Photoactive Injectable Hydrogel, Its Preparation Method and Application in Artificial Retina

Abstract

The present invention provides a method for preparing a photoactive injectable hydrogel. The preparation method includes: soaking the modified hydrogel gel-forming raw materials in a buffer solution, and then soaking the obtained treated product in an ionic solution to obtain the photoactive injectable hydrogel; the modified hydrogel gel-forming raw materials are obtained by grafting dopamine onto the hydrogel gel-forming raw materials; the buffer solution includes Tris buffer solution or Tris-HCl buffer solution; the ionic solution includes divalent manganese ion solution, divalent zinc ion solution or trivalent iron ion solution. The photoactive injectable hydrogel of the present invention has good photothermal effect and photoelectric effect; the raw materials are inexpensive, the preparation method is simple, and it has good prospects for popularization and application; the photoactive injectable hydrogel of the present invention is particularly suitable for use as an artificial retina and overcomes the disadvantages existing in the existing artificial retina technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering materials, and relates to a photoactive injectable hydrogel, and particularly to a photoactive injectable hydrogel that can be used as an artificial retina. Background Art

[0002] Vision plays a crucial role in the cognitive and behavioral processes of humans, and is the main channel for humans to obtain external information. About 80% of human perception, learning, cognition, and activities are regulated through vision. According to statistics from the World Health Organization, the total number of blind people in the world has reached 39 million, and the main causes of blindness include cataract, glaucoma, age-related macular degeneration, childhood blindness, corneal opacity, uncorrected refractive error, trachoma, diabetic retinopathy, and eye diseases of unknown cause. Among them, the vast majority of diseases can be cured by surgery, or regain vision through appropriate preventive or improvement measures. However, irreversible degenerative blinding eye diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) currently do not have good treatment methods.

[0003] When treating ophthalmic diseases including the above-listed eye diseases, due to the complexity of the eye structure, conventional drug delivery methods such as eye drops and eye ointments are difficult to achieve ideal effects in addition to surgery. As a material with multi-scale development, hydrogels have the advantages of high water content, good biocompatibility, good light transmittance, and controllable properties. Moreover, injectable hydrogels also have the advantage of easy operation during surgery. Therefore, they have broad research prospects in the treatment of ophthalmic diseases.

[0004] Currently, the applications of hydrogels in ophthalmic diseases mainly include being used as ophthalmic drug carriers, wound adhesives, vitreous substitutes, and cell scaffolds, etc.

[0005] When using hydrogels as drug carriers, in order to ensure the effectiveness and stability of drug active molecules, it is necessary to consider the drug loading method, drug dosage, polymer concentration, crosslinking density, and degradation rate, etc. For example, WO2014039012A1 discloses a novel hyaluronic acid (HA) hydrogel containing vesicles loaded with drugs or proteins or nucleic acids, which can be used for the treatment of ophthalmic diseases such as glaucoma, uveitis, vitreoretinal, and medical retinal diseases; another example is CN114042157A, which discloses a photosensitive injectable vitreous gel, which improves the treatment effect of AMD by a reasonable combination of natural drugs and assembling them into a suitable drug delivery system; and another example is Xianfang Rong [1]et al. developed a PLGA-PEG-PLGA hydrogel loaded with insulin nanoparticles, which can be used as a drug delivery carrier for related ophthalmic drugs.

[0006] When using a hydrogel as a vitreous substitute, the main considerations are high light transmittance, good viscoelasticity, effective pressing time, and no toxic or side effects on eye tissues. For example, CN113827779A discloses a biological polysaccharide hydrogel, which improves the anti-biological degradation property and the effective pressing time on the retina, and can be used as a vitreous substitute.

