Injectable KN93 / L-arg double-load hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention discloses injectable KN93 / L-arg double-loaded hydrogel microspheres as well as a preparation method and application thereof.The hydrogel microspheres take GelMA and HepMA as matrixes, target drug molecules KN93 and L-arg are doped into the hydrogel matrixes, a microfluidic technology and a photochemical cross-linking reaction are adopted to prepare the functionalized hydrogel microspheres, and the adhesiveness is remarkably improved through dopamine modification. Wherein the inner ear diseases comprise drug-induced deafness, such as sensorineural deafness caused by cis-platinum. The hydrogel microspheres synthesized by the method provided by the invention provide proper cavities capable of accommodating drug molecules, so that efficient loading and stable release of drugs are realized. In addition, the drug-loaded hydrogel microspheres can stay in the middle ear for a long time, so that drug molecules are promoted to permeate into internal lymph through a round window membrane and then are taken by sensory cells of the internal ear. The preparation method provided by the invention is simple, and the prepared injectable KN93 / L-arg double-loaded hydrogel microspheres can effectively remove ROS and protect the survival of cochlea sensory cells, and can play a good role in treating inner ear diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to an injectable KN93 / L-arg dual-loaded hydrogel microsphere and a preparation method and application thereof. Background Art

[0002] Among the hearing-impaired population, sensorineural hearing loss accounts for the majority. Clinically, sensorineural hearing loss can be caused by exogenous and endogenous factors. Exogenous factors include excessive noise, ototoxic drugs, aging, etc. For the treatment of patients with sensorineural hearing loss, hearing aids and cochlear implants are the current standard options. The absence or dysfunction of cochlear sensory cells and spiral ganglia is the main cause of sensorineural hearing loss. Therefore, finding new therapeutic targets to maintain the survival of hair cells and spiral ganglia is the core link in the treatment of sensorineural hearing loss.

[0003] The inner ear is a highly differentiated and very delicate peripheral sensory organ. The treatment of inner ear diseases is limited by the blood-membrane labyrinth barrier of the cochlea, and it is difficult for drug molecules to enter the endolymph to exert the corresponding therapeutic effect. Therefore, successful treatment of inner ear diseases depends on efficient drug delivery carriers. In recent years, local drug delivery has become an effective method for the treatment of inner ear diseases. For example, drugs are delivered by intratympanic injection to increase local drug concentrations and thereby reduce systemic adverse reactions. However, traditional intratympanic drug delivery is not efficient because it can be excreted through the Eustachian tube and stays in the middle ear for a short time. In recent years, more and more reports have been published on encapsulating drugs, genes, proteins, etc. into delivery carriers and transferring them to the inner ear to observe functional changes.

[0004] Hydrogel is a hydrophilic cross-linked polymer with good biocompatibility. Monodisperse polymer particles with different compositions are prepared by microfluidics technology, and the particle size of microgels ranges from tens of nanometers to hundreds of microns. The delivery of drug molecules through hydrogel microspheres provides a very promising strategy for inner ear diseases. Compared with bulk hydrogels, hydrogel microspheres have significant advantages. The surface area to volume ratio of hydrogel microspheres is large, which maximizes the loading rate. In addition, hydrogel microspheres can be evenly distributed in the solvent, and the drug release rate and duration after injection are more stable. After being modified in different ways, the hydrogel microspheres can increase their adhesion to the tissue, so that they can increase their residence time in the tissue and achieve the goal of sustained drug release.

[0005] In view of the above background, how to provide an injectable hydrogel microsphere prepared based on microfluidic technology and apply it to the treatment of inner ear diseases is an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide an injectable KN93 / L-arg dual-loaded hydrogel microsphere and a preparation method and application. The prepared hydrogel microsphere can efficiently load the nitric oxide (NO) precursor arginine (L-arg) and the calmodulin inhibitor KN93. After administration by tympanic injection, the controlled release of the drug can be achieved, and the concentration of the drug entering the inner ear can be increased. It can play a good role in repairing inner ear sensory cells and treating inner ear diseases, so as to solve the problems in the prior art raised in the above background technology.

[0007] To achieve the above-mentioned purpose, the present invention proposes, on the one hand, an injectable KN93 / L-arg dual-loaded hydrogel microsphere, comprising methacryloyl gelatin and methacryloyl heparin as a matrix, incorporating target drug molecules KN93 and L-arg into the hydrogel matrix, and preparing functionalized injectable KN93 / L-arg dual-loaded hydrogel microspheres by microfluidic technology and photochemical cross-linking reaction.

[0008] On the other hand, the present invention provides a method for preparing injectable KN93 / L-arg dual-loaded hydrogel microspheres, comprising the following steps:

[0009] Preparation of Gel MA: Take 20g gelatin, add 200ml carbonate buffer solution and heat to 50℃ to dissolve, then take 2ml methacrylic acid and inject into the gelatin solution with a micro syringe pump; after stirring at 50℃ for 3 hours, add 100ml phosphate buffered saline solution to terminate the reaction; after centrifugation at 7000rpm for 15 minutes at 25℃, remove the oily liquid, take the upper layer of Gel MA solution and put it into a regenerated cellulose dialysis bag, dialyze at 37℃ for 2 days, then freeze-dry and store at -20℃ for later use;

[0010] Synthesis of HepMA: 1 g of heparin was completely dissolved in 50 mL of deionized water under mechanical stirring at 4°C, and 8 mL of methacrylic anhydride (MA) was added dropwise to react for 2 hours; 6 mL of sodium hydroxide was then added to the reaction system to terminate the reaction, and stirred at 4°C overnight; after centrifugation at 7000 rpm for 15 minutes at 25°C, the oily liquid was removed, and the upper layer of HepMA solution was placed in a dialysis bag for 3 days, then freeze-dried and stored at -20°C;

[0011] Microfluidic emulsion method for synthesizing injectable drug-loaded hydrogel microspheres: The hydrogel microspheres are synthesized by microfluidic emulsion method and photochemical cross-linking reaction. First, the water phase and the oil phase are injected into the inlet of the modified microfluidic device respectively. The shear force and the hydrophobic contact at the cross-section produce pre-gel droplets. After UV curing, the organic solvent is removed and the hydrogel microspheres are obtained after washing with water.

