Encapsulated mesenchymal stem cell microneedle modified corneal stroma lens as well as preparation method and application thereof

Abstract

The invention discloses an encapsulated mesenchymal stem cell microneedle modified cornea stroma lens and a preparation method and application thereof, the cornea stroma lens is a cornea stroma lens generated from femtosecond laser small incision cornea microlens extraction (such as SMILE operation), and has excellent biocompatibility and transparency. The lens is modified by a microneedle structure, so that the lens can be accurately anchored to a cornea defect part, and meanwhile, the encapsulated mesenchymal stem cells locally and continuously release repair promoting factors to promote cornea cell regeneration and tissue repair. The preparation method is simple, high in universality and suitable for large-scale production, and based on reutilization of the myopia operation waste lens, the availability of materials is improved, waste of medical resources is reduced, and the myopia operation waste lens has important economic and clinical application prospects in the field of cornea repair.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a mesenchymal stem cell encapsulated microneedle modified corneal stroma lens and a preparation method and application thereof. Background Art

[0002] The cornea, as a protective barrier for the front part of the eye, is an important part of the refractive system that focuses light onto the retina. Corneal damage caused by trauma, foreign bodies, chemical exposure, and infection usually manifests as corneal scarring and stromal thinning, which can lead to impaired vision or even blindness in severe cases. It is estimated that 10 million patients worldwide are affected by corneal damage, but current treatment options are severely limited due to the lack of donor corneas, immune rejection, and the risk of infection. Therefore, many studies are devoted to the development of corneal substitutes or artificial corneas, such as the use of amniotic membranes, decellularized animal corneas, and synthetic hydrogel patches. Among them, hydrogels can provide a suitable microenvironment for mesenchymal stem cells and promote corneal regeneration. Although current corneal substitutes have certain therapeutic effects, they are mainly composed of synthetic materials, and there may be great challenges in their complex synthesis process and biocompatibility. In addition, there may be a risk of detachment when suturing the cell-laden hydrogel to the cornea. Therefore, there is an urgent need to develop new corneal substitutes for corneal transplantation.

[0003] Corneal stromal lenses from myopic refractive surgery, i.e., femtosecond laser small incision lenticule extraction, are abundant in source and have lower xenoimmunogenicity than animal tissue materials. Storage requirements can be further reduced and the risk of rejection can be reduced through the decellularization process. Decellularization can retain bioactive substances that promote cell growth and improve compatibility with the recipient cornea. In addition, microneedles, which are composed of micron-sized needles, have been widely used for painless, non-invasive and efficient transdermal drug delivery. They can also deliver active compounds (such as drug molecules, growth factors and stem cells) into the corneal stroma, thereby enhancing the stability of the implanted lens with the corneal stromal bed. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a mesenchymal stem cell encapsulated microneedle modified corneal stroma lens with good biocompatibility, optical transmittance and mechanical strength and a preparation method thereof in view of the deficiencies in the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a mesenchymal stem cell encapsulated microneedle-modified corneal stroma lens comprises the following steps: S1: adding sodium dodecyl sulfate to phosphate buffer solution to prepare a mixed solution; adding a fresh corneal stromal lens to the mixed solution and shaking it thoroughly; S2: washing the corneal stromal lens in step S1 in a sterile phosphate buffer solution to remove excess sodium dodecyl sulfate on the surface, thereby obtaining a decellularized corneal stromal lens; S3: dissolving hyaluronic acid in deionized water, adding methacrylic acid, stirring the reaction thoroughly, adjusting the pH value to weak alkalinity, removing unreacted monomers and other impurities, obtaining an aqueous solution of methacryloyl hyaluronic acid, and freeze-drying the purified solution to obtain methacryloyl hyaluronic acid powder; S4: dissolving the methacrylylated hyaluronic acid powder in step S3 in a sterile phosphate buffer solution, adding a photoinitiator, and adding the solution to a suspension containing mesenchymal stem cells to obtain a photocurable hydrogel precursor solution; S5: adding the photocurable hydrogel precursor solution prepared in step S4 to the microneedle mold, removing excess bubbles by centrifugation, absorbing excess methacryloyl hyaluronic acid solution from the microneedle base, and placing the acellular corneal stromal lens prepared in step S2 at the microneedle base; S6: The microneedle mold prepared in step S5 is cured under ultraviolet light, and the microneedle is demoulded.

