Injectable optoelectronic hydrogels for repairing retinal photoreceptor function damage and preparation method and application thereof

Abstract

Injectable photoelectrohydrogel for repairing retinal photoreceptor damage and its preparation method and application, photoelectroactive spirulina and conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) hydrogel are physically cross-linked to construct an injectable material with photoelectric response characteristics, and the function of damaged photoreceptor cells in the retina is repaired in situ. Sp as a biologically active component not only gives the material photoelectric signal conversion capability, but also can achieve local antioxidant stress function, PEDOT-based hydrogel forms a three-dimensional scaffold by physically wrapping Sp, which not only ensures the activity of algal cells but also has excellent charge and ion conduction performance. The hydrogel can be injected into the retina to construct a biomimetic electrochemical signal transmission interface, rebuild the signal transduction of damaged photoreceptor cells, and realize the in situ repair of retinal signal pathways and the partial recovery of visual function. This technology provides a new strategy for the curative treatment of retinal diseases such as blinding eye diseases, and has the advantages of minimally invasive intervention, excellent biocompatibility and customizable treatment area.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of retinal injury repair research and ophthalmic tissue engineering, and particularly relates to an injectable optoelectronic hydrogel for repairing retinal photoreceptor cell injury and a preparation method and application thereof. BACKGROUND

[0002] As the core part of visual signal transmission, the photoreceptor cells (rods / cones) of the retina trigger the visual pathway through light-electricity signal conversion. However, irreversible apoptosis of photoreceptor cells caused by genetic diseases (such as retinitis pigmentosa) and degenerative diseases (such as age-related macular degeneration) leads to the loss of vision of more than 10 million patients worldwide. Photoreceptor cells cannot regenerate after injury, while the downstream visual pathway (such as bipolar cells, ganglion cells) often remains intact. At present, the clinical treatment can only alleviate the deterioration of the disease through symptomatic intervention, and there is no effective radical solution so far. Artificial intervention such as gene therapy, stem cell therapy, drug therapy, and retinal implantation has significant bottlenecks: such as targeting specific mutant genes, the risk of immune rejection, the inability to reverse the damaged photoelectric signal transduction function, and the performance of the prosthetic device depending on the external camera and silicon-based electrode array, which has low resolution and small field of view, and the high mechanical modulus of the device material leads to interface stress concentration and causes chronic inflammation and fibrous wrapping.

[0003] In recent years, "artificial photoreceptors" based on conductive materials (such as PEDOT:PSS, nanowire arrays) have become a research hotspot, which provides a method to bypass the damaged photoreceptor cells and directly transmit signals to the remaining retinal neurons. However, it relies on the semiconductor heterojunction to construct the photoelectric conversion unit, and the mechanical mismatch between the rigid device and the soft tissue of the retina easily causes chronic inflammation and fibrous wrapping; the dependence on the external imaging system leads to limited field of view and low resolution; the geometric gap between the electrode and the curved surface of the retina hinders the exchange of nutrients and oxygen, aggravates the local hypoxia and reactive oxygen species (ROS)-mediated cytotoxicity, and limits its long-term efficacy. How to implant a flexible photoelectric conversion prosthesis in the subretinal space to replace the photoreceptor cells to transmit visual electrical signals has always been a bottleneck challenge in ophthalmology. SUMMARY

[0004] In order to solve the technical defects existing in the prior art, the present application provides an injectable optoelectronic hydrogel for repairing retinal photoreceptor cell injury and a preparation method and application thereof, which realizes the conversion and transmission of photoelectric signals and the recovery of visual function by injecting an optoelectronic hydrogel material into the subretinal space of the damaged photoreceptor cells. This in-vivo hydrogel material has integrated photoelectric signal transduction capability, injectability, high biocompatibility, mechanical compliance, antioxidant capacity, and the ability to repair damaged retinal tissue.

[0005] The technical solution adopted by the present application is: the injectable photoelectric hydrogel for repairing retinal photoreceptor function damage, the injectable PEDOT-Sp photoelectric hydrogel is prepared by pre-crosslinking a polymer solution of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate (PEDOT:PSS) and 4-dodecylbenzenesulfonic acid (DBSA), and then blending and reacting with modified spirulina (Sp), and forming the PEDOT-Sp photoelectric hydrogel in situ by adding physiological saline.

[0006] The physiological saline is phosphate buffered saline (PBS) or 0.9% NaCl solution.

