, and there is not enough strong evidence to support its wide application, so it is not recommended as a routine treatment. Recombinant keratin has good tissue repair effect, and we have screened out the human hair-derived recombinant keratin RK37 with the best effects of repairing cartilage and anti-inflammation, and prepared a hydrogel microsphere injection together with HAMA. This injection can not only protect the articular cartilage through lubrication and physical barrier, but also play a role in promoting the generation of cartilage matrix and anti-inflammation, and has good biocompatibility and good effect in treating knee osteoarthritis. SUMMARY

[0004] The present application aims at solving the problems in the prior art and provides a recombinant keratin microsphere injection and a preparation method thereof.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: A kind of recombinant keratin microsphere injection, comprising hydrogel microspheres prepared by light crosslinking reaction of recombinant keratin RK37 and methacrylated hyaluronic acid.

[0006] Preferably, in the hydrogel microspheres, the mass-volume concentration of methacrylated hyaluronic acid is 4%, the mass-volume concentration of recombinant keratin RK37 is 1%, and the mass-volume concentration of photoinitiator is 0.5%.

[0007] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0008] Preferably, the average particle size of the hydrogel microspheres is 400 μm, and the Zeta potential is -30 mV.

[0009] A preparation method of a recombinant keratin microsphere injection, comprising the following steps: S1: preparing recombinant keratin RK37: cloning the gene sequence encoding RK37 into pET-28a(+) plasmid, transforming into Escherichia coli BL21(DE3), inducing expression by IPTG, breaking the bacterial body by high-pressure homogenization, collecting the inclusion body by centrifugation, washing the inclusion body with buffer containing Triton X-100 and high-salt buffer, dissolving the inclusion body with buffer containing urea, purifying the protein by nickel column affinity chromatography, dialysis and freeze-drying to obtain recombinant keratin RK37; S2: preparing methacrylated hyaluronic acid: dissolving hyaluronic acid in deionized water, adding methacrylic anhydride for reaction, then adding sodium hydroxide solution for neutralization, freeze-drying after dialysis to obtain methacrylated hyaluronic acid; S3: preparing hydrogel microspheres; S4: post-processing.

[0010] Preferably, in S1, centrifuge at 4℃, 8000 rpm for 5 min to collect the bacteria, discard the supernatant, resuspend the bacterial body in 10 times the volume of buffer A, break the bacterial body by high-pressure homogenization, collect the inclusion body by centrifugation as follows: use a high-pressure homogenizer pre-cooled to 4℃, break three times at 700 bar pressure, centrifuge at 4℃, 10000 rpm for 20 min, collect the precipitate; wherein buffer A is: 50 mM Tris-HCl pH8.0, 150 mM NaCl, 1% Tween-20.

[0011] Preferably, in S1, the inclusion body is washed with buffer containing Triton X-100 and high-salt buffer, and the inclusion body is dissolved with buffer containing urea, as follows: Resuspend the precipitate with 10 times weight of buffer B and buffer C in sequence; centrifuge at 10000 rpm for 20 min at 4℃, collect the precipitate and dissolve it in appropriate amount of buffer D for 8 h; Buffer B is 50 mM Tris-HCl pH8.0; 150 mM NaCl; 5 mM EDTA; 10 mM β-mercaptoethanol; 1 mM urea; 0.5% TritonX-100; Buffer C is 50 mM Tris-HCl pH8.0; 2.5 M NaCl; 5 mM EDTA; 10 mM β-mercaptoethanol; Buffer D is 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 8 M urea, 10 mM β-mercaptoethanol.

[0012] Preferably, in S1, the protein is purified by nickel column affinity chromatography, specifically as follows: equilibrate the nickel column with buffer D for 10 min, add the supernatant, suspend at 4℃ for 2 h, sequentially wash with buffer D containing ddH2O, buffer D containing 5 mM and 10 mM imidazole to remove impurities and non-specifically bound proteins, and elute the target protein with buffer D containing 250 mM imidazole, repeat twice, and combine the eluate.