[0012] The produced hydrogel microspheres and dopamine were dissolved in 10 ml of tris(hydroxymethyl)aminomethane buffer respectively. After being fully dissolved, they were mixed and stirred in the dark for 10 hours. The products were collected by centrifugation at 1000 rpm for 10 minutes to obtain dopamine-modified KN93 / L-arg dual-loaded hydrogel microspheres, which were stored at 4°C.

[0013] Preferably, the methacrylic anhydride and gelatin contain abundant carboxyl groups, and the volume ratio of the methacrylic anhydride to the gelatin solution is 1:1 to 1:1.5.

[0014] Preferably, the concentration of heparin is 20 mg / mL, and the volume ratio of methacrylic anhydride to heparin solution is 10:1-5:1.

[0015] Preferably, KN93 is a competitive inhibitor of calmodulin-dependent kinase type II, and L-arginine is a NO precursor that can be rapidly decomposed to produce NO after entering the cell.

[0016] Preferably, the material concentration of the water phase is 5%, the injection speed is 10-15 μL / min, and the injection speed of the oil phase is 75-100 μL / min.

[0017] Preferably, the organic solvent is one of volatile isopropanol and methanol.

[0018] Preferably, the concentrations of the hydrogel microspheres and the dopamine solution are 1 mg / mL and 0.5 mg / mL, respectively, and the volume ratio of the hydrogel microspheres to the dopamine solution is 1:1-1:1.5.

[0019] Preferably, the final effective concentration of KN93 and L-arginine in the complex is 50-100 μg / ml.

[0020] On the other hand, the present invention provides an injectable KN93 / L-arg dual-loaded hydrogel microsphere, and uses the hydrogel microsphere in the preparation of a drug for inner ear diseases.

[0021] Technical effects and advantages of the present invention: The injectable KN93 / L-arg dual-loaded hydrogel microspheres and the preparation method and application thereof proposed in the present invention have the following advantages over the prior art:

[0022] The hydrogel microspheres synthesized by the present invention provide a suitable cavity that can accommodate drug molecules, achieving efficient loading and stable release of drugs. In addition, the drug-loaded hydrogel microspheres can stay in the middle ear for a long time, promoting the penetration of drug molecules into the endolymph through the round window membrane, and then being taken up by the inner ear sensory cells. The preparation method provided by the present invention is simple, and the prepared injectable KN93 / L-arg dual-loaded hydrogel microspheres can effectively remove ROS, protect the survival of cochlear sensory cells, and can play a good role in treating inner ear diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The synthetic flow chart of the injectable KN93 / L-arg dual-loaded hydrogel microspheres of the present invention;

[0024] Figure 2 is the nuclear magnetic hydrogen spectrum of Gel MA and HepMA in the present invention;

[0025] Figure 3 This is a general appearance picture of the injectable KN93 / L-arg dual-loaded hydrogel microspheres of the present invention under bright field;

[0026] Figure 4 This is a scanning electron micrograph of the injectable KN93 / L-arg dual-loaded hydrogel microspheres of the present invention;

[0027] Figure 5 It is the element (C, N, S, Cl) spectrum of the injectable KN93 / L-arg dual-loaded hydrogel microspheres in the present invention;

[0028] Figure 6 is a particle size distribution diagram of the injectable hydrogel microspheres of the present invention;

[0029] Figure 7 This is a result diagram of the degradation product detection of the injectable KN93 / L-arg dual-loaded hydrogel microspheres of the present invention;

[0030] Figure 8 This is an optical microscope picture of the degradation of the injectable KN93 / L-arg dual-loaded hydrogel microspheres of the present invention;

[0031] Fig. 9 This is a graph showing the expansion rate of injectable KN93 / L-arg dual-loaded hydrogel microspheres in the present invention;

[0032] Fig.10 This is a graph showing the drug loading rate of the injectable hydrogel microspheres of the present invention;

[0033] Fig.11 It is a fluorescence image of drug distribution of injectable hydrogel microspheres in the present invention;

[0034] Fig.12This is a diagram showing the results of the biosafety assessment of the injectable hydrogel microspheres of the present invention;

[0035] Fig.13 This is a diagram showing the distribution and tracing results of the injectable drug-loaded hydrogel microspheres in the present invention in the inner ear and hair cells during in vivo imaging;

[0036] Fig.14 This is a graph showing the results of the injectable KN93 / L-arg dual-loaded hydrogel microspheres protecting cells and cochlear tissue damage in the present invention;

[0037] Fig.15 This is a diagram showing the protection of rat hearing loss by injectable KN93 / L-arg dual-loaded hydrogel microspheres in the present invention;

[0038] Fig.16 This is a diagram showing the protection of rat cochlear hair cell damage by injectable KN93 / L-arg dual-loaded hydrogel microspheres in the present invention;

[0039] Fig.17 is the structural formula of Gel MA in the present invention;

[0040] Fig.18 It is the structural formula of HepMA in the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] The present invention provides an injectable KN93 / L-arg dual-loaded hydrogel microsphere, and the prepared hydrogel microsphere can efficiently load the NO precursor arginine (L-arg) and the calmodulin inhibitor KN93. After administration through tympanic injection, the controlled release of the drug can be achieved, the concentration of the drug entering the inner ear can be increased, and it can play a good role in repairing the inner ear sensory cells and treating inner ear diseases.