[0007] When using a hydrogel as a cell scaffold, it mainly utilizes the characteristics of the hydrogel that can provide a suitable microenvironment for cells to promote the adhesion, proliferation, and differentiation of related cells and play corresponding functions. For example, CN113766937A discloses a complex containing neural retina, retinal pigment epithelial cells, and a hydrogel, which is suitable for the transplantation of neural retina and retinal pigment epithelial cell sheets; another example is Nikolaos Mitrousis et al. [2] used a hydrogel to transplant retinal pigment epithelium (RPE) and photoreceptors, and to a certain extent achieved vision restoration in a retinal degenerative disease model; another example is that CN102258808A discloses a hydrogel cell scaffold for promoting the amplification of retinal pigment epithelial cells, which overcomes the disadvantages of the prior art such as being prone to aging, unable to effectively adhere to the retina, and the transplanted retinal pigment epithelial cells unable to adhere to the fovea centralis of the retina to form a cell layer, and realizes and promotes the adhesion, spreading, and proliferation of human retinal pigment epithelial cells, and can play a function of preventing their aging.

[0008] However, whether the hydrogel is used as a drug delivery carrier, a vitreous substitute, or a cell scaffold, it cannot simulate natural light stimulation to enable blind people to have visual sensations, which limits the application of hydrogels in the treatment of eye diseases such as AMD and RP. In patients with both AMD and RP, although the photoreceptors in the outer layer of the retina are damaged or lost, the inner layer neurons (bipolar cells and ganglion cells) of the retina remain relatively intact, which provides room for people to develop an artificial retina that can produce visual sensations.

[0009] Currently, traditional artificial retinas capable of generating light-stimulated sensations are mainly microelectrode devices. For example, CN111588984A discloses an implant of an implantable retinal stimulator, which can reduce the suture difficulty of the surgery and reduce surgical trauma; another example is that CN104873330A provides an artificial retina implant, the stimulating electrode array of which is fixed on the retina. The vitreous body can not only serve as a support for the electrode array but also as a radiator, and has less impact on the blood vessels of the retina, and is also easy to observe from outside the eye. However, the implantation surgery of such implants is complex and requires a high level of professional operation skills of doctors; the surgery usually requires a sufficiently large scleral incision to allow the electrode array to pass through, and the positioning of the electrode array must be very precise, and there is also a certain risk of tissue infection during the surgery. In addition, because metal or inorganic semiconductors are used as visual prosthesis materials, after implantation, since the implant needs to be in direct contact with eye tissues, these materials with a serious mismatch in mechanical modulus with eye tissues usually cause nerve tissue scars, affecting or even hindering signal transmission. At the same time, these materials do not have the adhesiveness of biological tissues and are difficult to maintain stable adhesion with eye tissues, thereby further affecting signal transmission and the exertion of their functions.

[0010] On this basis, in order to solve the defect that traditional hydrogels are difficult to provide visual sensations and the disadvantages of traditional artificial retinas, such as complex implantation surgery and a series of problems caused by all materials, and at the same time, due to the unique advantages of hydrogels in the treatment of eye diseases, people gradually consider introducing the function of realizing photosensitive stimulation into the hydrogel system. For example, Restrepo Schild V [3] provides a bilayer retinal tissue composed of a hydrogel and a cell membrane protein (bacteriorhodopsin). This artificial retina is composed of natural and biodegradable biological materials and does not contain foreign substances or living substances, making the retina less invasive and having a relatively low probability of causing adverse reactions and side effects; another example is that PCT / US2010 / 026362 provides a protein-based artificial retina, which uses a hydrogel as a substrate and contains multiple monolayers of natural bacteriorhodopsin or bacteriorhodopsin mutants. This patent also overcomes the problem of relatively large surgical invasiveness of traditional artificial retinas during transplantation. At the same time, the implant provided by it can replace damaged photoreceptor cells, so it can be used to treat any retinal disease or condition that has not damaged the bipolar or ganglion cell network.

[0011] As can be seen from the above introduction, the application research of hydrogels in the treatment of eye diseases has been relatively in-depth, and their advantages have been continuously demonstrated. At the same time, traditional artificial retinas have significant surgical invasiveness and postoperative problems such as scarring, which urgently need to be solved. Meanwhile, people have also noticed the theoretical advantages of using hydrogels as matrices or carriers to carry artificial retinas that can be used to generate light-stimulated nerve responses, and there have been a small number of research results in preparing corresponding products. However, the current technical solutions mainly rely on corresponding special proteins, which not only require high raw material costs but also need to design special schemes for maintaining the activity of the corresponding proteins in practical applications.