[0013] Preferably, in S3, the preparation of the hydrogel microspheres is specifically as follows: dissolve the methacrylated hyaluronic acid, recombinant keratin RK37 and photoinitiator in phosphate buffer to mix uniformly as an aqueous phase; dissolve Span 80 in paraffin oil to mix uniformly as an oil phase; form uniform emulsion droplets by the microfluidic device from the aqueous phase and the oil phase, and perform a photo-crosslinking reaction under ultraviolet irradiation to form the hydrogel microspheres.

[0014] Preferably, in S4, the post-treatment is specifically as follows: collect the hydrogel microspheres, wash with isopropyl alcohol and deionized water, and obtain the recombinant keratin microsphere injection after centrifugation.

[0015] The beneficial effects of the present application are: 1. The present application uses recombinant protein technology to synthesize recombinant human hair keratin RK37 for treating knee osteoarthritis, and this type of recombinant keratin can promote chondrocytes to generate cartilage matrix and inhibit inflammatory response.

[0016] 2. The present application combines the recombinant human hair keratin RK37 with HAMA to make hydrogel microspheres by photo-curing technology, the microspheres can be administered by injection, can lubricate the joint to reduce friction, and have the effects of promoting cartilage matrix formation and inhibiting inflammatory response.

[0017] 3. Compared with the existing sodium hyaluronate injection and medical chitosan injection, the recombinant keratin RK37 hydrogel microsphere injection can not only increase the lubricity of the knee joint and buffer the mechanical pressure stress of the joint, but also has the effects of promoting the formation of cartilage matrix and inhibiting inflammatory reaction, and has a stronger effect on treating osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of Rk37@HAMA after freeze-drying of the present application; Figure 2 It is a schematic diagram of Rk37@HAMA under a light microscope of the present application; Figure 3 It is a schematic diagram of Rk37@HAMA under a scanning electron microscope after freeze-drying of the present application; Figure 4 It is a particle size distribution diagram of Rk37@HAMA of the present application; Figure 5 It is an infrared spectrum diagram of recombinant human hair keratin RK37 of the present application; Figure 6 It is a schematic diagram of the coefficient of friction (COF) of Rk37@HAMA, HA, PBS and HAMA of the present application. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0020] Example 1: A synthesis method of recombinant human hair keratin RK37, RK37 is the best recombinant keratin species for treating knee osteoarthritis among 17 kinds of recombinant human hair keratin species screened by human cartilage cell experiments, and the specific steps are as follows: S1: The gene sequence of RK37 is cloned into pET-28a(+) plasmid, and then the plasmid is transformed into E.coli BL21(DE3) to express the protein.

[0021] S2: The transformed E.coli is placed in LB medium containing 50µg / mL kanamycin and cultured at 37℃, 200rpm. When the OD value reaches 0.6-0.8, 1mM IPTG is added, and the expression is induced at 16℃, 160rpm for 8 hours. The bacteria are collected by centrifugation at 4℃, 8000rpm for 5min, the supernatant is discarded, and the bacteria are resuspended in 10 times the volume of buffer A (50mM Tris-HCl pH8.0, 150mM NaCl, 1% Tween-20). A high-pressure homogenizer is used, and the lysate is lysed three times at 700bar pressure, and then centrifuged at 4℃, 10000rpm for 20min to collect the precipitate.

[0022] S3: The precipitate was resuspended with 10 times weight of buffer B (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 5 mM EDTA; 10 mM β-mercaptoethanol; 1 mM urea; 0.5% Triton X-100) and buffer C (50 mM Tris-HCl pH 8.0; 2.5 M NaCl; 5 mM EDTA; 10 mM β-mercaptoethanol) sequentially. The precipitate was collected by centrifugation at 10,000 rpm for 20 min at 4°C, and was dissolved in an appropriate amount of buffer D (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 8 M urea, 10 mM β-mercaptoethanol) for 8 h. After centrifugation at 10,000 rpm for 20 min at 4°C, the supernatant was taken.