[0044] The present invention discloses a KN93 / L-arg dual-loaded hydrogel microsphere that is injectable and can efficiently enter the inner ear, and can be injected into the local tympanic cavity to achieve efficient drug delivery to the inner ear. In the present invention, methacrylic anhydride, a polymer with good biocompatibility, is directly reacted with gelatin to obtain methacryloyl gelatin (Gel MA), and methacrylic anhydride is reacted with heparin to obtain methacryloyl heparin (HepMA). Using Gel MA and HepMA as matrices, L-arg, a NO precursor with antioxidant activity, and KN93, a calmodulin inhibitor, are incorporated into the microsphere matrix, and the preparation of functionalized hydrogel microspheres is achieved by microfluidic technology. In addition, KN93 / L-arg dual-loaded hydrogel microspheres can be administered systemically or locally by injection through a syringe needle, and can be used as a safe and efficient inner ear drug delivery carrier with drug tracing function.

[0045] Example 2

[0046] In this embodiment, a method for preparing injectable KN93 / L-arg dual-loaded hydrogel microspheres is proposed, comprising the following steps:

[0047] Preparation of Gel MA: Take 20g gelatin, add 200ml carbonate buffer solution, heat to 50℃ and dissolve, then take 2ml methacrylic acid and inject into the gelatin solution with a micro syringe pump. After stirring at 50℃ for 3 hours, add 100ml phosphate buffered saline (PBS) solution to terminate the reaction. After centrifugation at 7000rpm for 15 minutes at 25℃, remove the oily liquid, take the upper Gel MA solution and put it into a regenerated cellulose dialysis bag (MWCO8000), dialyze at 37℃ for 2 days (change the liquid three times a day). Then freeze-dry and store at -20℃ for later use.

[0048] Synthesis of HepMA (methacryloyl heparin): 1g of heparin was completely dissolved in 50mL of deionized water under mechanical stirring at 4°C, and 8mL of methacrylic anhydride was added dropwise for 2 hours. Then 6mL of sodium hydroxide (NaOH) was added to the reaction system to terminate the reaction, and stirred overnight at 4°C. After centrifugation at 7000rpm for 15 minutes at 25°C, the oily liquid was removed, and the upper HepMA solution was taken and placed in a MWCO8000 dialysis bag for dialysis for three days. Then it was freeze-dried and stored at -20°C.

[0049] Microfluidic emulsion method to synthesize injectable drug-loaded hydrogel microspheres (Gel MA, HepMA, KN93, L-arg): The hydrogel microspheres were synthesized by microfluidic emulsion method + photochemical cross-linking reaction. First, the aqueous phase (5wt% Ge l MA, 0.4wt% HepMA, 0.5wt% photoinitiator, 0.2wt% KN93, 1% L-arg) and the oil phase (95wt% paraffin and 5wt% sorbitan monooleate (Span80)) were respectively injected into the inlet of the modified microfluidic device. The shear force and the hydrophobic contact at the intersection produced pre-gel droplets, which were then cured by UV light, and the organic solvent was removed and washed with water to obtain hydrogel microspheres.

[0050] In order to attach a polydopamine coating on the surface of the hydrogel microspheres to increase adhesion, the produced hydrogel microspheres and dopamine were dissolved in 10 ml tris(hydroxymethylaminomethane) buffer respectively. After sufficient dissolution, they were mixed and stirred in the dark for 10 hours. The product was collected by centrifugation at 1000 rpm for 10 minutes to obtain dopamine (PDA)-modified KN93 / L-arg dual-loaded hydrogel microspheres, which were stored at 4°C.

[0051] Preferably, in the above-mentioned method for preparing injectable KN93 / L-arg dual-loaded hydrogel microspheres, the first solvent is sodium bicarbonate.

[0052] Preferably, in the above-mentioned method for preparing the injectable KN93 / L-arg dual-loaded hydrogel microspheres, the methacrylic acid and gelatin contain abundant carboxyl groups. The volume ratio of the methacrylic acid to the gelatin solution is 1:1-1:1.5.

[0053] Preferably, in the preparation method of the above-mentioned injectable KN93 / L-arg dual-loaded hydrogel microspheres, the concentration of heparin is 20 mg / mL, and the volume ratio of methacrylic anhydride to heparin solution is 10:1-5:1.

[0054] Preferably, in the preparation method of the above-mentioned injectable KN93 / L-arg dual-loaded hydrogel microspheres, KN93 is a competitive inhibitor of calmodulin-dependent kinase type II, and L-arginine is a NO precursor, which can be rapidly decomposed to produce NO after entering the cell.

[0055] Preferably, in the above-mentioned method for preparing the injectable KN93 / L-arg dual-loaded hydrogel microspheres, the material configuration concentration of the aqueous phase is 5%, the injection speed is 10-15 μL / min, and the injection speed of the oil phase is 75-100 μL / min;

[0056] Preferably, in the above-mentioned method for preparing injectable KN93 / L-arg dual-loaded hydrogel microspheres, the organic solvent is one of volatile isopropanol and methanol.

[0057] Preferably, in the above-mentioned method for preparing injectable KN93 / L-arg dual-loaded hydrogel microspheres, the concentrations of the hydrogel microspheres and the dopamine solution are 1 mg / mL and 0.5 mg / mL, respectively. The volume ratio of the hydrogel microspheres to the dopamine solution is 1:1-1:1.5.

[0058] Preferably, in the above-mentioned method for preparing injectable KN93 / L-arg dual-loaded hydrogel microspheres, the final effective concentration of KN93 and L-arginine in the complex is 50-100 μg / ml.

