Intelligent contact lens with controllable release and real-time monitoring and preparation method thereof

Abstract

The application discloses a kind of controllable release and real-time monitoring intelligent contact lenses and preparation method thereof.The preparation method of a kind of drug-loaded contact lenses provided by the application includes the following steps: S1, providing contact lenses;S2, providing drugs;Providing flexible carrier;The drug is loaded on the flexible carrier, and the flexible carrier loaded with drug is obtained;S3, the flexible carrier loaded with drug is compounded with the contact lenses, and the drug-loaded contact lenses are obtained.The application first develops flexible carrier such as paper substrate as drug carrier, and is combined with contact lenses, to form wearable drug release device;Flexible carrier can provide a variety of drug loading methods, and improve drug loading rate.In addition, the application connects ester peptide sensitive to matrix metalloproteinase 9 (MMP-9) related to a variety of pathological processes as drug to realize on-demand drug release;Dye labeling is carried out on the drug, and the progress of drug release is monitored in real time, to remind patients to replace contact lenses in time.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of an intelligent contact lens, in particular to a preparation method of an intelligent wearable device capable of realizing controllable release and real-time monitoring of drugs. BACKGROUND

[0002] At present, contact lenses are mainly used for correcting vision and playing a cosmetic effect, and can provide a unique wearable ophthalmic treatment platform, such as delivering sodium hyaluronate, cyclosporine A, timolol and the like, because they can continuously contact tears. However, the current drug loading method is mostly to soak the contact lens in the drug, and the loading rate is low. Secondly, since the method is physical loading, the half-life of drug release is short. Thirdly, the drug is not released on demand according to the severity of the disease. Finally, the function of real-time monitoring of the drug cannot be realized to remind the patient to replace the contact lens in time. SUMMARY

[0003] The application aims to provide an intelligent contact lens capable of realizing controllable release and real-time monitoring and a preparation method thereof. The drug is loaded on a flexible carrier, and then the flexible carrier loaded with the drug is combined with the contact lens to obtain the drug-loaded contact lens, so that the drug loading rate can be improved, and the functions of controllable release and real-time monitoring of the drug are realized on this basis.

[0004] The application provides a preparation method of a drug-loaded contact lens, which comprises the following steps:

[0005] S1, providing a contact lens;

[0006] S2, providing a drug;

[0007] providing a flexible carrier;

[0008] loading the drug on the flexible carrier to obtain a flexible carrier loaded with the drug;

[0009] S3, combining the flexible carrier loaded with the drug with the contact lens to obtain the drug-loaded contact lens.

[0010] In the above preparation method, in step S1, the contact lens can be a corneal lens or a scleral lens.

[0011] Further, the corneal lens can be a hydrogel contact lens.

[0012] Still further, the hydrogel lens is prepared by curing and polymerizing monomers under the action of an initiator.

[0013] The hydrogel contact lens material can be HEMA, HEMA mixed material (Hefilcon, Phemfilcon, Perfilcon, etc.) or non-HEMA material (Corfilcon, Lidofilcon, Atlafilcon, etc.), and the polymerization monomer can be specifically hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA) and methacrylic acid (MAA).

[0014] The initiator can be specifically azobisisobutyronitrile or BASF photoinitiator 1173.

[0015] Specifically, the contact lens is made of the following raw materials, with the total mass percentage of the raw materials being 100%:

[0016]

[0017]

[0018] In one embodiment of the present application, the contact lens is made of the following raw materials, with the total mass percentage of the raw materials being 100%:

[0019]

[0020] The curing can be photocuring or thermal curing, such as ultraviolet light curing.

[0021] In another embodiment of the present application, the contact lens is made of the following raw materials, with the total mass percentage of the raw materials being 100%:

[0022]

[0023] The curing can be photocuring or thermal curing, such as ultraviolet light curing.

[0024] The hydrogel contact lens can be specifically prepared in a contact lens mold; the mold is a fully commercialized complete mold.

[0025] In the above preparation method, in step S2, the drug can be extracted from cells or purchased from commercial channels. The drug can be cutting-edge materials such as exosomes and silver nanoparticles, or commercialized antibiotics (chloramphenicol, levofloxacin, tobramycin dexamethasone, etc.), steroids, immunosuppressants (cyclosporine, etc.). The exosomes can come from stem cells, macrophages, Treg cells, etc.