[0023] S4: The nickel column was equilibrated with buffer D for 10 min, and the supernatant was added and suspended at 4°C for 2 h. The impurities and non-specifically bound proteins were removed by washing with buffer D containing ddH2O, buffer D containing 5 mM and 10 mM imidazole, respectively. The target protein was eluted with buffer D containing 250 mM imidazole, and the eluate was collected twice. The eluate was placed in a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyzed against ddH2O at 4°C for 2 days. After lyophilization, the product was stored at -80°C.

[0024] Example 2: A recombinant keratin RK37 was administered by combining with HAMA to synthesize hydrogel microspheres by a photocuring technique, and the specific steps were as follows: S1: 5 g of HA was added to 250 mL of deionized water, heated to 60°C, and mechanically stirred until the solution was clear and transparent. 10 mL of MA (1.04 g / mL) was added dropwise with a syringe, and the reaction was continuously mixed for 1 h under mechanical stirring. 10 mL of NaOH (200 mg / mL) was added dropwise using a microsyringe pump, and the reaction was carried out in an ice water bath under light protection for 24 h. After the reaction was completed, the solution was transferred to a dialysis bag and dialyzed at room temperature for 3 days. Finally, the HAMA aqueous solution was lyophilized and stored.

[0025] S2: 1 wt% RK37 and 0.5 wt% photoinitiator were added to a PBS solution containing 4 wt% HAMA, and the mixture was stirred thoroughly until it was clear and transparent, which was the aqueous phase. 5 wt% Span 80 was added to paraffin oil and mixed thoroughly, which was the oil phase.

[0026] S3: RK37 was combined with hyaluronic acid methacrylate (HAMA) to prepare RK37@HAMA microspheres. HAMA was synthesized from hyaluronic acid and methacrylic anhydride. After synthesis was completed, HAMA (4%), RK37 (0.1 or 1%) and phenyl-2,4,6-trimethylbenzoyl phosphonic acid lithium (0.5%, LAP) were dissolved in PBS buffer, and stirred to form a uniform transparent aqueous phase. Span 80 was thoroughly mixed into paraffin oil to form an oil phase. The flow rates of the two phases were independently regulated by a microfluidic device, and the shear force generated at the intersection of the microchannels formed a monodisperse emulsion. Droplets were irradiated with ultraviolet light to initiate photo-crosslinking, and finally RK37@HAMA microspheres were prepared. The collected microspheres were washed with isopropyl alcohol and deionized water three times to remove residual reagents, and centrifuged for 10 minutes to obtain RK37 hydrogel microspheres.

[0027] In the formula, the RK37 hydrogel microspheres should have the following characteristics: the diameter of the microspheres is about 400 μm, the Zeta potential is about -30 mV, and the in vitro complete degradation time is about 70 days. By establishing a rat knee osteoarthritis animal model through a medial meniscus destabilization model, and injecting the hydrogel microspheres into the knee joint cavity for administration, the gait of the rat knee joint can be improved, and joint wear can be reduced, thereby playing a role in treating osteoarthritis.

[0028] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical solution and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A recombinant keratin microsphere injection solution, characterized in that, It contains hydrogel microspheres prepared by photocrosslinking recombinant keratin RK37 with methacrylamide hyaluronic acid.

2. The recombinant keratin microsphere injection solution according to claim 1, characterized in that, In the hydrogel microspheres, the mass-volume concentration of methacrylamide hyaluronic acid is 4%, the mass-volume concentration of recombinant keratin RK37 is 1%, and the mass-volume concentration of photoinitiator is 0.5%.

3. The recombinant keratin microsphere injection solution according to claim 2, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

4. The recombinant keratin microsphere injection solution according to claim 1, characterized in that, The average particle size of the hydrogel microspheres is 400 μm, and the Zeta potential is -30 mV.