[0012] In summary, there is an urgent need in this field for a technology that can utilize the advantages of hydrogels, has a simple preparation method, low cost, and can well generate signals for stimulating neurons.

[0013] Non-patent literature cited in this section:

[0014] [1] Rong, X.; Yang, J.; Ji, Y.; Zhu, X.; Lu, Y.; Mo, X. Biocompatibility and safety of insulin-loaded chitosan nanoparticles / PLGA-PEG-PLGA hydrogel (ICNPH) delivered by subconjunctival injection in rats. J. Drug Delivery Sci. Technol. 2019, 49, 556 - 562.

[0015] [2] Mitrousis, N.; Hacibekiroglu, S.; Ho, M. T.; Sauvé, Y.; Nagy, A.; van der Kooy, D.; Shoichet, M. S. Hydrogel-mediated co-transplantation of retinal pigmented epithelium and photoreceptors restores vision in an animal model of advanced retinal degeneration. Biomaterials 2020, 257, 120233.

[0016] [3]Restrepo Schild, V.; Booth, M. J.; Box, S. J.; Olof, S. N.; Mahendran, K. R.; Bayley, H. Light-Patterned Current Generation in a Droplet Bilayer Array. Sci. Rep. 2017, 7, 46585. Summary of the Invention

[0017] Aiming at the disadvantages of the prior art, the object of the present invention is to provide an injectable hydrogel that can generate electrical signals for stimulating neurons, avoid using traditional microelectrode devices or special proteins, and has a simple preparation method and can be used as an artificial retina.

[0018] To achieve the above object, the present invention provides the following technical solutions:

[0019] A preparation method of a photoactive injectable hydrogel, the preparation method comprising:

[0020] Soaking the modified hydrogel gelling raw material in a buffer solution, and then soaking the treated product in an ionic solution to obtain the photoactive injectable hydrogel;

[0021] The modified hydrogel gelling raw material is a hydrogel gelling raw material treated by including grafting dopamine; the buffer solution includes Tris buffer solution or Tris-HCl buffer solution; the ionic solution includes divalent manganese ion solution, divalent zinc ion solution or trivalent iron ion solution;

[0022] The hydrogel gelling raw material includes any one of hyaluronic acid, sodium alginate, and polyvinyl alcohol.

[0023] Preferably, the hydrogel gelling raw material is hyaluronic acid; preferably, the modified hydrogel gelling raw material is grafted with dopamine and then further modified to improve the crosslinking degree; more preferably, the method for improving the crosslinking degree is to modify with BSA. Preferably, the buffer solution is Tris buffer solution; the ionic solution is divalent manganese ion solution or trivalent iron ion solution.

[0024] As a feasible embodiment of the present invention, when preparing the modified hydrogel gelling raw material, first add sodium ascorbate, EDC, and NHS to the aqueous solution of the hydrogel gelling raw material and stir, and then add dopamine for coupling reaction to obtain.

[0025] As a feasible embodiment of the present invention, when modifying with BSA, mix BSA and the hydrogel gelling raw material grafted with dopamine in water, and then add EDC and NHS for crosslinking to obtain.

[0026] Preferably, the molecular weight of the hyaluronic acid is 400,000 Da; the grafting degree of dopamine is 12-15%.

[0027] Preferably, the concentration of the buffer solution is 1 mol / L -1 ; the molar ratio of the ionic concentration of the ionic solution to the catechol in the modified hyaluronic acid is 1:2.

[0028] The present invention also aims to provide a photoactive injectable hydrogel prepared by the above-mentioned scheme.

[0029] The present invention also aims to provide the application of the above photoactive injectable hydrogel in the preparation of biomedical implants. The biomedical implants include artificial retinas or neural interfaces of man-machine systems.