[0007] The modified spirulina is positively charged modified spirulina with a modified layer of positive charges on the surface.

[0008] The mass percentage concentration of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate in the polymer solution of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate (PEDOT:PSS) is 0.1%.

[0009] A preparation method of an injectable photoelectric hydrogel for repairing retinal photoreceptor function damage, comprising the following steps:

[0010] (1) Sp positive modification: the negatively charged Sp is added dropwise into a 1.0 w / v% positively charged polyethyleneimine (PEI) solution, and modified for 3-5 h under continuous magnetic stirring to obtain the positively modified Sp. When PEI contacts with Sp, due to the interaction of positive and negative charges, the cationic part of PEI is electrostatically combined with part of the negative charges on the surface of Sp. This combination enables PEI to be closely adsorbed on the surface of Sp, forming a modified layer with positive charges;

[0011] (2) Preparation of precursor solution of injectable PEDOT-Sp hydrogel: a 0.1 w / v% PEDOT:PSS solution is prepared, and is uniformly mixed with 1 v / v% DBSA under continuous magnetic stirring. The product is dialyzed in a cellulose dialysis bag for 72 h, and the sample obtained by dialysis is evaporated and dried to obtain a PEDOT hydrogel precursor ink. The negatively charged PEDOT hydrogel precursor ink is combined with the positively modified Sp through electrostatic attraction to obtain an injectable PEDOT-Sp hydrogel precursor injection;

[0012] (3) In situ crosslinking of PEDOT-Sp hydrogel: the precursor solution of PEDOT-Sp hydrogel is slowly injected into a multivalent cation salt solution to form PEDOT-Sp hydrogel in situ.

[0013] The mass ratio of the mixed positive electric modified Sp and PEDOT hydrogel precursor ink in step (2) is 1:1.

[0014] The molecular weight cut-off of the dialysis in step (2) is 100-500 Da.

[0015] The application of the injectable photoelectric hydrogel in the preparation of artificial photoreceptors, the injectable PEDOT-Sp photoelectric hydrogel is directly injected into the subretinal space, and a hydrogel with higher crosslinking strength is formed after contacting the physiological environment, which can replace the photoreceptor to convert the light signal into an electric signal.

[0016] The application of the injectable photoelectric hydrogel in the preparation of a repair material for retinal photoreceptor cell damage.

[0017] The repair material is that the injectable PEDOT-Sp hydrogel precursor ink is directly injected into the subretinal space to form a hydrogel for repairing the damage of the retinal photoreceptor cell layer.

[0018] The application of the injectable PEDOT-Sp photoelectric hydrogel in the treatment of retinal function damage diseases, the retinal function damage diseases include age-related macular degeneration, retinal pigment epithelium degeneration, diabetic retinopathy, hypertensive retinopathy and other diseases caused by retinal photoreceptor damage, which lead to visual function loss or even blindness.

[0019] The application provides an injectable photoelectric hydrogel for repairing retinal photoreceptor function damage and a preparation method and application thereof, the PEDOT-Sp photoelectric hydrogel is injected into the subretinal space of the photoreceptor function damaged retina, the photoelectric signal conversion and visual function recovery are realized, the preparation method of the hydrogel is simple and efficient, the product has good photoelectric response performance, good mechanical fluidity and excellent biocompatibility, the process of converting the light signal into an electric signal of the photoreceptor cell is simulated through the electron transfer process of the Sp photosynthesis, the photoelectric signal transduction is reconstructed, and the gel is formed at the subretinal space; the three-dimensional porous hydrogel structure can provide reliable current conduction, the damaged photoreceptor is successfully replaced in the body to recover the visual function, a new idea and way are provided for the clinical treatment of the retinal damage diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The technical roadmap of the injectable PEDOT-Sp hydrogel prepared in the application.

[0021] Figure 2 (a), (b) and (c) are micro scanning electron microscope images of the prepared PEDOT hydrogel, Sp and PEDOT-Sp hydrogel, respectively.

[0022] Figure 3 The image shows the photoelectric properties of the prepared PEDOT-Sp hydrogel.

[0023] Figure 4 The biocompatibility characterization diagram of the prepared PEDOT-Sp hydrogel is shown.

[0024] Figure 5 Electroretinograms after treatment of an animal model of retinal photoreceptor layer damage with the prepared PEDOT-Sp hydrogel. The SI group was the animal model group of retinal photoreceptor layer damage induced by sodium iodate.