[0026] In the above preparation method, in step S2, the flexible carrier can be a flexible film made of flexible material. The drug can be loaded by physical loading, chemical loading or by compounding the drug with nanoparticles.

[0027] Preferably, the drug is covalently loaded on the flexible carrier; the flexible carrier is a flexible film capable of providing grafting groups (such as modified grafting groups), such as a polymer fiber film, a gel film or a plastic film; the polymer fiber film can be a nylon, a nitrocellulose film, a polyacrylonitrile fiber film, a polylactic acid or other materials capable of chemical grafting modification. The polymer fiber film can be a commercially available polymer fiber film, or a polymer fiber film prepared by electrospinning or melting method.

[0028] More preferably, in step S2, the loading step can be as follows:

[0029] grafting streptavidin to the surface of the flexible carrier to obtain a flexible carrier / streptavidin;

[0030] connecting biotin to the drug to obtain a drug / biotin.

[0031] specifically binding biotin in the drug / biotin to streptavidin in the flexible carrier / streptavidin to obtain a drug-loaded flexible carrier.

[0032] In a specific example of the present application, the method further comprises a step of chemically hydrolyzing the flexible carrier to expose functional groups for grafting before grafting the streptavidin to the surface of the flexible carrier, such as exposing carboxyl groups by hydrolysis under alkaline conditions, and grafting the streptavidin to the surface of the flexible carrier using EDC / NHS reaction. The grafting step can be specifically as follows: incubating the flexible carrier with exposed grafting functional groups with the streptavidin (such as incubating at 37℃ for 1h), and then blocking the free sites on the flexible carrier (such as using 3% bovine serum albumin at 37℃ for 30min).

[0033] In a specific example of the present application, the specific binding step is as follows: incubating the drug / biotin with the flexible carrier / streptavidin (such as co-incubating at 37℃ for 1h), which can be achieved.

[0034] Further preferably, on the one hand, in order to further achieve the function of controllable release of the drug, the biotin is connected with an ester peptide, and the ester peptide contains a fragment interval that can be specifically recognized by matrix metalloproteinase;

[0035] In step S2, the loading step is as follows:

[0036] connecting biotin to the ester peptide to obtain an ester peptide-biotin; and connecting the ester peptide-biotin to the drug to obtain a drug / ester peptide-biotin;

[0037] Specifically binding the biotin in the drug / ester peptide-biotin to the streptavidin in the flexible carrier / streptavidin, to obtain a flexible carrier loaded with drugs and capable of controlled release of drugs.

[0038] The matrix metalloproteinase can be any matrix metalloproteinase having "click" chemical breaking performance, such as MMP-9 or MMP-2.

[0039] The ester peptide contains a fragment interval that can be specifically recognized by a matrix metalloproteinase, such as a fragment interval that can be specifically recognized by MMP-9 (PLG↓VR); in specific embodiments of the present application, the biotin-modified ester peptide can specifically be bio-GGGPLGVRGKGGC, wherein bio represents biotin.

[0040] Further preferably, in another aspect, in order to achieve the function of real-time monitoring, the method further comprises the step of dye labeling the drug for real-time monitoring of the release of the drug.

[0041] In specific embodiments of the present application, the drug is dye labeled by any one of the following A1) and A2):

[0042] A1) dye labeling the drug; connecting biotin to the dye-labeled drug;

[0043] A2) dye labeling the drug; connecting the drug to biotin through the dye.

[0044] The substance used for the dye labeling can be a fluorescent dye or a dye of a color recognizable by the naked eye;

[0045] The fluorescent dye can be an Alex series dye, a Cy series dye, or fluorescein isothiocyanate (FITC);

[0046] The dye recognizable by the naked eye can specifically be a gold nanoparticle.

[0047] In the present application, drug carriers are designed by taking advantage of the significant difference in biochemical parameters between the tear fluid of healthy people and patients, and these carriers can respond to these changes and release drugs according to the severity. For example, matrix metalloproteinase 9 (MMP-9) enzyme is involved in the pathological process of many ophthalmic diseases, such as dry eye, keratitis, cataract, glaucoma, etc., and overexpression of MMP-9 can trigger the release of drugs from the carrier. In specific embodiments of the present application, the controlled release of drugs can be achieved according to the content of MMP-9 in the tear fluid of patients.

