Artificial cornea with bionic structure carrying PRP (platelet rich plasma) microsphere sustained-release system and preparation method of artificial cornea

Abstract

The invention relates to the technical field of keratoprosthesis preparation, in particular to a biomimetic structure keratoprosthesis carrying a PRP microsphere slow release system and a preparation method of the biomimetic structure keratoprosthesis. PRP is encapsulated through PLGA microspheres, and stepped release of growth factors is achieved through degradation controllability of PLGA; the PRP-PLGA microspheres still keep a stable slow-release effect within 14 days in vitro, the problem that growth factors are rapidly inactivated due to direct application of traditional PRP is effectively solved, the treatment window period is remarkably prolonged, and continuous biological activity stimulation needed by cornea repair is met; by utilizing the degradation controllability of the PLGA, the stepped release of the growth factor is realized, so that the stable release effect of the PRP can be maintained for a long time after the artificial cornea is transplanted, the treatment window period after the artificial cornea is transplanted is prolonged, and the continuous biological activity stimulation required by cornea repair is met.

Description

Technical Field

[0001] This application relates to the technical field of artificial cornea preparation, in particular to a bionic artificial cornea with a PRP microsphere sustained-release system and a preparation method thereof. Background Art

[0002] Corneal transplantation is the only means to treat corneal blindness, but the donors available for transplantation are extremely scarce. More than 98.5% of patients worldwide cannot obtain donor tissues, and there are severe inflammatory reactions and high rejection rates after corneal transplantation. In recent years, researchers have developed several bioengineered artificial corneas in an attempt to replace natural corneal donors. Our research group previously constructed a bionic collagen (Col) corneal graft with good biocompatibility and biomechanics using natural collagen as the raw material.

[0003] Platelet rich plasma (PRP) is a blood product of autologous origin, rich in active substances such as growth factors and anti-inflammatory factors, and has the functions of anti-inflammation and promoting rapid repair of damaged tissues.

[0004] In the prior art, an artificial cornea and its preparation method with the publication number CN102580147A are composed of a porous peripheral support part formed by polyvinyl alcohol, 6-O-carboxymethyl chitosan, nano-phosphate, etc. and an optical center part formed by polyvinyl alcohol hydrogel. This artificial cornea has good biocompatibility and anti-infectivity, and has the effect of promoting rapid healing of the surgical wound.

[0005] The above prior art can also achieve the function of preparing artificial corneas, but the corneas prepared by the above prior art have severe inflammatory reactions and high rejection rates after transplantation, affecting the transplantation effect. Secondly, when the artificial corneas of the above prior art are used, they cannot carry active substances such as growth factors and anti-inflammatory factors, and the retention time of the active substances is short, requiring frequent medication, and the bioavailability is limited. Summary of the Invention

[0006] In order to solve the problems in the background art, the present invention proposes a bionic artificial cornea with a PRP microsphere sustained-release system and a preparation method thereof.

[0007] The bionic artificial cornea with a PRP microsphere sustained-release system and a preparation method thereof provided by this application adopt the following technical solutions:

[0008] The bionic structured artificial cornea with a PRP microsphere sustained-release system includes PRP-PLGA microspheres and a collagen matrix; the PRP-PLGA microspheres are formed by emulsifying and centrifuging a poly(lactic-co-glycolic acid) copolymer solution and platelet-rich plasma through an emulsifier; adding the PRP-PLGA microspheres into the collagen matrix solution and continuously stirring until the microspheres are uniformly dispersed; crosslinking and curing the collagen matrix solution containing the PRP-PLGA microspheres through a crosslinking agent to form a transparent artificial cornea implant; the diameter of the artificial cornea is 2.5-3.5 mm, and the thickness is 250-350 μm.

[0009] Further, the crosslinking agent is a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the mass ratio of it to the collagen matrix content is 1:1:6.

[0010] A preparation method of a bionic structured artificial cornea with a PRP microsphere sustained-release system includes the following steps:

[0011] (1) PRP preparation: Extract and concentrate platelet-rich plasma from animal whole blood by two-step gradient centrifugation, and adjust the final platelet concentration to (1000-1500)×10 9 / L;

[0012] (2) PLGA solution preparation: Dissolve poly(lactic-co-glycolic acid) copolymer in dichloromethane to prepare a 5%-10% (w / v) PLGA solution;

[0013] (3) PRP-PLGA microsphere preparation: Slowly add PRP to the PLGA solution, gently stir to prevent the growth factors in PRP from being inactivated, and then slowly drop the mixed PRP-PLGA mixed solution into the water phase containing an emulsifier, emulsify after mixing with the PLGA solution, and form PRP-PLGA microspheres through solvent evaporation and curing;