[0025] Figure 6 The cliff test behavior was evaluated after treatment of an animal model of retinal photoreceptor layer damage with the prepared PEDOT-Sp hydrogel.

[0026] Figure 7 This is a schematic diagram illustrating the repair of retinal photoreceptor cell layer damage by the prepared PEDOT-Sp hydrogel. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Preparation of Injectable Conductive Polymer Hydrogel

[0029] like Figure 1 As shown in the first step, dissolve 1 mg of PEDOT:PSS solid particles in 1 mL of deionized water and mix thoroughly with magnetic stirring at 500-800 r / min. Then add 1 μL of DBSA to the 0.1% PEDOT:PSS solution and mix well. In the PEDOT solution, PSS... - Chains surround hydrophobic PEDOT via electrostatic interactions. + DBSA provides H + Make PSS - Protonation, and the attraction of the corresponding PEDOT through π-π stacking and hydrophobic interactions. + Chain physical cross-linking. The product was dialyzed using a cellulose dialysis bag (molecular weight cutoff of 100-500 Da) for 72 h. The dialyzed sample was evaporated and dried to 1 mL to obtain PEDOT hydrogel precursor ink.

[0030] like Figure 1As shown in the second step, the negatively charged Sp is added dropwise to the 1.0 w / v% positively charged PEI solution, and modified for 4 h under continuous magnetic stirring. Since PEI is a cationic polymer with multiple positively charged amino groups, and the cell wall surface of Sp contains some negative charges, such as phosphorylation or carboxyl groups, etc. This enables Sp to be electrostatically attracted to the cationic PEI, thereby achieving modification, and ultimately obtaining positively modified Sp;

[0031] As shown in Figure 1 As shown in the third step, the PEDOT-Sp hydrogel precursor injection is injected into the PBS solution, including but not limited to phosphate buffer, interstitial fluid, etc. to form a hydrogel. Figure 2 The scanning electron microscopy results of (a) show that the PEDOT hydrogel presents a reticular structure, forming a uniform three-dimensional cross-linked network. Figure 2 The scanning electron microscopy results of (b) show that Sp presents a typical spiral shape, with a uniform overall structure and a slightly rough surface, which may be due to the natural structural characteristics of the cell wall. Figure 2 The scanning electron microscopy results of (c) show that the PEDOT-coated Sp still maintains its spiral shape, with a good overall structure and no obvious damage or cracks, and is uniformly distributed in the three-dimensional cross-linked network formed by PEDOT.

[0032] Figure 3 (a) is the photocurrent detection of different concentrations of hydrogels under light and dark alternation for 6 hours and 30 minutes under white light stimulation. The results show that 0.1% PEDOT-Sp hydrogel can produce the largest photocurrent, about 0.53 μA / cm 2 Therefore, 0.1% PEDOT-Sp hydrogel is used. Experiments are carried out using 460 nm, 520 nm, 630 nm and 400-700 nm visible light, and the results are shown in Figure 2 (b) shows that PEDOT-Sp hydrogel can produce the largest photocurrent (1.0 μA / cm 2 ) under 460 nm light.

[0033] Figure 4 The cell survival rate experiment results show that 0.1% PEDOT, 0.1% PEDOT-Sp (dark) and 0.1% PEDOT-Sp (460 nm) have similar cell proliferation activity on the 1st day, 3rd day and 5th day, and the cell survival rate is above 85%. 460 nm light for 1 hour does not cause damage to the cells, and the prepared 0.1% PEDOT-Sp hydrogel has good biocompatibility.

[0034] Example 2

[0035] To verify the therapeutic effect of 0.1% PEDOT-Sp hydrogel in vivo, we injected 0.1% PEDOT-Sp precursor solution into the subretinal space of the photoreceptor layer damaged animals to replace the photoreceptor cells, and stimulated 0.1% PEDOT-Sp to generate photocurrent by 460 nm light, so as to realize the conversion of optical signal to electrical signal, thereby recovering the electroretinogram of the photoreceptor layer damaged mice to achieve the effect of improving vision.