[0048] In the present application, in order to achieve the functions of controlled release of drugs and / or real-time monitoring of drugs of the drug-loaded contact lenses, the connection order of the functional groups can be adjusted according to the actual situation in the preparation method.

[0049] For example, in one specific example of the present application, the drug is loaded on the flexible carrier by the following steps:

[0050] 1) grafting streptavidin SA to the surface of polyacrylonitrile nanofiber membrane PAN to obtain a flexible carrier-streptavidin PAN-SA;

[0051] 2) providing drug exosomes EXO and connecting a biotin-modified ester peptide bio-GGGPLGVRGKGGC to the drug; more specifically, connecting the exosomes to the biotin-modified ester peptide by modifying gold nanoparticles on the surface of the exosomes using Au-S bonds to obtain EXO-Au-bio-GGGPLGVRGKGGC;

[0052] 3) specifically binding biotin in EXO-Au-bio-GGGPLGVRGKGGC to streptavidin in PAN-SA to obtain PAN-SA / EXO-Au-bio-GGGPLGVRGKGGC.

[0053] In this example, the modification of the drug with gold nanoparticles can achieve the connection of biotin-ester peptide on one hand, and can monitor the release of the drug in real time by observing the color change of the gold nanoparticles on the other hand.

[0054] For example, in another specific example of the present application, the drug is loaded on the flexible carrier by the following steps:

[0055] 1) grafting streptavidin SA to the surface of polyacrylonitrile nanofiber membrane PAN to obtain a flexible carrier-streptavidin PAN-SA;

[0056] 2) providing drug exosomes EXO and labeling the drug with a fluorescent dye such as DID to obtain dye-labeled drug DID-EXO; connecting a biotin-modified ester peptide bio-GGGPLGVRGKGGC to the exosomes (such as by modifying 6-maleimide at the end of the ester peptide to achieve the connection of the biotin-modified ester peptide to the exosomes through the bonding of 6-maleimide to the exposed sulfhydryl group of the exosomes) to obtain DID-EXO-bio-GGGPLGVRGKGGC;

[0057] 3) specifically binding biotin in DID-EXO-bio-GGGPLGVRGKGGC to streptavidin in PLA-SA to obtain PLA-SA / DID-EXO-bio-GGGPLGVRGKGGC.

[0058] In the above preparation method, in step S3, the complexing step is as follows:

[0059] Cutting a micro-channel on the inner side of the contact lens; cutting a flexible carrier into a shape corresponding to the size of the micro-channel, and fitting it in the micro-channel.

[0060] Preferably, the thickness of the flexible carrier is 5-30 μm;

[0061] Preferably, the micro-channel is one or more annular micro-channels distributed outside the center of the contact lens.

[0062] Preferably, the diameter of the micro-channel is 0.5-2 mm (such as 1 mm).

[0063] Preferably, the plurality of annular micro-channels can be concentric; the distance between two adjacent annular micro-channels can be 1-5 mm.

[0064] The drug-loaded contact lens prepared by the preparation method of any one of the above is also within the protection scope of the present application.

[0065] The present application has the following beneficial effects:

[0066] 1. The present application first develops a flexible carrier such as a paper substrate as a drug carrier, and combines it with a contact lens to form a wearable drug release device. Its advantage is that the flexible carrier can provide multiple drug loading methods, including physical loading, chemical loading or compounding with nanoparticles, which improves the drug loading rate.

[0067] 2. The present application designs an ester peptide (bio-GGGPLGVRGKGGC) sensitive to matrix metalloproteinase 9 (MMP-9) related to various pathological processes as a drug connection. Compared with the existing slow-release method, this design can realize on-demand drug release according to the patient's condition, thereby realizing personalized treatment. In addition, in addition to MMP-9, other collagenases with "click" breaking performance such as MMP-2 can also be selected.

[0068] 3. The present application is marked with a dye such as a dye or a fluorescent dye on the drug, which can monitor the progress of drug release in real time to remind the patient to replace the contact lens in time. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 Transmission electron microscope photos of the exosome drug MSC-EXO prepared in Example 1 and Example 2.