[0014] (4) Composite crosslinking: Disperse the PRP-PLGA microspheres in the collagen solution, and add a crosslinking agent for low-temperature crosslinking reaction;

[0015] (5) Molding treatment: Inject the crosslinked mixed solution into a mold for molding to obtain an artificial cornea implant

[0016] Further, the specific steps of PRP preparation in step (1) include:

[0017] a. Centrifuge the whole blood at 1500 rpm for 10 min, take the upper layer of concentrated platelets without the buffy coat layer into a sterile centrifuge tube A, and aspirate the reserved concentrated platelets from the sterile centrifuge tube A into a sterile centrifuge tube B;

[0018] b. Centrifuge the sterile centrifuge tube A at 3000 rpm for 20 min, pour the supernatant of the upper layer of the sterile centrifuge tube A into the centrifuge tube C, and label it as PPP for standby;

[0019] c. The bottom of the sterile centrifuge tube A is platelet aggregates. Gradually aspirate the concentrated platelets in the sterile centrifuge tube B into the sterile centrifuge tube A, and adjust the final platelet concentration to (1000 - 1500)×10 9 / L, which is PRP.

[0020] Furthermore, in step (3), the emulsification process uses an aqueous solution containing 1% polyvinyl alcohol, the magnetic stirring speed is 500 - 1000 rpm, and the emulsification time is 10 - 15 minutes.

[0021] Furthermore, the emulsification condition in step (3) is: stir at room temperature for 4 hours to volatilize the organic solvent.

[0022] Furthermore, after the solvent volatilizes in step (3), collect the solidified PRP - PLGA microspheres by centrifugation, and wash the PRP - PLGA microspheres with sterile PBS to remove the residual emulsifier and organic solvent.

[0023] Furthermore, the cross - linking reaction condition in step (4) is: continuously stir at 4°C for 4 hours.

[0024] Furthermore, the mold forming parameters in step (5) are: diameter 3 ± 0.5 mm, thickness 300 ± 50 μm, and after forming, store it in sterile PBS for standby.

[0025] Beneficial effects

[0026] Compared with the prior art, the present invention provides a bionic - structure artificial cornea carrying a PRP microsphere sustained - release system and its preparation method, which has the following beneficial effects:

[0027] 1. In this invention, PRP is encapsulated by PLGA microspheres, and the step - by - step release of growth factors is achieved by using the controllable degradation of PLGA; the PRP - PLGA microspheres still maintain a stable sustained - release effect within 14 days in vitro, effectively overcoming the problem of rapid inactivation of growth factors caused by the direct application of traditional PRP, significantly extending the treatment window period, and meeting the continuous bioactive stimulation required for corneal repair.

[0028] 2. In this invention, a low - temperature emulsification and gentle stirring process are adopted during the microsphere preparation process to avoid the denaturation and inactivation of growth factors such as VEGF and PDGF in PRP; at the same time, the PLGA outer shell isolates the external enzymatic hydrolysis environment, protects the active components of PRP, and ensures that the released growth factors have a bio - efficacy equivalent to that of fresh PRP.

[0029] 3. In this invention, the artificial cornea formed by crosslinking the collagen matrix with EDC / NHS highly matches the natural cornea. It can not only provide mechanical support but also avoid implant rejection or mechanical damage caused by hardness differences. The uniform dispersion of microspheres in the collagen network further simulates the extracellular matrix microenvironment of the natural cornea, promotes the migration and adhesion of host cells, and reduces the rejection rate after artificial cornea transplantation. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] The preparation method of the bionic structural artificial cornea carrying the PRP microsphere sustained-release system provided in the embodiments of the present invention includes:

[0032] (1) Preparation of PRP

[0033] 1) Take a healthy New Zealand rabbit, and draw 10 mL of whole blood using a blood collection tube containing an anticoagulant (sodium citrate), and immediately gently mix to prevent coagulation.

[0034] 2) First centrifugation: Centrifuge the whole blood at 1500 rpm (405×g) for 10 minutes. After separation, it is divided into three layers (the lower layer of red blood cells, the middle layer of buffy coat, and the upper layer of plasma). Use a pipette to aspirate the platelet-rich plasma without the buffy coat in the upper layer and transfer it to a sterile centrifuge tube A, record its volume V1, and perform platelet counting C1 (using a fully automatic blood cell analyzer).

[0035] 3) Calculation of reserved platelet volume: Calculate the required reserved volume V of concentrated platelets according to the formula V=(C1×V1) / 1000. For example, if C1 = 800×10 9 / L and V1 = 2 mL, then V = 1.6 mL. Aspirate the reserved concentrated platelets from the sterile centrifuge tube A and transfer them to a sterile centrifuge tube B for standby.