[0030] The present invention also aims to provide an artificial retina, which comprises the above photoactive injectable hydrogel.

[0031] As shown in a comparative example of the present invention, when the buffer solution is replaced with PBS buffer solution, the obtained hydrogel not only fails to produce an obvious photothermal effect, but also fails to produce an obvious photoelectric effect.

[0032] As shown in another comparative example of the present invention, when not soaked in the ionic solution, the obtained hydrogel also fails to produce an obvious photothermal effect and photoelectric effect.

[0033] It can be seen from the above two comparative examples that in the present invention, the selection of the buffer solution and the ionic solution is crucial for the generation of the photothermal effect and the photoelectric effect.

[0034] On this basis, as shown in a comparative example of the present invention, the inventors also investigated adding a certain concentration of polypyrrole (PPy) to the Tris buffer solution and found that the obtained hydrogel can only produce an obvious photothermal effect, but the obtained photoelectric effect is very weak.

[0035] As shown in the examples of the present invention, the hydrogel obtained by the present invention has good photothermal and photoelectric effects. The preparation method of the obtained hydrogel is very simple, overcoming the defects of traditional artificial retinas using traditional microelectrode devices. Compared with the existing technologies for preparing hydrogels that can generate electrical signals, it also does not need to use expensive special proteins. It is a photoactive injectable hydrogel with low cost, simple process and excellent performance.

[0036] It is not difficult for those skilled in the art to know that although the photoactive injectable hydrogel obtained by the present invention is particularly suitable for use as an artificial retina, its application is not limited thereto. The photoactive injectable hydrogel obtained by the present invention can also be used as other biomaterials that require photoelectric effects.

[0037] Advantages of the present invention:

[0038] 1. The photoactive injectable hydrogel of the present invention has good photothermal effect and photoelectric effect;

[0039] 2. The raw materials of the photoactive injectable hydrogel of the present invention are inexpensive, and the preparation method is simple, having good prospects for popularization and application;

[0040] 3. The photoactive injectable hydrogel of the present invention is particularly suitable for use as an artificial retina and overcomes the disadvantages existing in the existing artificial retina technology. Description of the Drawings

[0041] Figure 1 and Figure 2 are the photothermal and photoelectric result diagrams obtained from Examples 1-6 and Comparative Examples 1-3 of the present invention; wherein Figure 1 in, part A shows the photothermal and photoelectric results of the hydrogel obtained in Example 1, part B shows the photothermal and photoelectric results of the hydrogel obtained in Example 2, part C shows the photothermal and photoelectric results of the hydrogel obtained in Example 3, part D shows the photothermal and photoelectric results of the hydrogel obtained in Example 4, and part E shows the photothermal and photoelectric results of the hydrogel obtained in Example 5; Figure 2 in, part A and part B respectively show the photoelectric and photothermal results of the hydrogel obtained in Example 3; part C and part D respectively show the photoelectric results and photothermal results of the hydrogel obtained in Example 6; part E and part F respectively show the photoelectric and photothermal effects of the hydrogels obtained in Comparative Examples 1-3.

[0042] Figure 3 are the ophthalmological observation results of mice injected with the photoelectric hydrogel prosthesis PEH (using the hydrogel of Example 1 as the prosthesis for in vivo partial research): (a) fundus image of a mouse 7 weeks after injection of the hydrogel prosthesis; (b) cross-sectional images of the retinas of normal mice (control group) and mice injected with the hydrogel prosthesis (experimental group) on the 2nd day after injection by optical coherence tomography (OCT); the left representative OCT scan in part (b) of the figure; the right side of part (b) of the figure is a magnified image of the area indicated by the square.

[0043] Figure 4 are the fluorescence co-imaging observations of the retina and the hydrogel prosthesis; (a) fluorescence image of a cross-section of the retina of a c57 mouse, including the hydrogel located in the subretinal space and DAPI nuclear staining, scale bar: 100 μm; (b) whole retina of a c57 mouse injected with the hydrogel prosthesis analyzed by confocal z-stack scanning, scale bar: 500 μm.