[0070] Figure 2 Transmission electron microscope photos of the gold nanoparticle-modified exosome EXO-Au in Example 1 (the scale is ~ 150 nm).

[0071] Figure 3This is a photograph of the smart contact lens with controllable release and real-time monitoring prepared in Example 1.

[0072] Figure 4 These are photographs of the gold-labeled drug and the flexible carrier before and after drug loading, prepared in Example 1. Figure 4 (a) is a photograph of a gold-labeled drug. Figure 4 (b) The left image in the middle is a photo of the flexible carrier before drug loading, which is white, and the right image is a photo of the flexible carrier after drug loading, which is red.

[0073] Figure 5 The curve shows the change in drug release rate over co-incubation time when the flexible carrier carrying the drug in Example 1 is co-incubated with MMP-9.

[0074] Figure 6 The image shows the red color fading after the flexible carrier loaded with the drug in Example 1 was co-incubated with MMP-9 (2 μg / mL) for 48 hours.

[0075] Figure 7 These are fluorescein sodium staining images taken 5 and 9 days after the mice with dry eye were treated with exosomes by instilling them into their eyes in Example 1.

[0076] Figure 8 The images show confocal fluorescence images of the DID-labeled drug before and after PLA-SA in Example 2. The left image is a confocal fluorescence image of PLA-SA, and the right image is a confocal fluorescence image of PLA-SA / DID-EXO-bio-GGGPLGVRGKGGC.

[0077] Figure 9 The left image shows the fluorescence intensity of PLA-SA / DID-EXO-bio-GGGPLGVRGKGGC co-incubated with 100 μL of MMP-9 (2 μg / mL) for 24 h, and the right image shows the fluorescence on the PLA membrane after incubation. Detailed Implementation

[0078] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0079] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0080] The sources of raw materials used in the following examples are as follows:

[0081] Streptavidin (SA) was purchased from Beijing Solabio Technology Co., Ltd., product name: Streptavidin, product number or product catalog number: S9171.

[0082] Mesenchymal stem cells (MSCs) were purchased from the National Biomedical Experimental Cell Resource Library, item number: 5301RAT-KCB1917BMS.

[0083] Biotin-labeled ester peptide (bio-GGGPLGVRGKGGC) was customized by Shanghai Qiangyao Biotechnology Co., Ltd.

[0084] Fluorescent dye DID was purchased from Thermo, product name: Vybrant TM DiD cell labeling solution, product number: V22887.

[0085] Example 1, preparation of smart contact lenses with controllable release and real-time monitoring

[0086] I. Preparation

[0087] The smart contact lenses with controllable release and real-time monitoring were prepared according to the following steps:

[0088] S1, preparation of contact lenses

[0089] Material selection: the composition of each raw material is as follows in mass percentage: hydroxyethyl methacrylate (HEMA) 60%, methacrylic acid (MAA) 0.6%, ethylene glycol dimethacrylate (EGDMA) 0.6%, water 38.2%, BASF photoinitiator 1173 0.6%.

[0090] Lens mold parameters: lens thickness 0.10 mm, base curve radius 8.60 mm, diameter 14 mm. Light curing time 3-5 minutes.

[0091] The specific steps are as follows: mix each material uniformly according to the above proportion, take 150 μL of the mixed solution and add it to the lens mold, use a UV lamp with a wavelength of 365 nm (50 W) to irradiate for 5 min, and the curing is complete. Then soak in water above 80°C for 24h to complete the hydration process.

[0092] S2, preparation and modification of flexible carrier

[0093] Polyacrylonitrile nanofiber membrane (PAN) prepared by electrospinning: Polyacrylonitrile powder was added into dichloromethane with a concentration of 8 w / v%, and magnetically stirred for 8 h. A direct current output of +13 kV was applied at a distance of 25 cm under the condition of 25 °C and 35% relative humidity. The flow rate of the solution was set to 3 mL / h, and the electrospinning time was 2 h, resulting in a polyacrylonitrile nanofiber membrane (PAN) of ~100 μm. The collected nanofiber membrane was dried at 60 °C for 8 h, followed by hot roller pressing (60 °C). The prepared PAN had a thickness of 5-30 μm, specifically 20 μm in this embodiment, and a fiber diameter of 200-800 nm.