[0006] The corneal stromal lens modified by the microneedle encapsulating mesenchymal stem cells of the present invention has good biocompatibility, optical permeability and mechanical strength, and is a good source of biological materials for corneal biological patches; mesenchymal stem cells have self-renewal and multidirectional differentiation capabilities, can promote the regeneration and repair of corneal tissue, and improve the healing of corneal injuries or diseases. Through microneedle technology, the precise targeted release of growth factors secreted by mesenchymal stem cells can be achieved, the therapeutic effect can be enhanced, and the side effects can be reduced. Compared with traditional corneal stromal lens transplantation, the microneedle-modified stromal lens has significant advantages in tissue integration, drug delivery ability and healing effect. The microneedle structure can improve the fit of the lens to the corneal tissue, and at the same time reduce the damage to the surrounding tissue during the transplantation process through a minimally invasive method. In addition, the microneedle can also be used as a drug release carrier to achieve local and continuous drug delivery, thereby effectively inhibiting postoperative inflammatory reactions and promoting the repair and regeneration of corneal tissue.

[0007] Specifically, in step S1, the dosage ratio of the sodium dodecyl sulfate to the phosphate buffer solution is 0.04-0.06 g / 30-60 mL; the corneal stromal lens is added to the mixed solution and the oscillation temperature is controlled at 3-5° C., and the oscillation condition is 150-180 rpm / min.

[0008] Specifically, in step S2, the washing conditions are 3-5°C, an oscillation frequency of 150-180 rpm / min, for 10-30 min, and repeated twice or more.

[0009] Specifically, in step S3, unreacted monomers and other impurities are removed by dialysis or ultrafiltration, the dialysis frequency is 400-600 rpm / min, and the dialysis is performed at room temperature for more than 5 days.

[0010] Specifically, in step S3, the concentration of the aqueous solution of methacryloyl hyaluronic acid obtained is (2-4) g / 100 mL.

[0011] Specifically, in step S4, the usage ratio of methacryloyl hyaluronic acid powder to sterile phosphate buffer solution is (0.05-0.15) g / (1-2) mL.

[0012] Specifically, in step S4, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and its added amount is 1 / 40 to 1 / 30 of the mass of the methacrylated hyaluronic acid powder.

[0013] Specifically, in step S6, the UV lamp curing condition is to cure for 10s to 30s under UV light with a wavelength of 400-410 nm.

[0014] Furthermore, the mesenchymal stem cell encapsulated microneedle-modified corneal stroma lens prepared by the above preparation method is also within the protection scope of the present invention.

[0015] Furthermore, the present invention also claims to protect the use of the above-mentioned mesenchymal stem cell encapsulated microneedle modified corneal stroma lens in the preparation of drugs for treating corneal diseases. Beneficial Effects

[0016] (1) The present invention encapsulates mesenchymal stem cell microneedle-modified corneal stroma lens. The corneal stroma lens derived from SMILE surgery is rich in source and has lower xenogeneic immunogenicity than animal tissue materials. Through the decellularization process, storage requirements can be further reduced and the risk of rejection can be reduced. Furthermore, decellularization can retain bioactive substances that promote cell growth and improve compatibility with the recipient cornea. In addition, microneedles are composed of micron-sized needles and have been widely used for painless, non-invasive and efficient transdermal drug delivery. They can also deliver active compounds (such as drug molecules, growth factors and stem cells) into the corneal stroma, thereby enhancing the adhesion of the implanted lens to the corneal stroma bed. Therefore, the combination of decellularized corneal lens and microneedle system may be a new method to promote corneal regeneration.

[0017] (2) The present invention uses a template method as the basis for preparing a microneedle-modified corneal stromal lens encapsulating mesenchymal stem cells. The method is simple, easy to operate, low in price, and does not require very high technical requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 It is a schematic diagram of the decellularization process of the corneal stromal lens of the present invention.

[0020] Figure 2 It is a schematic diagram of the process of microneedle-modified corneal stroma lens and a light microscope diagram of the present invention.

[0021] Figure 3 This is a photograph of the anterior segment after the microneedle-modified corneal stroma lens of the present invention was used for corneal transplantation in a rabbit.

[0022] Figure 4 The invention relates to corneal slice staining after corneal transplantation.

[0023] Figure 5 This is a photo of the anterior segment after traditional corneal stromal lens transplantation. DETAILED DESCRIPTION

[0024] The present invention can be better understood with reference to the following examples.

