CN104644545A - Controlled-release and slow-release silk fibroin gel preparation for treating inner ear disease - Google Patents

Abstract

The present invention relates to a preparation for treating inner ear diseases, in particular to a controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases; the controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases of the present invention includes preparations The main body, the main body of the preparation includes a gel carrier and a drug dispersed or adsorbed in the carrier, the drug is a hormone drug for treating inner ear diseases, and the carrier is a silk fibroin gel. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases of the present invention has a long drug release time, has no effect on hearing after use, and is safe and harmless.

Description

A controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases

technical field

The invention relates to a preparation for treating inner ear diseases, in particular to a controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases.

Background technique

In recent years, the treatment of sudden hearing loss (SSNHL) by administering hormonal drugs to the inner ear through the tympanic cavity (IT) has been increasingly applied, covering from first-line treatment to refractory sudden hearing loss (RSSNHL). ) first aid and other clinical fields. Compared with systemic administration, IT injection can not only make the perilymph contain a higher concentration of drugs, but also enhance the blood flow and ion gradient of the cochlea, which all contribute to better hearing recovery. However, the IT administration of water-soluble hormone drugs is very limited clinically, because some drugs may be absorbed by the mucous membrane of the tympanic cavity, or flow away from the tympanic cavity through the eustachian tube, so they cannot fully interact with the round window membrane (RWM). ) contact, and then enter the inner ear through the round window membrane. The round window membrane is the main barrier from the tympanic cavity to the inner ear, and the amount of drug absorbed by it directly determines the drug concentration in the inner ear. Drugs injected directly may be absorbed by the mucous membrane in the tympanic cavity, or flow away from the tympanic cavity through the Eustachian tube, and only a very small amount can be absorbed by the round window membrane. Tympanic injections alone do not deliver sufficient drug to the inner ear because diffusion of the drug to the inner ear is limited. In clinical practice, repeated or continuous administration can be used to maintain the drug concentration in the inner ear, that is, a catheter connected to the pump is implanted in the tympanic cavity, so that the continuous administration of the fully implanted microcatheter is connected to the micropump. Compared with repeated injections, pump administration can achieve the effect of continuous drug delivery. However, implantable micropumps have not gained widespread clinical acceptance due to tissue damage during insertion and removal.

Degradable biomaterials, which have received widespread attention recently, can be used as an alternative to multiple injections and implantable micropumps to control the sustained release of drugs. Companies and research institutes at home and abroad are actively researching a variety of synthetic or natural materials to establish a clinically applicable slow-release and controlled-release system for inner ear drugs. The basic idea is to embed therapeutic molecules with degradable materials and place them around the round window membrane. The material can adhere to the round window membrane and continuously release the embedded therapeutic molecules to the inner ear.

Silk fibroin is a natural protein polymer isolated from silkworm silk, which has all the characteristics required for tissue repair and drug delivery carriers, including biodegradability, biocompatibility, stable drug embedding, and material form diversity And processing is simple, without organic solvents and the like. Studies have confirmed the feasibility of clinical application of silk fibroin gel for injection. Silk fibroin is a biological material approved by the FDA, so silk fibroin gel is safe for clinical application. These characteristics enable silk fibroin gel to be used as a drug carrier to deliver drugs to the inner ear, prolong the residence time of drugs in the tympanic cavity through IT, and reduce the loss of drugs caused by flowing into the eustachian tube. However, how to form drugs with silk fibroin gel Stable controlled-release and sustained-release preparations are still a difficult problem.

In view of the above-mentioned defects, the designer, actively researches and innovates, in order to create a controlled-release and slow-release silk fibroin gel preparation for the treatment of inner ear diseases, so that it has more industrial value.

Contents of the invention

In order to solve the above-mentioned technical problems, the object of the present invention is to provide a controlled-release and slow-release silk fibroin gel preparation for the treatment of inner ear diseases, which has a long drug release time, has no effect on hearing, and is safe and harmless.

The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases of the present invention includes a preparation body, the preparation body includes a carrier in a gel state, and a drug dispersed or adsorbed in the carrier, and the drug is used for treating inner ear diseases Hormonal drugs, the carrier is silk fibroin gel.

Specifically, the silk fibroin gel is made by inducing gelation with a silk fibroin solution.

Specifically, the methods of inducing gelation include pH value change method, ultrasonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H ₂ O ₂ (hydrogen peroxide) blending method, and low molecular weight PEG (polyethylene oxide) Ethylene glycol) blending method.

Preferably, the way of inducing gelation is a low molecular weight PEG (polyethylene glycol) blending method.

Specifically, the main body of the preparation is prepared by inducing gelation after the drug is mixed with the silk fibroin solution in the form of an aqueous solution or in the form of insoluble microspheres.

Specifically, the concentration of silk fibroin in the main body of the preparation is 1-30%.