[0094] The PAN membrane was hydrolyzed at 60 °C for 1 h using a 0.1 M sodium hydroxide solution to expose carboxyl groups, and a 0.1 M HC1 solution was added to neutralize the base solution to terminate the hydrolysis. The PAN membrane after surface hydrolysis treatment was washed with deionized water and dried at room temperature. The hydrolyzed PAN membrane was placed in a 10 mL EDC / NHS ethanol solution (EDC 30 mM, NHS 10 mM). After 15 min at 37 °C, the carboxyl groups were activated, and then washed thoroughly with PBS. The activated PAN membrane was incubated with 1 mg / mL streptavidin at 37 °C for 1 h. Then, the free sites on the membrane were blocked with a 3% bovine serum albumin solution for 30 min at 37 °C. After each reaction step, the PAN-SA was washed thoroughly with PBST solution and the surface residual moisture was absorbed with filter paper. The resulting PAN-SA was stored at 4 °C.

[0095] S3, drug preparation, fluorescent labeling, and ester peptide connection

[0096] Mesenchymal stem cells were cultured, and the culture process was as follows: culture medium conditions: DMEM high glucose medium (Solebao, 11995), 1% double antibody (Solebao, P1400), 10% fetal bovine serum (Gibco, 1921005PJ). A 10 cm diameter dish was used, 10 mL of the above culture medium was added, and then 5 x 10 6 mesenchymal stem cells were added. It was placed in a 37 °C, 5% CO2 incubator. The cells were observed, and the cells were passaged at a ratio of 1:2 every 2-3 days. The supernatant was collected during the passage for subsequent collection of exosomes.

[0097] MSC-EXO was collected by differential centrifugation. The specific method is as follows: collect 200 mL of culture supernatant of MSC, centrifuge at 300 g for 10 min at 4°C, remove impurities and take the supernatant. Centrifuge at 2000 g for 10 min at 4°C, remove dead cells and take the supernatant. Centrifuge at 10000 g for 30 min at 4°C, remove cell debris and take the supernatant. Centrifuge at 100000 g for 70 min at 4°C, discard the supernatant, and the exosomes are contained in the precipitate. Resuspend the precipitate with PBS, centrifuge again at 4°C for 70 min, and discard the supernatant. Resuspend the precipitate with 200 μL of PBS, collect it in a sterile EP tube, and store it at -80°C for standby use (MSC-EXO). The TEM image is shown in Figure 1 .

[0098] Gold nanoparticles were modified on the surface of exosomes, and biotin-modified ester peptide (bio-GGGPLGVRGKGGC) was connected to the exosomes by Au-S bond to form EXO-Au-bio-GGGPLGVRGKGGC drugs. The specific steps are as follows:

[0099] Preparation: Take 4 mL of MSC-EXO (2 mg / mL), add 20 μL of DSPE-PEG-SH (10 mg / mL), and incubate at 4°C for 20 min. Add 400 μL of HAuCl4 (29 mM), incubate at 37°C for 40 min. Transfer the solution to an ultrafiltration tube (10 kD), centrifuge at 4500 g for 10 min, and remove the ungrafted HAuCl4. Supplement PBS to 4 mL, and adjust the pH to 9 with 0.1 M K2CO3. Add 400 μL of NaBH4 (20 mM). Centrifuge the ultrafiltration tube at 4500 g for 10 min, and remove the NaBH4. Continue to wash with PBS (pH 9) for two more times. Determine the protein concentration of EXO@Au using a BCA kit, and then dilute it with PBS (pH 9) to a protein concentration of 2 mg / mL to obtain gold nanoparticle-modified exosomes (EXO-Au). The TEM image is shown in Figure 2 .

[0100] Labeling: Add 0.1 mL of linker (bio-GGGPLGVRGKGGC, pH 9, 1 mg / mL) (the solvent is PBS (0.01 M, pH 7.2-7.4), and the pH is adjusted with 0.1 M K2CO3) to 1 mL of EXO@Au (pH 9), incubate at 37°C for 60 min. Add 200 μL of BSA (1%), incubate at 37°C for 30 min. Centrifuge at 10000 rpm at 4°C for 20 min. Remove the supernatant, resuspend with 200 μL of 1% BSA to obtain MMP-9 specific ester peptide-labeled nanodrugs (EXO-Au-bio-GGGPLGVRGKGGC), and store them at -80°C for standby use.