[0025] Example 1 Corneal stromal lens decellularization Combination Figure 1 As shown in the figure, the corneal stromal lens was removed by VisuMax femtosecond-assisted SMILE surgery. The surgical parameters were: corneal cap diameter 7.5mm, corneal cap thickness 110μm, and side incision position 130°. The energy of the lens, corneal cap, and side incision was set to 130nJ. The decellularization process was: treatment with 0.1% sodium dodecyl sulfate (SDS) at 4°C for 24 hours (170 rpm / min), and then washed with sterile PBS at 4°C for 15 minutes 3 times (170 rpm / min) to obtain a decellularized corneal stromal lens.

[0026] Figure 1 Figure b is a microscopic photo of the corneal stroma lens before and after decellularization. It can be seen that the lens before decellularization retains complete cell nuclei and cell structures, while after decellularization, the cell components are obviously removed, leaving only the three-dimensional structure of the extracellular matrix (ECM). This shows that the decellularization process effectively removes cell components while retaining the basic structure of the matrix, providing a suitable biological scaffold for subsequent applications. Example 2 Preparation of microneedle-modified corneal stroma lens Combination Figure 2, dissolve hyaluronic acid in deionized water, add methacrylic acid, stir the reaction thoroughly, adjust the pH to weak alkalinity, remove unreacted monomers and other impurities, obtain an aqueous solution of methacryloyl hyaluronic acid, and freeze-dry the purified solution to obtain methacryloyl hyaluronic acid (HAMA) powder.

[0027] HAMA powder was then dissolved in sterile deionized water to achieve concentrations of 2%, 5%, and 10% (w / v). 0.025% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was also added to the mixture as a photoinitiator. HAMA was then cast into a polydimethylsiloxane (PDMS) mold and centrifuged at 3000g for 5 min to remove bubbles. The excess HAMA solution was then removed. A piece of lens was placed on the back of the microneedle as a supporting substrate. The filled mold was exposed to 405nm UV light for 30 seconds. After photopolymerization, the HAMA hydrogel will solidify to form microneedles. The microneedles were then carefully peeled off the mold. For MSC-encapsulated microneedles, MSCs were first digested and suspended in cell culture medium. After centrifugation, the cell pellet was first mixed with the HAMA solution. The MSC@HAMA solution was then used to make MSC-encapsulated microneedle-modified lenses.

[0028] Figure 2 The optical microscope images of the microneedle-modified corneal stroma lens are given. (a) illustrates the preparation process. In (b) and (c), it can be observed that the microneedles are evenly arranged on the lens surface, showing a regular array structure, indicating that the preparation process has good accuracy and repeatability. (d) provides a magnified view of the microneedle, further showing the sharp morphology and high consistency of each microneedle. This structure helps penetrate the corneal epithelium and achieve more efficient function, enhancing drug delivery or tissue binding performance. Example 3: Application of corneal stroma lens modified with microneedles encapsulating mesenchymal stem cells in corneal damage repair New Zealand rabbits weighing 2.5-3.5 kg were selected for animal experiments. Rabbits were anesthetized with a combination of intramuscular ketamine (35-50 mg / kg) and xylazine (5-10 mg / kg). A femtosecond laser was used to create a corneal incision of approximately 2-3 mm in length at the corneoscleral junction, and a stromal pocket was made in the corneal stroma using a VisuMax laser. The stromal pocket was formed by removing and cutting excess corneal tissue. Then, the microneedle-modified corneal stromal lens encapsulated with mesenchymal stem cells was slowly implanted into the stromal pocket. Corneal repair was observed on the day of surgery, 1 week, 2 weeks, 4 weeks, and 8 weeks after surgery. The severity of corneal inflammation, transparency, and epithelial smoothness were observed by slit lamp and fluorescent staining. On the day of surgery, both the cornea and conjunctiva showed severe swelling and congestion, accompanied by white secretions. The incision site could be observed around the implantation area. One week after surgery, the inflammation was relieved and the implanted lens remained transparent. The lens was successfully implanted into the stroma and remained stable during the observation period. The modified microneedles were still visible, indicating that the microneedles did not fall off during implantation and were able to adhere to the stromal bed. Eight weeks after surgery, there was no significant inflammation, the cornea retained the lens, and the lens had good biocompatibility with the surrounding host cornea.

[0029] Figure 3 This is a photo of the anterior segment of a rabbit cornea transplanted with a microneedle-modified corneal stroma lens. It can be seen that (a) shows a slit lamp image of the cornea, where the cornea maintains transparency and is accompanied by mild edema. (b) is an optical coherence tomography image of the anterior segment of the cornea, which shows the hierarchical structure of the cornea in more detail, including the epithelium, anterior elastic layer, stroma, posterior elastic layer, and endothelium. The lens is effectively implanted between the corneal stroma layers, and the modified microneedles can be seen, which helps to improve the biocompatibility and transplant stability of the lens.