[0059] The present invention aims at the problems of low effective concentration of inner ear drugs, many systemic adverse reactions, short residence time of local administration in the middle ear, and low drug delivery efficiency. Based on microfluidic chip technology, hydrogel microspheres of specific sizes can be prepared. The hydrogel microspheres have suitable cavities that can accommodate guest molecules, which can greatly increase the drug loading capacity of the hydrogel sustained-release system and prolong the effective release time of the drug in vivo, thereby improving the stability of the drug loading and having high light efficiency to facilitate the tracing of the drug carrier.

[0060] In addition, the injectable hydrogel microspheres are modified with dopamine, which can give the material better adhesion and retention in the body, allowing it to stay in the middle ear for a long time after local administration through the tympanic cavity, further ensuring local, long-term and stable release of drugs and increasing the concentration of drugs that penetrate into the inner ear.

[0061] By optimizing the preparation method and process of the injectable hydrogel microsphere drug delivery system, specific pharmacodynamic functions are conferred by loading targeted drugs. The preparation method of the injectable KN93 / L-arg dual-loaded hydrogel microsphere developed has mild conditions, a non-toxic and harmless preparation process, good biosafety and bioactivity, and can effectively remove reactive oxygen species (ROS) and reduce damage to inner ear hair cells, and can effectively treat and protect inner ear damage and hearing loss caused by chemotherapy drugs.

[0062] Example 2

[0063] In this embodiment, the preparation of injectable drug-loaded hydrogel microspheres is proposed, comprising the following steps:

[0064] Take 10g gelatin and add it to 100ml carbonate buffer solution, stir at 50℃ until fully dissolved; then take 1ml methacrylic acid and inject it into the prepared gelatin solution using a micro syringe pump. Stir and react at 50℃ for 3 hours, then add an equal volume of PBS solution to terminate the reaction. Centrifuge at 7500rpm for 15 minutes at 25℃, take the upper Gel MA solution and put it into MWCO8000, dialyze at 37℃ for 2 days (change the liquid three times a day), freeze-dry, and the product Gel MA is stored at -20℃ for use.

[0065] 1g of heparin was added to 50mL of deionized water, and mechanically stirred at 4°C until completely dissolved. Then 8mL of methacrylic anhydride was added dropwise and fully reacted for 2 hours. Then 6mL of NaOH was added to the reaction system to terminate the reaction, and stirred at 4°C overnight. After centrifugation at 7000rpm for 15 minutes at 25°C, the upper HepMA solution was taken and placed in a MWCO8000 dialysis bag for 3 days. Freeze-dried, the product Hep l MA was obtained and stored at -20°C for future use.

[0066] Weigh 25 mg of the prepared Gel MA and Hep l MA, add them to 1 ml of deionized water, heat to 45 ° C and stir for 1 hour until fully mixed. Then use a coaxial capillary microfluidic device to prepare hydrogel microspheres. When the continuous phase fluid shears the dispersed phase fluid, when the shear force is greater than the interfacial tension of the two phases, the dispersed phase is sheared into a single dispersed microsphere of a specific size at the junction of the two phases, and the hydrogel microspheres are obtained after UV curing, removal of organic solvents and water washing.

[0067] Example 3

[0068] This example proposes the preparation of injectable KN93 / L-arg dual-loaded hydrogel microspheres, including:

[0069] Microfluidics technology and photochemical cross-linking reaction are used to incorporate highly dispersed small molecule drugs with biological reactivity into hydrogel microspheres to construct hydrogel microsphere carriers that can efficiently load small molecule drugs. The synthesis flow chart is as follows: Figure 1 As shown. Take 10g gelatin and add it to 100ml carbonate buffer solution, stir at 50℃ until fully dissolved; then take 1ml methacrylic acid and inject it into the prepared gelatin solution using a micro syringe pump. Stir and react at 50℃ for 3 hours, then add an equal volume of PBS solution to terminate the reaction. After centrifugation at 7500rpm for 15 minutes at 25℃, take the upper layer of Gel MA solution and put it into a MWCO8000 dialysis bag, dialyze at 37℃ for 2 days (change the liquid three times a day), freeze-dry, and the product Gel MA is stored at -20℃ for use.

[0070] 1g of heparin was added to 50mL of deionized water, and mechanically stirred at 4°C until completely dissolved. Then 8mL of methacrylic anhydride (MA) was added dropwise and fully reacted for 2 hours. Then 6mL of 5M NaOH was added to the reaction system to terminate the reaction, and stirred overnight at 4°C. After centrifugation at 7000rpm for 15 minutes at 25°C, the upper HepMA solution was taken and placed in a MWCO8000 dialysis bag for 3 days. Freeze-dried, the product Hep l MA was obtained and stored at -20°C for future use.

[0071] Weigh 25 mg of the prepared Gel MA and Hep l MA, add 0.5 wt% photoinitiator, 0.2 wt% KN93, 1% L-arginine, and fully dissolve into the external water phase. Paraffin and Span80 are used as the oil phase. Under different injection speed conditions, the microfluidic emulsion method + photochemical cross-linking reaction is used to synthesize hydrogel drug-loaded microspheres with uniform size, and the hydrogel microspheres are obtained after UV curing, and the oil phase is removed by an organic solvent and washed with water.

[0072] The above hydrogel microspheres and dopamine were dissolved in 10 ml of tr is buffer respectively. After being fully dissolved, they were mixed and stirred in the dark for 10 hours. The products were collected by centrifugation at 1000 rpm for 10 minutes to obtain PDA-modified KN93 / L-arg dual-loaded hydrogel microspheres, which were stored at 4°C.