[0036] 4) Second centrifugation: Centrifuge the remaining concentrated platelets in the sterile centrifuge tube A at 3000 rpm (1620×g) for 20 minutes. After centrifugation, it is divided into the upper layer of PPP (platelet-poor plasma, transferred to centrifuge tube C) and the bottom layer of platelet aggregates.

[0037] 5) Remove and reserve the upper layer of PPP: Pour the upper layer of PPP in the sterile centrifuge tube A into the centrifuge tube C and label it as PPP for standby.

[0038] 6) Final concentration adjustment: Slowly and stepwise add the reserved concentrated platelets in sterile centrifuge tube B to the bottom platelet aggregates in sterile centrifuge tube A, gently pipette and mix evenly to adjust the final platelet concentration to 1200×10 9 / L, thus obtaining the PRP solution.

[0039] During the above preparation process, PRP is rich in active substances such as growth factors and inflammation inhibitory factors, and has the functions of anti-inflammation and promoting rapid repair of damaged tissues.

[0040] (2) Preparation of PLGA solution

[0041] Dissolve poly(lactic-co-glycolic acid) (PLGA) in an organic solvent (dichloromethane) to prepare a 5%-10% (w / v) solution, and magnetically stir until completely dissolved.

[0042] During the above preparation process, by adjusting the PLGA molecular weight, microsphere particle size and crosslinking density, the PRP release period (7 - 15 days) can be accurately regulated to adapt to the corneal injury repair requirements of different severities (such as chronic ulcers requiring long-term sustained release); the degradation products of PLGA are lactic acid and glycolic acid, which can be normally metabolized, avoiding inflammatory reactions and reducing the rejection rate after artificial corneal transplantation.

[0043] (3) Preparation of PRP-PLGA microspheres

[0044] 1) Solution mixing: Slowly add PRP to the PLGA solution and gently stir to form a uniform mixed solution, taking care to avoid vigorous stirring to prevent the inactivation of growth factors in PRP.

[0045] 2) Aqueous phase emulsification: Slowly drip the PRP-PLGA mixed solution into the aqueous phase containing an emulsifier (1% PVA solution), magnetically stir for about 10 - 15 minutes, and the rotation speed is 500 - 1000 rpm.

[0046] 3) Solvent evaporation and curing: Transfer the emulsion to a magnetic stirrer and stir at room temperature for 4 hours to volatilize the organic solvent and cure the PRP-PLGA mixture, gradually forming PRP-PLGA microspheres.

[0047] 4) Microsphere purification: Centrifuge the emulsion at 1000×g for 5 minutes, discard the supernatant, collect the precipitated PRP-PLGA microspheres, and wash them 3 times with sterile PBS to remove the residual PVA and organic solvents.

[0048] During the above preparation process, PRP is encapsulated inside PLGA microspheres. Meanwhile, PLGA has controllable degradation, enabling the gradual release of PRP inside the PLGA microspheres. By utilizing the controllable degradation of PLGA, a stepwise release of growth factors is achieved, which can then meet the requirement that PRP can maintain a stable release effect for a long time after artificial corneal transplantation, extend the treatment window period after artificial corneal transplantation, and meet the continuous bioactive stimulation required for corneal repair.

[0049] (4), Collagen composite crosslinking

[0050] 1) Under continuous stirring, add PRP-PLGA microspheres into the collagen solution and continue stirring until the PRP-PLGA microspheres are evenly dispersed.

[0051] 2) Crosslinking reaction: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) to the above mixed solution according to the ratio of EDC∶NHS∶collagen solid content of 1∶1∶6, and stir at 4°C for 4 h to make it undergo a crosslinking reaction.

[0052] (5), Artificial corneal molding

[0053] 1) Inject the crosslinked gel into a special mold (inner diameter 3 mm, thickness 300 μm), and let it stand at 37°C for 30 minutes for shaping.

[0054] 2) After demolding, place the artificial corneal implant in sterile PBS for storage and keep it for use in a 4°C environment; the size of the implant can be flexibly customized according to the corneal defect area of the patient to improve the surgical adaptability.

[0055] The collagen matrix forms a high degree of matching between the artificial cornea and the natural cornea through EDC / NHS crosslinking, which can not only provide mechanical support but also avoid implant rejection or mechanical damage caused by differences in hardness. The uniform dispersion of PRP-PLGA microspheres in the collagen network further simulates the extracellular matrix microenvironment of the natural cornea and promotes the migration and adhesion of host cells.

[0056] By combining the active repair ability of PRP with the biomimetic structural characteristics of collagen, a long-acting and controllable release of growth factors is achieved through a microsphere sustained-release system, providing a new treatment plan with both biological activity, mechanical adaptability, and operational feasibility for corneal injury repair, especially suitable for the regenerative medicine treatment of intractable corneal ulcers, chemical burns and other diseases.