5. A method for preparing the recombinant keratin microsphere injection solution according to any one of claims 1-4, characterized in that, Includes the following steps: S1: Preparation of recombinant keratin RK37: The gene sequence encoding RK37 was cloned into the pET-28a(+) plasmid, transformed into Escherichia coli BL21(DE3), and expressed by IPTG. The bacterial cells were homogenized by high pressure, and inclusion bodies were collected by centrifugation. The inclusion bodies were washed with buffer containing Triton X-100 and high-salt buffer, dissolved with buffer containing urea, purified by nickel column affinity chromatography, dialyzed and lyophilized to obtain recombinant keratin RK37. S2: Preparation of methacrylamide hyaluronic acid: Hyaluronic acid was dissolved in deionized water, methacrylic anhydride was added to react, then sodium hydroxide solution was added to neutralize, dialyzed and lyophilized to obtain methacrylamide hyaluronic acid; S3: Preparation of hydrogel microspheres; S4: Post-processing.

6. The method for preparing a recombinant keratin microsphere injection solution according to claim 5, characterized in that, In S1, the bacteria are collected by centrifugation for 5 min at 4℃ and 8000 rpm, the supernatant is discarded, the bacterial cells are resuspended in 10 times the volume of buffer A, and the bacterial cells are lysed by high-pressure homogenization. The inclusion bodies are collected by centrifugation as follows: using a high-pressure homogenizer pre-cooled to 4℃, the cells are lysed three times at 700 bar pressure, centrifuged at 4℃ and 10000 rpm for 20 min, and the precipitate is collected. Buffer A consists of: 50mM Tris-HCl pH 8.0, 150mM NaCl, and 1% Tween-20.

7. The method for preparing a recombinant keratin microsphere injection solution according to claim 6, characterized in that, In step S1, the inclusion bodies are washed with a buffer containing Triton X-100 and a high-salt buffer, and dissolved with a buffer containing urea, as follows: The precipitate was resuspended in buffer B and buffer C, which were 10 times its weight in volume, in sequence; centrifuged at 4°C and 10,000 rpm for 20 min, the precipitate was collected, and dissolved in an appropriate amount of buffer D for 8 h. Buffer B consisted of: 50 mM Tris-HCl pH 8.0; 150 mM NaCl; 5 mM EDTA; 10 mM β-mercaptoethanol; 1 mM urea; and 0.5% Triton X-100. Buffer C consists of: 50 mM Tris-HCl pH 8.0; 2.5 M NaCl; 5 mM EDTA; 10 mM β-mercaptoethanol; Buffer D is: 50mM Tris-HCl, pH 8.0, 150mM NaCl, 8M murrea, 10mM β-mercaptoethanol.

8. The method for preparing a recombinant keratin microsphere injection solution according to claim 7, characterized in that, In S1, the protein is purified by nickel column affinity chromatography as follows: equilibrate the nickel column with buffer D for 10 min, add the supernatant, suspend at 4°C for 2 h, wash successively with buffer D containing ddH2O, 5 mM, and 10 mM imidazole to remove impurities and non-specifically bound proteins, elute the target protein with buffer D containing 250 mM imidazole, repeat twice, and combine the eluents.

9. The method for preparing a recombinant keratin microsphere injection solution according to claim 5, characterized in that, In step S3, the preparation of hydrogel microspheres specifically involves: dissolving methacrylamide hyaluronic acid, recombinant keratin RK37, and a photoinitiator in phosphate buffer and mixing them evenly to form the aqueous phase; dissolving Span 80 in paraffin oil and mixing them evenly to form the oil phase; and using a microfluidic device to form uniform emulsion droplets between the aqueous and oil phases, followed by photocrosslinking under ultraviolet irradiation to form hydrogel microspheres.

10. The method for preparing a recombinant keratin microsphere injection solution according to claim 9, characterized in that, In step S4, the post-processing specifically involves: collecting hydrogel microspheres, washing them with isopropanol and deionized water, and centrifuging them to obtain the recombinant keratin microsphere injection solution.

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