[0073] Example 4

[0074] The characterization of injectable KN93 / L-arg dual-loaded hydrogel microspheres is presented in this example, including:

[0075] The successful synthesis of the two biomacromolecules, Gel MA and HepMA, was confirmed by 1HNMR. Figure 2 ), where in the 1HNMR spectrum of Gel MA, two new signals at δ = 5.41ppm and δ = 5.65ppm appeared, indicating the presence of methacryloyl groups. For HepMA, the signals at δ = 6.20ppm and δ = 5.77ppm in the 1HNMR spectrum indicated the newly generated vinyl methylene groups. This proves the successful synthesis of Gel MA and HepMA. The structural formula of Gel MA is as follows Fig.17 As shown, the structure of HepMA is as follows Fig.18 shown.

[0076] The morphology of the hydrogel before and after polydopamine (PDA) encapsulation was observed by optical microscopy ( Figure 3 ), Gel MA / HepMA hydrogel microspheres are transparent and uniformly spherical, with a particle size of about 150 μm ( Figure 3 , i). The PDA coating with increased bioadhesion was black, and the hydrogel morphology did not change significantly before and after encapsulation, proving the successful synthesis of hydrogel microspheres ( Figure 3 , ii).

[0077] The morphology of the freeze-dried hydrogel microspheres was observed by scanning electron microscopy, and it was found that there was a three-dimensional porous structure inside the hydrogel microspheres. This structure indicates that the hydrogel has good drug loading potential ( Figure 4 ,i). After PDA modification, the three-dimensional porous structure on the surface of the hydrogel microspheres can also be seen, and the PDA coating on the surface of the hydrogel can be clearly seen ( Figure 4, ii).

[0078] In order to analyze the uniformity of drug loading in hydrogel microspheres, NO precursor L-arginine and KN93 were loaded, and the distribution of C, N, S, and Cl elements in the hydrogel was observed by high-angle annular dark field scanning transmission. It can be seen that each element is evenly distributed inside the hydrogel, which proves the stability and uniformity of drug release in hydrogel microspheres ( Figure 5 ).

[0079] In addition, the particle size of the hydrogel microspheres before and after PDA coating was detected by dynamic light scattering technology, and it was found that the particle size increased by an average of 24 μm after PDA coating. Figure 6 ).

[0080] The hydrogel microspheres, drug-loaded hydrogel microspheres, and drug-loaded hydrogel microspheres modified with PDA were immersed in PBS buffer, taken out within 1-8 weeks, dried, and the remaining weight was weighed to evaluate the degradability of the hydrogel microspheres. The hydrogel microspheres were almost completely degraded in about 8 weeks ( Figure 7 ). Optical microscopy of hydrogels at different times is similar to the degradation curve. The hydrogel microspheres are almost completely degraded in the 8th week ( Figure 8 ).

[0081] We evaluated the swelling rates of hydrogel microspheres, drug-loaded hydrogel microspheres, and PDA-modified drug-loaded hydrogel microspheres. After the freeze-dried hydrogel microspheres were left in PBS for 6 hours, the weighing results of the excess water absorption showed that the swelling rate reached equilibrium at around 150%, indicating good drug loading capacity ( Fig. 9 ).

[0082] Embodiment 5;

[0083] This embodiment proposes an evaluation of the drug loading efficiency of injectable drug-loaded hydrogel microspheres, including: drug loading is achieved by loading drug molecules into the interior of the hydrogel microspheres by immersion. The prepared hydrogel microspheres are immersed in PBS dissolved with drug molecules, rhodamine B (RHB) is selected as a simulated drug with a concentration of 10 mg / mL, and further stirred fully to accelerate the drug molecules to enter the cavity of the hydrogel microspheres so that the drug is fully loaded. The process is operated in the dark to prevent quenching of the fluorescence signal.

[0084] After the drug loading is completed, the supernatant is collected after standing, and the hydrogel microspheres loaded with drug molecules are washed with PBS solution. The washing liquid is also collected and mixed with the supernatant. After constant volume, 400 μL of liquid is aspirated into a 96-well plate, and the RHB absorbance and fluorescence intensity are measured respectively using a multifunctional microplate reader. The drug encapsulation rate of the hydrogel microspheres is calculated according to the standard curve. Among them, the standard curve drawing strategy is: construct a drug solution with a gradient concentration, use an microplate reader to measure the absorbance and fluorescence intensity of the solution at each concentration, and use software to draw a concentration-absorbance / fluorescence intensity correlation curve. At the same time, a time gradient is set to detect the drug loading rate at different times after loading. The results show that within 4 hours, the hydrogel microspheres can quickly load drugs ( Fig.10 In addition, the drug-loaded hydrogel microspheres were scanned layer by layer using a laser confocal microscope to observe the distribution of drug molecules in the hydrogel microspheres ( Fig.11 ).

[0085] Example 6

[0086] This example proposes a biosafety assessment of injectable drug-loaded hydrogel microspheres:

[0087] HE I-OC1 cells were seeded in a 96-well plate at a density of about 1500 cells / well, cultured in a high-glucose medium containing 10% volume of fetal bovine serum for 24 hours, the medium was removed, and the HE I-OC1 cells were evaluated under an optical microscope to be in good growth condition. The medium containing injectable hydrogel microspheres at a final concentration of 100ug / mL was added to the culture medium, and the fresh medium without injectable hydrogel microspheres was used as the control group. Five independent wells were set up for each group and cultured in a 37°C incubator.