[0044] Figure 5Electroretinogram test on mice injected with hydrogel prosthesis (PEH); (a) ERG recordings of mice in the control group and the experimental group under 30 Hz light pulses; (b) ERG recordings of mice in the experimental group under near-infrared light pulses of 10 Hz / 20 Hz / 30 Hz; (c) ERG signal statistics of mice in the control group and the experimental group under 30 Hz white light and 30 Hz near-infrared light. The ratio of the peak near 30 Hz to the peak near 50 Hz was used as the signal-to-noise ratio (S / N). n = 4 or 3. ns, no statistically significant difference; *p < 0.05; the control group was injected with BSA-HA-Dopa hydrogel, and the experimental group was injected with BSA-HA-Dopa-Mn 2+ (Tris) Photoelectric hydrogel (hydrogel of Example 1).

[0045] Figure 6 Is the optical image of the injectable hydrogel of the present invention. Detailed implementation manners

[0046] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still fall within the protection scope of the present invention.

[0047] Source of raw materials and explanation of abbreviated terms:

[0048] The molecular weight of hyaluronic acid (HA) is 400 kDa and it is purchased from Tianjin Sinechem Optode Technology Co., Ltd. Sodium ascorbate is purchased from Shanghai Meyer Chemical Technology Co., Ltd. (Shanghai, China). N-hydroxysuccinimide (NHS) is purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) is purchased from Shanghai Merck Chemical Technology Co., Ltd. (Shanghai, China). Manganese chloride anhydrous is purchased from Anychem (Anhui, China). Zinc chloride, ferric chloride (III) anhydrous and dopamine hydrochloride are purchased from Macklin (Shanghai, China). Tris(hydroxymethyl)aminomethane (Tris) is purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Phosphate buffered saline (PBS) is purchased from Gibco. Indium tin oxide coated polyethylene terephthalate (ITO-PET) film is purchased from South China Xiangcheng Technology Co., Ltd. (Hunan, China). Polyimide (PI) film is purchased from Shenzhen Zhenpai Adhesive Co., Ltd. (Guangdong, China).

[0049] HA: Hyaluronic acid

[0050] BSA: Bovine serum albumin

[0051] NHS: N-hydroxysuccinimide

[0052] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0053] PPy: polypyrrole

[0054] ITO-PET: Indium Tin Oxide Coated Polyethylene Terephthalate

[0055] PI: Polyimide

[0056] Tris buffer: 6.055 g of tris(hydroxymethyl)aminomethane was dissolved in 50 mL of deionized water to obtain a 1-molar Tris buffer.

[0057] Tris-HCl buffer: 6.055 g of tris(hydroxymethyl)aminomethane was dissolved in 45 mL of deionized water, and concentrated HCl was added to adjust the pH to 8. Deionized water was added to make the volume to 50 mL to obtain a Tris-HCl buffer with a concentration of 1 molar.

[0058] Example 1

[0059] (1) Hyaluronic acid polymer grafted with dopa groups:

[0060] HA-Dopa was synthesized by the EDC / NHS coupling amide bond reaction between the carboxylic acid group of HA and the amine group of dopamine. First, 1 g of HA (MW: 400 kDa) was dissolved in deionized water. Then, 2.95 g of sodium ascorbate, 3.82 g of EDC and 2.3 g of NHS were added to the HA solution and stirred for 1 hour. -1 The NaOH solution was adjusted to pH 7.8 and stirred, sealed and purged with nitrogen for about 15 minutes with a balloon to remove oxygen; after that, 2.97 g of dopamine was transferred to the EDC / HA / NHS solution to couple dopamine with HA, and the reaction was carried out overnight at room temperature under magnetic stirring. The solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3.5 kDa, and the final product was lyophilized and stored at 4 °C.