[0101] S4, drug loading

[0102] Drug loading to flexible carrier: EXO-Au-bio-GGGPLGVRGKGGC was co-incubated with PAN-SA at 37℃ for 1h to obtain PAN-SA / EXO-Au-bio-GGGPLGVRGKGGC, and drug loading to flexible carrier was achieved through specific binding of streptavidin and biotin.

[0103] S5, drug and flexible carrier complexing

[0104] Using laser cutting technology, microchannels with a diameter of 0.5-2mm (the depth of the microchannels is 5-30μm, and the distance between the microchannels is 1-5mm) were cut in the contact lenses, and the corresponding flexible carriers were cut and trimmed, and the two were complexed to obtain intelligent contact lenses with controllable release and real-time monitoring, the photos of which are shown in Figure 3 , the thickness of which is 20μm, the diameter of the microchannels is 1mm, and the distance between the adjacent two microchannels is 3mm.

[0105] II. Experimental results and analysis

[0106] A. Successful drug loading

[0107] As shown in Figure 4 , the gold-labeled drug (EXO-Au) prepared in the above step S3 is red ( Figure 4 a), the flexible carrier prepared in step S2 is white before drug loading, and the flexible carrier after drug loading prepared in step S5 is red ( Figure 4 b), proving the successful loading of the drug.

[0108] B. Controllable release of ester peptide sensitive to MMP-9

[0109] As shown in Figure 5 , the drug-loaded flexible carrier was co-incubated with MMP-9 (2μg / mL), and with the increase of co-incubation time, the release amount of the drug increased significantly, proving the controllable release of ester peptide sensitive to MMP-9.

[0110] C. Real-time monitoring of drug

[0111] In this embodiment, the drug labeled with gold nanoparticles can give a color signal to prove that the drug can be monitored in real time. As shown in Figure 6 , the drug-loaded flexible carrier was co-incubated with MMP-9 (2μg / mL) for 48h, and the red color on the carrier faded, so the labeling of gold nanoparticles played a role in visual perception of the residual amount of the drug, prompting people to replace the contact lenses in time.

[0112] D. Effectiveness of the drug

[0113] In C57BL mice for 14 consecutive days (C57BL purchased from Beijing VitoLiu Experimental Animal Technology Co., Ltd., mouse age 42-48 days, gender: male mice), every day at 9 o'clock in the morning and 9 o'clock in the evening, a total of twice, 10 μL of benzalkonium chloride (0.2%) was added to the mouse eye. Then the mouse was added with the exosome (EXO) drug prepared in step S3 (10 μL each time, 1 mg / mL, once in the morning and once in the evening), after 9 days of administration, it can be clearly seen from the fluorescein sodium staining picture ( Figure 7 ) that the dry eye of the mouse is almost completely recovered, while the control group (dry eye group-DED is not treated). It is proved that the EXO drug has superior therapeutic effect.

[0114] Example 2, preparation of controllable release and real-time monitoring of smart contact lenses

[0115] The controllable release and real-time monitoring of smart contact lenses are prepared according to the following steps:

[0116] S1, preparation of contact lenses

[0117] Material selection: the composition of each raw material is as follows in mass percentage: hydroxyethyl methacrylate (HEMA) 75%, methacrylic acid (MAA) 0.75%, ethylene glycol dimethacrylate (EGDMA) 0.75%, water 22.75%, BASF photoinitiator 1173 0.75%.

[0118] Lens mold parameters: lens thickness 0.10 mm, base curve radius 8.60 mm, diameter 14 mm. Light curing time 3-5 minutes.

[0119] The specific steps are as follows: mix each material uniformly according to the above proportion, take 150 μL of mixed solution and add it to the lens mold, use a 365 nm (50 W) wavelength ultraviolet lamp to irradiate for 5 min, and solidify completely. Then soak in water above 80℃ for 24 h to complete the hydration process.