[0088] On the 1st, 2nd and 3rd day after treatment, the culture medium was removed, the cells were collected, fixed with 4% paraformaldehyde for 1 hour, the fixative was removed, and the cells were washed 3 times with 0.01M PBS, each time for 10 minutes. Cell fluorescent probe was used for staining, the culture medium containing the probe was incubated for 4 hours, and the cells were washed 3 times with 0.01M PBS, each time for 5 minutes. After washing, the anti-fluorescence quencher was added and the coverslip was covered, and the slides were sealed for testing. Images were collected under laser confocal microscope, counted, and quantitatively analyzed for cell density ( Fig.12 ). The results showed that the injectable hydrogel microspheres did not affect the growth of cells after treatment and had good biosafety.

[0089] On the third day of culture, the culture medium was removed, and after centrifugation at 6000 rpm for 10 minutes, the supernatant was removed, and HEI-OC1 cells were resuspended in 0.01 M PBS. Cell viability was determined using a cell viability detection (CCK8) kit. Fresh culture medium containing CCK8 reaction solution was used, incubated in the dark for 1.5 hours, and the absorbance of cells in different treatment groups was determined using a plate reader. The quantitative analysis results showed that a certain concentration of injectable hydrogel microspheres had almost no cytotoxicity to HEI-OC1 cells, and had good biosafety. Fig.12 shown.

[0090] Example 7

[0091] This example proposes dynamic detection of inner ear tracing using injectable hydrogel microspheres:

[0092] In order to dynamically evaluate the distribution of hydrogel microstructures in the inner ear and the efficiency of drug uptake in the inner ear, the fluorescent material cyanine 5.5 (Cy5.5) was selected as the loading material to evaluate the drug uptake in the inner ear.

[0093] Rats were intraperitoneally injected with ketamine 100 mg / kg and sodium thiazide 25 mg / kg. After deep anesthesia, 0.5 mL of hydrogel microspheres loaded with Cy5.5 at a concentration of 100 ug / mL were injected into the tympanic cavity using a microinjection pump under a microscope. The distribution of Cy5.5 in the inner ear was detected using an in vivo imaging system (IVIS) on the 1st, 3rd, 5th, 7th, 11th, and 14th day after injection. The results of in vivo imaging of small animals showed that the injectable hydrogel microspheres could stay in the tympanic cavity for a longer time, promoting the penetration of loaded drug molecules into the inner ear through the round window membrane. Fluorescent signals could still be detected in the cochlear region 7 days after injection, and almost no fluorescent signals were detected on the 11th day. Fig.13 , B). In isolated rat cochlea, compared with simple injection of Cy5.5, injectable hydrogel microspheres can significantly increase the uptake rate of Cy5.5 in the cochlea.

[0094] To further evaluate the uptake of Cy5.5 by inner ear hair cells, rats were anesthetized successfully, killed by cervical dislocation, and the rat cochlea was isolated. After being fully fixed with 4% paraformaldehyde, the fixative was removed, and the rats were soaked and washed 3 times with 0.01M PBS, each time for 10 minutes. Then, they were decalcified with ethylenediaminetetraacetic acid for 72 hours. After the bone softened, the cochlear basilar membrane was carefully separated under a microscope. Immunofluorescence staining was performed to mark the basilar membrane hair cells, and the membrane was blocked and broken at room temperature with 5% bovine serum albumin (BSA) solution containing 1% Triton-X100 (Triton-X100) for 45 minutes. After the blocking was completed, the blocking solution was removed by rinsing with 0.1% PBS, and a certain proportion of primary antibody was added according to the experimental requirements. The cells were incubated at 4°C overnight, and then washed 4 times with 0.1% PBS at room temperature for 10 minutes each time.

[0095] After washing, add fluorescent secondary antibodies of specific species according to experimental requirements, transfer to a dark box and incubate at room temperature for 90 minutes. Wash 4 times with 0.1% PBS at room temperature, 10 minutes each time.

[0096] After rinsing, phalloidin dye was added and incubated at room temperature for 20 minutes. After sealing, the dynamic distribution of Cy5.5 in the cochlear basal membrane and fluorescence quantification were detected under laser confocal microscope.

[0097] The results showed that Cy5.5 signals were detected in the cochlear sensory hair cells one day after administration, strong fluorescence signals were still present on the fifth day, and weak signals were still detectable on the seventh day, indicating that the hydrogel microspheres can penetrate the cochlear blood-membrane labyrinth barrier, enter the endolymph, and be successfully taken up by the cochlear sensory cells and can be distributed in the sensory cells for a long time.

[0098] In addition, in the cochlear basilar membrane, the uptake of Cy5.5 by cochlear sensory cells is mainly concentrated in the outer hair cells ( Fig.13 ,C). This lays the foundation for the application of the invention in the treatment of inner ear related diseases.

[0099] Example 8

[0100] This example proposes the protective effect of injectable KN93 / L-arg dual-loaded hydrogel microspheres on cell and cochlear tissue damage:

[0101] The protective effect of injectable KN93 / L-arg dual-loaded hydrogel microspheres against HEI-OC1 cells and cochlear basal membrane damage was further evaluated.

[0102] HEI-OC1 cells were inoculated on a cell slide at a density of about 1500 cells / well and cultured in a high culture medium containing 10% volume of fetal bovine serum for 12 hours. The culture medium was removed and the HEI-OC1 cells were evaluated under an optical microscope to be in good growth condition. The cisplatin injury model was constructed by adding a culture medium containing 10 μM final concentration of cisplatin, and injectable KN93 / L-arg dual-loaded hydrogel microspheres were added to evaluate cell damage.

[0103] Apoptosis (Tune l) staining was used to evaluate the apoptosis of HE I-OC1 cells. After treatment, the culture medium was removed from the cells, the cells were collected, fixed with 4% paraformaldehyde for 1 hour, the fixative was removed, and the cells were washed 3 times with 0.01 M PBS, each time for 10 minutes. The culture medium containing Tune l dye probe was added and incubated in the dark for 8 hours, and then washed 3 times with 0.01 M PBS, each time for 5 minutes. After rinsing, the cell nucleus was stained and marked, and then the anti-fluorescence quencher was added and the coverslip was covered, and the slides were sealed for testing.