[0061] (2) BSA modification:

[0062] BSA-HA-Dopa was synthesized by EDC / NHS coupling amide bond reaction between carboxylic acid groups of HA and amine groups of BSA. 400 mg BSA and 400 mg HA-Dopa were dissolved in 20 mL deionized H 2 O, 0.383 g EDC and 0.230 g NHS were added to the solution to cross-link BSA and HA-Dopa, after which the hydrogel was transferred to deionized water to achieve swelling equilibrium for 24 h; during this period, deionized water was replaced more than 6 times to remove unreacted reactants.

[0063] (3) Formation of the optoelectronic hydrogel:

[0064] Immerse the product BSA-HA-Dopa obtained in step (2) in 1 mol L -1 Tris buffer solution, and then determine the concentration of the immersed MnCl 2+ solution according to the grafting rate of catechol on the main chain, with the molar ratio of catechol to Mn 2 being 1:2 for immersion. Finally, wash with deionized water to obtain the optoelectronic hydrogel, BSA-HA-Dopa-Mn 2+ (Tris) hydrogel.

[0065] Example 2

[0066] Except for not performing the BSA modification in step (2) and directly proceeding to step (3) after completing step (1), the rest is the same as in Example 1. The specific preparation process is as follows: Dissolve 30 mg of HA-Dopa in 45 μL of Tris buffer solution, add 5 μL of MnCl 2 solution (2.4 mol L -1 ), to obtain HA-Dopa-Mn 2+ (Tris) hydrogel.

[0067] Example 3

[0068] Except for replacing the Tris buffer solution in the step with Tris-HCl buffer solution, the rest is the same as in Example 2. The specific preparation process is as follows: Dissolve 30 mg of HA-Dopa in 45 μL of Tris-HCl buffer solution (pH = 8), add 5 μL of MnCl 2 solution (2.4 mol L -1 ), to obtain HA-Dopa-Mn 2+ (Tris-HCl) hydrogel.

[0069] Example 4

[0070] Except for replacing the MnCl 2 solution in the step with ZnCl 2 solution, the rest is the same as in Example 2. The specific preparation process is as follows: Dissolve 30 mg of HA-Dopa in 45 μL of Tris buffer solution, add 5 μL of ZnCl 2 solution (2.4 mol L -1 ), to obtain HA-Dopa-Zn 2+ (Tris) hydrogel.

[0071] Example 5

[0072] Except for replacing the Tris buffer in the steps with Tris-HCl buffer, the rest is the same as in Example 4. The specific preparation process is as follows: 30 mg of HA-Dopa is dissolved in 45 μL of Tris-HCl buffer (pH = 8), and 5 μL of ZnCl 2 solution (2.4 mol L -1 ) is added to obtain HA-Dopa-Zn 2+ (Tris-HCl) hydrogel.

[0073] Example 6

[0074] Except for replacing the MnCl 2 solution with FeCl 3 solution and adding metal ions according to the molar ratio of catechol to Mn 2+ changed from 1:2 to 1:3, the rest is the same as in Example 3, and HA-Dopa-Fe 3+ (Tris-HCl) hydrogel is obtained.

[0075] Comparative Example 1

[0076] Except for replacing the Tris buffer in the steps with PBS solution, the rest refers to Example 2. The specific preparation process is as follows: 30 mg of HA-Dopa is dissolved in 45 μL of PBS, and 5 μL of Mn 2+ solution (2.4 mol L -1 ) is added for crosslinking to obtain HA-Dopa-Mn 2 + (PBS) hydrogel.

[0077] Comparative Example 2

[0078] Except for not adding Mn 2+ solution, the rest is the same as in Example 3. The specific preparation process is as follows: 30 mg of HA-Dopa is dissolved in 50 μL of Tris-HCl buffer (pH = 8) to obtain HA-Dopa(Tris-HCl) hydrogel.

[0079] Comparative Example 3

[0080] Except for replacing the PBS solution in the steps with Tris-HCl buffer containing 500 μg / mL PPy and not adding Mn 2+ solution, the rest is the same as in Comparative Example 1. The specific preparation process is as follows: 30 mg of HA-Dopa is dissolved in 50 μL of Tris-HCl buffer (pH = 8, containing 500 μg / mL PPy) to obtain HA-Dopa-Mn 2+ (Tris-HCl, with PPy) hydrogel.