[0057] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in all respects, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Accordingly, all changes that fall within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims involved.

Claims

1. A bionic structural artificial cornea equipped with a PRP microsphere sustained-release system, characterized in that, Comprising: PRP-PLGA microspheres and a collagen matrix; The PRP-PLGA microspheres are formed by emulsifying and centrifuging a poly(lactic-co-glycolic acid) copolymer solution and platelet-rich plasma through an emulsifier; Adding the PRP-PLGA microspheres into a collagen matrix solution and continuously stirring until the microspheres are uniformly dispersed; Crosslinking and curing the collagen matrix solution containing the PRP-PLGA microspheres through a crosslinking agent to form a transparent artificial cornea implant; The diameter of the artificial cornea is 2.5 - 3.5 mm, and the thickness is 250 - 350 μm.

2. The bionic structural artificial cornea equipped with the PRP microsphere sustained-release system according to claim 1, wherein: The crosslinking agent is a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the mass ratio of it to the collagen matrix content is 1:1:

6.

3. A preparation method of a bionic structural artificial cornea carrying a PRP microsphere sustained-release system as described in any one of claims 1-2, characterized in that, Including the following steps: (1)PRP preparation: Extract and concentrate platelet-rich plasma from animal whole blood by double gradient centrifugation, and adjust the final platelet concentration to (1000 - 1500)×10 9 / L; (2) Preparation of PLGA solution: Dissolving poly(lactic-co-glycolic acid) copolymer in dichloromethane to prepare a 5% - 10% (w / v) PLGA solution; (3) Preparation of PRP-PLGA microspheres: Slowly adding PRP into the PLGA solution, gently stirring to prevent the growth factors in PRP from being inactivated, and then slowly dropping the mixed PRP-PLGA mixed solution into an aqueous phase containing an emulsifier, emulsifying after mixing with the PLGA solution, and forming PRP-PLGA microspheres through solvent evaporation and curing; (4) Composite crosslinking: Dispersing the PRP-PLGA microspheres in a collagen solution, and adding a crosslinking agent for a low-temperature crosslinking reaction; (5) Molding treatment: Injecting the crosslinked mixed solution into a mold for molding to obtain an artificial cornea implant.

4. The preparation method of the bionic structural artificial cornea carrying the PRP microsphere sustained release system according to claim 3, characterized in that: The specific steps of PRP preparation in step (1) include: a. Centrifuging whole blood at 1500 rpm for 10 min, taking the upper-layer concentrated platelets without the buffy coat layer into a sterile centrifuge tube A, and sucking the reserved concentrated platelets from the sterile centrifuge tube A into a sterile centrifuge tube B; b. Centrifuging the sterile centrifuge tube A at 3000 rpm for 20 min, pouring the upper-layer supernatant of the sterile centrifuge tube A into a centrifuge tube C, and labeling it as PPP for standby; c. The bottom of the sterile centrifuge tube A is platelet aggregates. The concentrated platelets in the sterile centrifuge tube B are stepwise aspirated and placed into the sterile centrifuge tube A, and the final platelet concentration is adjusted to (1000 - 1500)×10 9 / L, which is PRP.

5. The preparation method of the bionic structural artificial cornea equipped with the PRP microsphere sustained-release system according to claim 4, characterized in that: In the emulsification process in step (3), an aqueous solution containing 1% polyvinyl alcohol is used, the magnetic stirring speed is 500 - 1000 rpm, and the emulsification time is 10 - 15 minutes.

6. The preparation method of the bionic structural artificial cornea equipped with the PRP microsphere sustained-release system according to claim 4, characterized in that: The emulsification conditions in step (3) are: Stirring at room temperature for 4 hours to volatilize the organic solvent.

7. The preparation method of the bionic structured artificial cornea equipped with the PRP microsphere sustained-release system according to claim 4, characterized in that: After the solvent volatilizes in step (3), the solidified PRP-PLGA microspheres are collected by centrifugation, and the PRP-PLGA microspheres are washed with sterile PBS to remove the residual emulsifier and organic solvent.

8. The preparation method of the bionic structural artificial cornea equipped with the PRP microsphere sustained-release system according to claim 4, wherein: The crosslinking reaction conditions in step (4) are: Continuously stirring for 4 hours in a 4°C environment.

9. The preparation method of the bionic structural artificial cornea equipped with the PRP microsphere sustained-release system according to claim 4, characterized in that: The mold molding parameters in step (5) are: diameter 3 ± 0.5 mm, thickness 300 ± 50 μm, and after molding, it is stored in sterile PBS for standby.