[0104] Images were collected under laser confocal microscope, the number of Tune l-positive cells was counted, and the apoptosis ratio of cells in different treatment groups was quantitatively analyzed.

[0105] The results showed that the apoptosis rate of HE I-OC1 cells was significantly decreased after treatment with injectable KN93 / L-arg dual-loaded hydrogel microspheres, indicating that injectable KN93 / L-arg dual-loaded hydrogel microspheres can protect cells from cisplatin-induced cell damage. Dichlorofluorescein diacetate (DCFH-DA) and mitochondrial reactive oxygen species probe (M itoSox) staining were used to evaluate the level of intracellular reactive oxygen species. The culture medium was removed from the treated cells, the cells were collected, and 4% paraformaldehyde was added for fixation for 1 hour. The fixative was removed and washed 3 times with 0.01MPBS, each time for 10 minutes. The culture medium containing DCFH-DA dye probe and MitoSox was added and incubated overnight in the dark, and washed 3 times with 0.01MPBS, each time for 5 minutes.

[0106] After rinsing, the cell nucleus was stained and marked, and then the anti-fluorescence quencher was added and the coverslip was covered. The images were collected under laser confocal and the fluorescence intensity was quantitatively analyzed. The results showed that the intracellular reactive oxygen species decreased significantly after treatment with the injectable KN93 / L-arg dual-loaded hydrogel microspheres, indicating that the injectable KN93 / L-arg dual-loaded hydrogel microspheres can remove the reactive oxygen species produced in the cells, thereby protecting the cells from damage caused by cisplatin ( Fig.14 ).

[0107] Rats aged 3-5 days were deeply anesthetized and killed by cervical dislocation. After cutting off the skull, the temporal bone tissue of the mouse was quickly separated using vascular forceps, and the temporal bone tissue was transferred to the pre-prepared PBS. The cochlea was quickly found under a dissecting microscope, and the mouse cochlea was separated and removed. The excess tissue around the cochlea was carefully removed and the cochlear basement membrane was separated, transferred to the pre-prepared glass slide and flattened.

[0108] Add to the culture medium containing 10% bovine serum albumin, glutamine, and 20% glucose. The explants were cultured at 37°C overnight and different interventions were performed according to the corresponding experimental requirements. After treatment, the basement membrane was collected, fixed with 4% paraformaldehyde for 1.5 hours, the fixative was removed, and the 0.01M PBS was soaked and washed 3 times, each time for 10 minutes. Subsequently, a 5% bovine serum albumin (BSA) solution containing 1% Triton-X100 was used to block and permeabilize the membrane at room temperature for 45 minutes. After the blocking was completed, the blocking solution was removed by rinsing with 0.1% PBST. Myosin 7A was added according to the experimental requirements, incubated at 4°C overnight, and washed 4 times with 0.1% PBST at room temperature, each time for 10 minutes.

[0109] After washing, add fluorescent secondary antibodies of specific species according to experimental requirements, transfer to a dark box and incubate at room temperature for 90 minutes. Wash 4 times with 0.1% PBST at room temperature, 10 minutes each time. After rinsing, stain the nuclei and incubate at room temperature for 10 minutes.

[0110] After sealing, images were collected under laser confocal microscope to count the number of cochlear basilar membrane hair cells. The results showed that the cochlear capillary loss rate was significantly reduced after treatment with injectable KN93 / L-arg dual-loaded hydrogel microspheres, indicating that injectable KN93 / L-arg dual-loaded hydrogel microspheres can protect cochlear hair cell damage caused by cisplatin. Fig.14 shown.

[0111] Example 9

[0112] This example proposes the protective effect of injectable KN93 / L-arg dual-loaded hydrogel microspheres on rat hearing and cochlear hair cells:

[0113] In vivo (rat) experiment: A rat model of chronic hearing loss induced by cisplatin was constructed, and 3.5 mg / kg cisplatin injection was injected intraperitoneally for 3 consecutive days, and 0.5 mL of injectable KN93 / L-arg dual-loaded hydrogel microspheres with a concentration of 100 ug / mL were injected intratympanically. On the 5th day, after deep anesthesia, auditory brainstem potential detection was performed on rats in each group to evaluate the auditory function of the rats. The results of auditory brainstem potential detection showed that the hearing of rats in the control group and the group with simple hydrogel microsphere injection was normal, indicating that intratympanic injection of injectable KN93 / L-arg dual-loaded hydrogel microspheres had no significant effect on the hearing of rats. The hearing of the group injected with injectable KN93 / L-arg dual-loaded hydrogel microspheres was significantly better than that of the group with simple cisplatin, suggesting that injectable KN93 / L-arg dual-loaded hydrogel microspheres can protect hearing loss caused by cisplatin, such as Fig.15 shown.

[0114] To further evaluate the damage of hair cells in rats in different treatment groups, rats were anesthetized successfully, and the rat cochlea was isolated by cervical dislocation. After being fully fixed with 4% paraformaldehyde, the fixative was removed, and the rats were soaked and washed 3 times with 0.01M PBS for 10 minutes each time. Then, the cochlear basilar membrane was carefully separated under a microscope after decalcification with ethylenediaminetetraacetic acid for 72 hours. After the bone softened, the basilar membrane was carefully separated under a microscope. Immunofluorescence staining was performed to mark the basilar membrane hair cells, and the membrane was blocked and broken at room temperature for 45 minutes with 5% bovine serum albumin (BSA) solution containing 1% Triton-X100. After the blocking was completed, the blocking solution was removed by rinsing with 0.1% PBST, and Myosin7a antibody was added according to the experimental requirements, and incubated at 4°C overnight. At room temperature, it was washed with 0.1% PBST for 4 times, each time for 10 minutes. After the washing was completed, the fluorescent secondary antibody of the specific species was added according to the experimental requirements, and the cells were transferred to a dark box and incubated at room temperature for 90 minutes.