[0081] Experimental Example

[0082] Characterize Examples 1-6 and Comparative Examples 1-3:

[0083] 1. Photoactivity Characterization of Hydrogels

[0084] Preparation of hydrogel devices: The hydrogel was thinned and flattened, and the thickness was controlled at about 300 μm. Indium tin oxide-coated polyethylene terephthalate (ITO-PET) films were used as the upper and lower electrodes of the hydrogel, and a polyimide film was placed between the upper and lower electrodes as an insulating material to prevent short circuits caused by the connection of the upper and lower electrodes. A 1 cm hole was cut in the middle of the polyimide film material to allow the middle-layer hydrogel to contact the upper and lower electrodes. 2 hole to enable the middle-layer hydrogel to contact the upper and lower electrodes.

[0085] Performance testing: Photoelectric measurements were performed using a CHI 660E electrochemical workstation (CH Instruments, Inc., USA), and photothermal measurements were performed using a FOTRIC thermal imager (Beijing Laser Wave Optoelectronic Technology Co., Ltd., China). The device was periodically irradiated with an 808 nm laser, an infrared imager was used to measure the surface temperature, and the electrochemical workstation was used to test the open-circuit voltage of the hydrogel device.

[0086] Note: Figure 1 The data shown were obtained by testing the device preparation and photo-response testing methods described above. Regarding Figure 2 testing, different from the method described above, in the preparation of the device, ITO glass was used instead of ITO-PET film as the upper and lower electrodes for photoelectric testing; in addition, the photoelectric and photothermal properties were tested separately. For photothermal testing, it was directly tested without processing the hydrogel material into a device.

[0087] As Figure 1 and Figure 2 shown, the hydrogels obtained in Examples 1-6 all had good photothermal and photoelectric effects, while Comparative Examples 1-3 did not have the corresponding photothermal and photoelectric effects.

[0088] 2. Injection Surgery and Intraocular Characterization of Photoactive Hydrogel Prostheses (Taking the Hydrogel Obtained in Example 1 as an Example)

[0089] (1) The hydrogel prosthesis was injected into the subretinal space

[0090] For subretinal injection, tropicamide eye drops were used to dilate the pupils, and the animals were anesthetized with ketamine (80 mg / kg) and xylazine (4 mg / kg). The hydrogel was injected into the subretinal space through an angled 34-gauge subcutaneous injection needle (Hamilton, Switzerland). During and after the injection, the animals were placed on a warm blanket and the eyes were kept moist to avoid cataracts.

[0091] (2) Fundus imaging

[0092] At the 7th week after injection, the mice injected with the hydrogel were anesthetized as described above. After anesthesia, the mice were fixed and the pupils were dilated with tropicamide eye drops. Fundus imaging of the mice was performed using a Zeiss Clarus 500 fundus camera (Germany).

[0093] (3) OCT observation

[0094] At the 7th week after injection, the mice in the normal control group and the hydrogel injection group were anesthetized as described above. After anesthesia, the mice were fixed and the pupils were dilated with tropicamide eye drops. The cornea was protected with medical sodium hyaluronate gel. The mice were placed in front of a 90D front lens and the incident angle of light was adjusted, and optical coherence tomography was performed using a Zeiss HD-OCT Cirrus 5000 (Germany).

[0095] (4) Co-imaging of the fluorescently labeled hydrogel prosthesis and the retina

[0096] FITC fluorescently labeled hydrogel: During the BSA enhancement step in the preparation process of the gel, an appropriate amount of fluorescein isothiocyanate-polyethylene glycol-amine (FITC-PEG-NH 2 ) was added to fluorescently label the gel.