[0120] S2, preparation and modification of flexible carrier

[0121] Poly lactic acid nanofiber membrane (PLA) prepared by electrospinning: Poly lactic acid powder was added into dichloromethane with a concentration of 12 w / v%, and magnetically stirred for 8 h. A direct current output of +17 kV was applied at a distance of 25 cm under the condition of 25 °C and 35% relative humidity. The flow rate of the solution was set to 1 mL / h, and the electrospinning time was 6 h, producing a poly lactic acid nanofiber membrane (PLA) of ~100 μm. The collected nanofiber membrane was dried at 60 °C for 8 h, followed by hot roller pressing (60 °C). The prepared PLA had a thickness of 5-30 μm, and the thickness of the embodiment was 20 μm, and the fiber diameter was 200-800 μm.

[0122] Carboxyl groups were exposed by hydrolysis using 0.1 M sodium hydroxide solution at 60 °C for 1 h, and the alkaline solution was neutralized by adding 1.1 mL of 0.1 M HCl to terminate the hydrolysis. The PLA membrane after surface hydrolysis treatment was washed with deionized water and dried at room temperature. The hydrolyzed PLA membrane was placed in a 10 mL EDC / NHS ethanol solution (EDC 30 mM, NHS 10 mM). After 15 min at 37 °C, the carboxyl groups were activated, and then washed thoroughly with PBS. The activated PLA membrane was incubated with 1 mg / mL streptavidin at 37 °C for 1 h. Then the free sites on the membrane were blocked with a 3% bovine serum albumin solution for 30 min at 37 °C. After each reaction step, the membrane was washed thoroughly with PBST solution and the surface residual moisture was absorbed with filter paper. The obtained PLA-SA was stored at 4 °C.

[0123] S3, drug preparation, fluorescent labeling and ester peptide connection

[0124] Mesenchymal stem cells were cultured, and the culture process was as follows: culture medium conditions: DMEM high glucose medium (Solebao, 11995), 1% double antibody (Solebao, P1400), 10% fetal bovine serum (Gibco, 1921005PJ). A 10 cm diameter dish was used, 10 mL of the above culture medium was added, and then 5 x 10 6 mesenchymal stem cells were added. It was placed in a 37 °C, 5% CO2 incubator. The cells were observed, and the cells were passaged at a ratio of 1:2 every 2-3 days. The supernatant was collected during the passage for subsequent collection of exosomes.

[0125] MSC-EXO was collected by differential centrifugation. The specific method is as follows: collect 200 mL of culture supernatant of MSC, centrifuge at 300 g for 10 min at 4°C, remove impurities and take the supernatant. 2000 g, 4°C centrifugation for 10 min, remove dead cells and take the supernatant. 10000 g, 4°C centrifugation for 30 min, remove cell debris and take the supernatant. 100000 g, 4°C centrifugation for 70 min, discard the supernatant, and the exosomes are contained in the precipitate. Resuspend the precipitate with PBS, centrifuge at 4°C for 70 min again, discard the supernatant. Resuspend the precipitate with 200 μL PBS, collect it in a sterile EP tube, and store it at -80°C for standby use (MSC-EXO), and the TEM map is as shown in Figure 1 .

[0126] Labeling exosomes with fluorescent dye DID: take 100 μL of 5 mg / mL, add 1 μL of DID dye, incubate at 37°C for 1 h, then filter the excess dye with an exosome filter column to obtain DID dye-labeled exosomes (DID-EXO), and connect the bio-modified ester peptide (bio-GGGPLGVRGKGGC, ester peptide ordered from Shanghai Qiangyao Biotechnology Co., Ltd., and the ends are modified with 6-maleimide) to the exosomes: treat the EXO solution with 1 mM TECP solution at 37°C for 30 min to break the disulfide bonds on the surface of the exosomes, exposing the sulfhydryl group on the 6-maleimide on the ester peptide. Add ester peptide to the 1 mg / mL EXO solution and react at 25°C for 1 h to form DID-EXO-bio-GGGPLGVRGKGGC drugs.

[0127] S4, drug loading

[0128] Drug loading on flexible carrier: co-incubate DID-EXO-bio-GGGPLGVRGKGGC with PLA-SA at 37°C for 1 h to obtain PLA-SA / DID-EXO-bio-GGGPLGVRGKGGC, achieving drug loading on the flexible carrier.

[0129] S5, drug and flexible carrier complexing

[0130] Using laser cutting technology, microchannels with a diameter of 0.5-2 mm (the depth of the microchannels is 5-30 μm, and the distance between the microchannels is 1-5 mm) are cut in the contact lenses, and the corresponding flexible carriers are cut out, and the two are complexed to obtain intelligent contact lenses for controlled release and real-time monitoring.