[0115] At room temperature, wash with 0.1% PBST 4 times, 10 minutes each time. After rinsing, add phalloidin dye and incubate at room temperature for 20 minutes. After sealing, collect pictures under laser confocal to count the number of hair cells of the cochlear basilar membrane. The results show that the cochlear capillary loss of rats treated with injectable KN93 / L-arg dual-loaded hydrogel microspheres is significantly reduced, indicating that injectable KN93 / L-arg dual-loaded hydrogel microspheres can protect the cochlear hair cells from damage caused by cisplatin, thereby protecting hearing loss ( Fig.16 ).

[0116] In summary, the hydrogel microspheres synthesized in the present invention provide a suitable cavity that can accommodate drug molecules, achieving efficient negative and stable release of drugs. In addition, the drug-loaded hydrogel microspheres can stay in the middle ear for a long time, promote the penetration of drug molecules into the endolymph through the round window membrane, and then be taken up by the inner ear sensory cells. The preparation method provided by the present invention is simple, and the prepared injectable KN93 / L-arg dual-loaded hydrogel microspheres can effectively remove ROS, protect the survival of cochlear sensory cells, and can play a good role in treating inner ear diseases.

[0117] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An injectable KN93 / L-arg dual-loaded hydrogel microsphere, characterized in that: The method includes using methacryloylated gelatin and methacryloylated heparin as matrices, incorporating target drug molecules KN93 and L-arg into the hydrogel matrix, and using microfluidic technology and photochemical cross-linking reaction to prepare functionalized injectable KN93 / L-arg dual-loaded hydrogel microspheres.

2. A method for preparing the injectable KN93 / L-arg dual-loaded hydrogel microspheres according to claim 1, characterized in that: The steps include: Preparation of GelMA: 20 g of gelatin was added to 200 ml of carbonate buffer solution and heated to 50°C to dissolve, and then 2 ml of methacrylic acid was injected into the gelatin solution using a micro-syringe pump; after stirring at 50°C for 3 hours, 100 ml of phosphate buffered saline solution was added to terminate the reaction; after centrifugation at 7000 rpm for 15 minutes at 25°C, the oily liquid was removed, and the upper layer of GelMA solution was placed in a regenerated cellulose dialysis bag, dialyzed at 37°C for 2 days, then freeze-dried and stored at -20°C for later use; Synthesis of HepMA: 1 g of heparin was completely dissolved in 50 mL of deionized water under mechanical stirring at 4°C, and 8 mL of methacrylic anhydride was added dropwise to react for 2 hours; then 6 mL of sodium hydroxide was added to the reaction system to terminate the reaction, and stirred at 4°C overnight; after centrifugation at 7000 rpm for 15 minutes at 25°C, the oily liquid was removed, and the upper layer of HepMA solution was placed in a dialysis bag for 3 days, then freeze-dried and stored at -20°C; Synthesis of injectable drug-loaded hydrogel microspheres by microfluidic emulsion method: The hydrogel microspheres are synthesized by microfluidic emulsion method and photochemical cross-linking reaction. First, the water phase and the oil phase are respectively injected into the inlet of the modified microfluidic device. The shear force and the hydrophobic contact at the cross-section produce pre-gel droplets. After UV curing, the organic solvent is removed and washed with water to obtain the hydrogel microspheres; the water phase includes GelMA 5wt%, HepMA 0.4wt%, photoinitiator 0.5wt%, KN93 0.2wt%, L-arg 1wt%; the oil phase includes paraffin 95wt% and sorbitan monooleate 5wt%; The produced hydrogel microspheres and dopamine were dissolved in 10 ml of tris(hydroxymethyl)aminomethane buffer respectively. After being fully dissolved, they were mixed and stirred in the dark for 10 hours. The products were collected by centrifugation at 1000 rpm for 10 minutes to obtain dopamine-modified KN93 / L-arg dual-loaded hydrogel microspheres, which were stored at 4°C.

3. The method according to claim 2, characterized in that: The methacrylic anhydride and gelatin contain carboxyl groups.

4. The method according to claim 2, characterized in that: The concentration of heparin is 20 mg / mL.

5. The method according to claim 2, characterized in that: KN93 is a competitive inhibitor of calmodulin-dependent kinase type II, with a concentration of 20-40 mg / mL. L-arginine is a NO precursor, which can be quickly decomposed to produce NO after entering the cell.

6. The method according to claim 2, characterized in that: The material concentration of the water phase is 5%, the injection speed is 10-15 μL / min, and the injection speed of the oil phase is 75-100 μL / min.

7. The method according to claim 2, characterized in that: The organic solvent is one of volatile isopropanol and methanol.

8. The method according to claim 2, characterized in that: The concentrations of the hydrogel microspheres and the dopamine solution are 1 mg / mL and 0.5 mg / mL respectively, and the volume ratio of the hydrogel microspheres to the dopamine solution is 1:1-1:1.

5.

9. The method according to claim 2, characterized in that: The final effective concentration of KN93 and L-arginine in the complex is 50-100 μg / ml.

10. An injectable KN93 / L-arg dual-loaded hydrogel microsphere as claimed in claim 1, wherein the hydrogel microsphere is used in the preparation of a drug for inner ear diseases.

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