[0030] Figure 4 This is a stained picture of the cornea section 8 weeks after corneal transplantation. It can be seen that the corneal collagen is arranged neatly, and there is no obvious infiltration of inflammatory factors. No cell nuclei are observed inside the transplanted lens, but some cell nuclei are observed around the edge of the lens, indicating that the decellularized lens has good biocompatibility with the surrounding tissues and the cells can be integrated at the edge. Comparative Example In contrast, the lack of microneedle modification in traditional corneal stromal lens transplantation may lead to loose integration of the lens and the stromal bag, affecting the restoration of transparency, and may trigger a stronger local inflammatory response, resulting in corneal edema and inflammatory cell infiltration.

[0031] Figure 5These are photos of the anterior segment after traditional corneal stroma lens transplantation. It can be seen that (a) shows the slit lamp image of the cornea, and it can be observed that the transparency of the corneal graft has decreased, the cornea is edematous, and there is conjunctival congestion. (b) The image is an optical coherence tomography image of the anterior segment of the cornea, and it can be observed that the cornea is obviously edematous and the transplanted lens graft is not tightly attached. (b) The image is a corneal section staining image 8 weeks after surgery, which shows that the corneal collagen fiber structure is disordered, a large number of inflammatory factors are infiltrated, and the lens graft is dissolved, which is also accompanied by a large number of inflammatory cell infiltrations. The present invention provides a concept and method for preparing a microneedle-modified corneal stroma lens encapsulating mesenchymal stem cells. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a microneedle-modified corneal stromal lens encapsulating mesenchymal stem cells, characterized in that: The steps include: S1: adding sodium dodecyl sulfate to phosphate buffer solution to prepare a mixed solution; adding a fresh corneal stromal lens to the mixed solution and shaking it thoroughly; S2: washing the corneal stromal lens in step S1 in a sterile phosphate buffer solution to remove excess sodium dodecyl sulfate on the surface, thereby obtaining a decellularized corneal stromal lens; S3: dissolving hyaluronic acid in deionized water, adding methacrylic acid, stirring the reaction thoroughly, adjusting the pH value to weak alkalinity, removing unreacted monomers and other impurities, obtaining an aqueous solution of methacryloyl hyaluronic acid, and freeze-drying the purified solution to obtain methacryloyl hyaluronic acid powder; S4: dissolving the methacrylylated hyaluronic acid powder in step S3 in a sterile phosphate buffer solution, adding a photoinitiator, and adding the solution to a suspension containing mesenchymal stem cells to obtain a photocurable hydrogel precursor solution; S5: adding the photocurable hydrogel precursor solution prepared in step S4 to the microneedle mold, removing excess bubbles by centrifugation, absorbing excess methacryloyl hyaluronic acid solution from the microneedle base, and placing the acellular corneal stromal lens prepared in step S2 at the microneedle base; S6: The microneedle mold prepared in step S5 is cured under ultraviolet light, and the microneedle is demoulded.

2. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S1, the dosage ratio of sodium dodecyl sulfate to phosphate buffer solution is 0.04-0.06 g / 30-60 mL; the corneal stromal lens is added to the mixed solution and the shaking temperature is controlled at 3-5° C., and the shaking condition is 150-180 rpm / min.

3. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S2, the washing conditions are 3-5°C, an oscillation frequency of 150-180 rpm / min, for 10-30 min, and repeated twice or more.

4. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S3, unreacted monomers and other impurities are removed by dialysis or ultrafiltration, the dialysis frequency is 400-600 rpm / min, and the dialysis is performed at room temperature for more than 5 days.

5. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S3, the concentration of the aqueous solution of methacryloyl hyaluronic acid obtained is (2-4) g / 100 mL.

6. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S4, the usage ratio of methacryloyl hyaluronic acid powder to sterile phosphate buffer solution is (0.05-0.15) g / (1-2) mL.

7. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S4, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and the amount of the photoinitiator added is 1 / 40 to 1 / 30 of the mass of the methacrylated hyaluronic acid powder.

8. The method for preparing the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 1, characterized in that: In step S6, the UV lamp curing condition is to cure under UV light with a wavelength of 400-410 nm for 10s-30s.

9. The corneal stroma lens encapsulated with mesenchymal stem cells and modified by the preparation method according to any one of claims 1 to 8.

10. Use of the mesenchymal stem cell encapsulated microneedle modified corneal stroma lens according to claim 9 in the preparation of drugs for treating corneal diseases.

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