[0131] II. Experimental results and analysis

[0132] A. Successful loading of drugs

[0133] Experimental data demonstrating the attachment of DID-labeled drugs to PLA-SA (d = 2 mm, thickness 30 μm): (e.g.) Figure 8 As shown, the left image is a fluorescence confocal image of PLA-SA, and the right image is a fluorescence confocal image of PLA / SA / DID-EXO-bio-GGGPLGVRGKGGC, which proves that the DID-EXO drug was successfully loaded onto PLA.

[0134] B. Controlled release of drugs

[0135] Demonstrating the gradual release of DID-labeled EXO: PLA-SA / DID-EXO-bio-GGGPLGVRGKGGC was co-incubated with 100 μL of MMP-9 (2 μg / mL) for 24 h, and the fluorescence value in the MMP-9 solution was detected at different time points. Figure 9 (Left image) demonstrates the gradual release of EXO. After incubation, the fluorescence intensity on the PLA membrane was detected, showing a significant decrease. Figure 9 (See right figure) This allows for real-time monitoring of drug residue levels by observing the intensity of fluorescence signals.

[0136] C. Drug loading rate: One PLA-SA tablet with a diameter of 2 mm and a thickness of 30 μm was co-incubated with 20 μL of DID-EXO-bio-GGGPLGVRGKGGC (1 mg / mL) at 37℃ for 1 h. The fluorescence intensity of the DID-EXO-bio-GGGPLGVRGKGGC solution before and after incubation was detected using a microplate reader, and the drug loading rate was calculated.

[0137] Drug loading rate (%) = (F b -F a ) / F b ×100%

[0138] Where F b - Fluorescence intensity value before incubation, F a - Fluorescence intensity value after incubation. In this embodiment, F b =30625,F a =21417, drug loading rate is 30%.

[0139] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A method for preparing a drug-loaded contact lens, comprising the following steps: S1, providing a contact lens; S2, providing a drug; providing a flexible carrier; the flexible carrier is a polymer fiber membrane, a gel membrane or a plastic membrane; the polymer fiber membrane is a nylon fiber membrane, a nitrocellulose fiber membrane, a polyacrylonitrile fiber membrane or a polylactic acid fiber membrane; loading the drug on the flexible carrier to obtain a drug-loaded flexible carrier; In step S2, the loading step is as follows: grafting streptavidin to the surface of the flexible carrier to obtain a flexible carrier / streptavidin; connecting biotin with an ester peptide to obtain an ester peptide-biotin; connecting the ester peptide-biotin with the drug to obtain a drug / ester peptide-biotin; the ester peptide contains a fragment interval that can be specifically recognized by matrix metalloproteinase; the matrix metalloproteinase is MMP-9; specifically binding biotin in the drug / ester peptide-biotin with streptavidin in the flexible carrier / streptavidin to obtain a drug-loaded flexible carrier that can controllably release the drug; In step S2, the method further comprises a step of dye labeling the drug for real-time monitoring of drug release; S3, compounding the drug-loaded flexible carrier with the contact lens to obtain the drug-loaded contact lens; In step S3, the compounding step is as follows: cutting a microchannel on the inside of the contact lens; cutting the flexible carrier into a shape corresponding to the size of the microchannel and fitting it in the microchannel; the thickness of the flexible carrier is 5-30 μm; the microchannel is one or more annular microchannels distributed outside the center of the contact lens; the diameter of the microchannel is 0.5-2 mm; the plurality of annular microchannels are concentric; the distance between two adjacent annular microchannels is 1-5 mm.

2. The method of claim 1, wherein: In step S2, the drug is loaded on the flexible carrier in a covalent bond manner.

3. The method of claim 1, wherein: The substance used for the dye labeling is a fluorescent dye or a dye with a color that can be recognized by the naked eye.

4. The method of claim 3, wherein: The fluorescent dye is an Alex series dye, a Cy series dye or fluorescein isothiocyanate; the dye with a color that can be recognized by the naked eye is a gold nanoparticle. 5.The drug-loaded contact lens prepared by the method of any one of claims 1-4.

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