[0036] Figure 5 The recovery of the photoelectric conversion function of 0.1% PEDOT-Sp hydrogel for photoreceptor layer damaged mice was evaluated by electroretinogram (ERG) detection. The electrophysiological results show that in the photoreceptor layer damaged group (SI), the efficiency of converting optical signal to electrical signal is low. After subretinal injection of 0.1% PEDOT-Sp hydrogel, the amplitudes of a wave and b wave of ERG detected under Photopic 3.0 (a, b), Scotpic 3.0 (c, d) and Scotpic 10.0 (e, f) conditions are increased compared with the damaged group, and the amplitudes of a wave and b wave of 0.1% PEDOT-Sp (460 nm) group are more obviously increased than those of 0.1% PEDOT-Sp (dark) group, which confirms that the 0.1% PEDOT-Sp hydrogel synthesized in the present study can be used to replace photoreceptor cells to convert optical signal to electrical signal, and realize the recovery of part of visual function.

[0037] Figure 6 The visual cliff test was used to verify whether injection of PEDOT-Sp hydrogel can help SI rats recover light perception behavior. The experiment evaluates the depth perception ability by creating a platform with different depths of visual cliff. The data shows that injection of PEDOT-Sp hydrogel can significantly prolong the time of rats staying in the "shallow side" under natural light and 460 nm light (d). The staying time of PEDOT-Sp group rats in the shallow side is significantly higher than that of the SI group: 87% (SI group 63%) on the first day, 67% (SI group 50%) on the 21st day. Under light stimulation, the staying time is further increased to 87% on the first day and 77% on the 21st day, indicating that light regulation can synergistically enhance the effect of visual recovery.

[0038] Technical personnel should know: although the present application has been described in the above specific embodiments, the inventive idea of the present application is not limited to this application, and any modification using the inventive idea will be included in the scope of protection of the present patent.

[0039] The above merely describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solution falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as falling within the protection scope of the present application.

Claims

1. An injectable opto-electric hydrogel for repairing damage to retinal photoreceptor function, characterized in that, The injectable photoelectric hydrogel is prepared by pre-crosslinking a polymer solution of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate (PEDOT:PSS) and 4-dodecylbenzenesulfonic acid (DBSA) and then blending with modified spirulina (Sp) and forming the PEDOT-Sp photoelectric hydrogel in situ by adding phosphate buffered saline (PBS).

2. The injectable optoelectronic hydrogel for repairing retinal photoreceptor function impairment according to claim 1, wherein, The mass percentage concentration of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate in the polymer solution of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate (PEDOT:PSS) is 0.1%.

3. A method of preparing the injectable optoelectronic hydrogel for repairing retinal photoreceptor function impairment according to claim 1, characterized in that, The method comprises the following steps: (1) Sp positive modification: the negatively charged Sp is added dropwise into a positively charged polyethyleneimine (PEI) solution, and modified for 3-5 h under continuous magnetic stirring to obtain the positively modified Sp; (2) preparation of the precursor solution of the injectable PEDOT-Sp hydrogel: the polymer solution of poly 3,4-ethylenedioxythiophene and polystyrene sulfonate (PEDOT:PSS) is uniformly mixed with 4-dodecylbenzenesulfonic acid (DBSA) under continuous magnetic stirring, the product is dialyzed by using a cellulose dialysis bag, the dialyzed sample is evaporated and dried to obtain a PEDOT hydrogel precursor ink, the positively modified Sp is mixed with the PEDOT hydrogel precursor ink to obtain an injectable PEDOT-Sp hydrogel precursor injection solution through electrostatic attraction; (3) in-situ crosslinking of the PEDOT-Sp hydrogel: the precursor solution of the PEDOT-Sp hydrogel is slowly injected into the phosphate buffered saline (PBS) to form the PEDOT-Sp hydrogel in situ.

4. The production method according to claim 3, characterized by, The mass ratio of the positively modified Sp to the PEDOT hydrogel precursor ink mixed in step (2) is 1:

1.

5. The preparation method according to claim 3, characterized in that, The molecular weight cut-off for dialysis in step (2) is 100-500 Da.

6. Use of the injectable opto-hydrogel according to claim 1 for the preparation of artificial photoreceptors. The injectable photoelectric hydrogel is directly injected into the subretinal cavity, and a hydrogel with higher crosslinking strength is formed after contacting the physiological environment, which can replace the photoreceptor cells to convert the light signal into an electrical signal.

7. The injectable photoelectric hydrogel of claim 1 is used for preparing a repair material for retinal photoreceptor cell damage.

8. Use according to claim 7, characterized in that, The repair material is prepared by directly injecting the injectable PEDOT-Sp hydrogel precursor injection solution into the subretinal cavity to form a hydrogel for repairing the damage of the retinal photoreceptor cell layer in situ.

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