[0097] The C57 mice injected with the fluorescently labeled optoelectronic hydrogel were decapitated. The eyeballs were removed with curved forceps and placed in physiological saline. After removing the cornea, the eyeballs were fixed overnight at 4 °C with an eyeball fixative. After removing the lens, the eyeballs were dehydrated with 10% (1 h, room temperature), 20% (1 h, normal temperature), and 30% (overnight, 4 °C) sucrose solution gradients. After embedding the eyeballs with OCT embedding medium, the eye axis was longitudinally cut with a cryostat, and the section thickness was 8 μm. The nuclear blue fluorescent dye DAPI was added and incubated in the dark for 5 minutes to stain the nuclei. The sections were washed twice with PBS for 10 minutes each. Finally, fluorescent images were taken with a confocal microscope.

[0098] 3. Visual electrophysiological response of mice injected with the hydrogel prosthesis to near-infrared light (taking the hydrogel obtained in Example 1 as an example)

[0099] After anesthetizing the mice, the pupils were dilated with compound tropicamide eye drops. During the experiment, the eyes were kept moist to avoid cataracts. The recording electrode was placed tightly against the center of the cornea. The ground electrode was inserted into the subcutaneous space of the tail, and the reference electrode was inserted into the subcutaneous space of the head. An 808 nm near-infrared (NIR) light with a spot diameter of 9 mm was placed in front of the pupil for stimulation. A multifunctional precision electronic timer was used to execute the millisecond NIR light blocking switch to obtain pulsed light at 10, 20, and 30 Hz. Data acquisition was performed using a visual electrophysiological detection device (Roland RETI-Port / Scan 21, Germany). Electroretinograms (ERGs) were collected at the 2nd week after injection of the hydrogel prosthesis.

Claims

1. A preparation method of a photoactive injectable hydrogel, characterized in that, the preparation method includes: soaking the modified hydrogel gelling raw material in a buffer solution, and then soaking the obtained product in an ionic solution to obtain the photoactive injectable hydrogel; the modified hydrogel gelling raw material is obtained by treating the hydrogel gelling raw material including grafting dopamine; the buffer solution includes Tris buffer solution or Tris-HCl buffer solution; the ionic solution includes divalent manganese ion solution, divalent zinc ion solution or trivalent iron ion solution; the hydrogel gelling raw material includes any one of hyaluronic acid, sodium alginate, and polyvinyl alcohol.

2. The preparation method according to claim 1, characterized in that, the hydrogel gelling raw material is hyaluronic acid.

3. The preparation method according to claim 1, characterized in that, the modified hydrogel gelling raw material is obtained by grafting dopamine onto hyaluronic acid and then performing a modification to increase the crosslinking degree.

4. The preparation method according to claim 3, characterized in that, the method for modifying to increase the crosslinking degree is to modify with BSA.

5. The preparation method according to any one of claims 1 to 4, characterized in that, the buffer solution is Tris buffer solution; the ionic solution is divalent manganese ion solution or trivalent iron ion solution.

6. The preparation method according to claim 1, characterized in that, when preparing the modified hydrogel gelling raw material, first add sodium ascorbate, EDC and NHS to the aqueous solution of the hydrogel gelling raw material and stir, and then add dopamine for a coupling reaction to obtain it.

7. The preparation method according to claim 4, characterized in that, when modifying with BSA, mix BSA and the hydrogel gelling raw material grafted with dopamine in water, and then add EDC and NHS for crosslinking to obtain it.

8. The preparation method according to claim 3, characterized in that, the molecular weight of the hyaluronic acid is 400,000 Da; the grafting degree of dopamine is 12-15%.

9. The preparation method according to claim 1, characterized in that, The concentration of the buffer solution is 1 mol / L -1 ; the molar ratio of the ionic concentration of the ionic solution to the catechol in the modified hyaluronic acid is 1:

2.

10. A photoactive injectable hydrogel, characterized in that, the photoactive injectable hydrogel is prepared by the preparation method according to any one of claims 1 to 9.

11. The application of the photoactive injectable hydrogel according to claim 10 in the preparation of biomedical implants, characterized in that, the biomedical implants include artificial retina or neural interface of a man-machine system.

12. An artificial retina, characterized in that, the artificial retina contains the photoactive injectable hydrogel according to claim 10.

Images