Preferably, the concentration of silk fibroin in the main body of the preparation is 7.5-15%.

Specifically, the diseases include Meniere's disease, sudden deafness (SSNHL), Meniere's syndrome, sensorineural hearing loss, and autoimmune inner ear disease (AEID), and the hormone drugs include Dexamethasone, Betamethasone, Prednisolone, Methylprednisolone, Deoxycorticosterone, 11-Deoxycorticosterone, 18-H-11-Deoxycorticosterone, Beclomethasone, Triamcinolone acetonide And one or more of its chemically synthesized derivatives.

The present invention also provides a method for preparing the above-mentioned controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases, which includes the following steps:

1) Mixing: mix the hormone drugs for the treatment of inner ear diseases with the silk fibroin solution in the form of aqueous solution or in the form of insoluble microspheres to obtain a drug suspension;

2) Gelling: The drug suspension is made into the main body of the preparation by inducing gelation.

Further, the methods of inducing gelation include pH value change method, ultrasonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H ₂ O ₂ (hydrogen peroxide) blending method, and low molecular weight PEG (polyethylene oxide) Ethylene glycol) blending method.

Further, in the mixing step, before the hormone drugs are uniformly mixed with the silk fibroin solution in the form of insoluble microspheres, coating treatment is also carried out, and the coating treatment includes the following steps:

a) suspending the hormone drug microspheres in a low-concentration silk fibroin solution, mixing them uniformly, and ultrasonically treating them for a period of time to disperse the agglomerated microspheres;

b) The solution obtained in step a) is vibrated, centrifuged, supernatant removed, washed with water and dried to obtain hormone drug microspheres coated with silk fibroin.

To obtain hormone drug microspheres coated with multiple layers of silk fibroin, repeat steps a-b.

The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to the present invention has two methods of use, one is in-situ gelation, the other is pre-gelation, in-situ gelation and pre-gelation The gelation method is the same as the gelation method, but there are differences in the specific use method. The difference between the two is that the pre-gelation is injected or implanted into the inner ear after gelation in vitro, while the in-situ gelation is injected into the round window. The entire space of the niche, and then form a semi-solid gel with a suitable shape, which can contact the surface of the round window membrane as much as possible; the preformed gel does not require the patient to maintain a certain posture, so it is more convenient, but the disadvantage is that it is difficult to pass through the cell at high concentration. Needle injection.

By means of the above scheme, the present invention has at least the following advantages: the drug of the present invention can treat inner ear diseases as a controlled-release and sustained-release preparation. Injectable silk fibroin gel is used as a drug carrier, and the gelation time of silk fibroin-PEG solution is highly controllable (Wang X, Partlow B, Liu J, et al.Injectable silk-polyethylene glycolhydrogels.Acta Biomater.2015; 15(12 ):51-61); the preparation scheme of in-situ gelation can reach the standard of zero-order release; the drug release time can reach at least 10 days; and it has no effect on hearing. At the same time, as a drug carrier, silk fibroin-PEG gel is safe, non-toxic and slow to degrade. It shows that the injectable silk fibroin-PEG gel is an effective and safe drug carrier, which can be used for the treatment of inner ear diseases by hormone sustained-release drugs.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Fig. 1 is a scanning electron microscope result figure of silk fibroin-PEG-mDEX gel in Example 6 of the present invention;

Fig. 2 is the result figure of the in vitro release test of silk fibroin-PEG-mDEX gel in Example 7 of the present invention;

Fig. 3 is the result figure of the concentration of DEX in the perilymph under different time points in the eighth embodiment of the present invention;

Fig. 4 is the comparison chart of the influence of the sham operation group and the round window membrane injection silk fibroin-PEG-mDEX gel group on the auditory threshold in the eighth embodiment of the present invention;

Fig. 5 is the histological section figure of guinea pig tympanum and inner ear sample in the eighth embodiment of the present invention;

Fig. 6 is a diagram of fluorescence staining results of basement membrane stained by rhodamine-phalloidin in Example 8 of the present invention;

Fig. 7 is the in vivo biodegradation diagram of silk fibroin-PEG-mDEX gel in Example 8 of the present invention.

Detailed ways

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Embodiment one

The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases of the present invention includes a preparation body, the preparation body includes a carrier in a gel state, and a drug dispersed or adsorbed in the carrier, and the drug is a hormone drug for treating inner ear diseases , the carrier is silk fibroin gel. Among them, the silk fibroin gel is made by inducing gelation with silk fibroin solution; the induction gelation methods include pH value change method, ultrasonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H ₂ O ₂ (hydrogen peroxide) blending method, and low molecular weight PEG (polyethylene glycol) blending method.

Hormonal drugs are divided into water-soluble drugs and water-insoluble drugs. Therefore, the main body of the preparation is made by inducing gelation after mixing the drug with the silk fibroin solution in the form of an aqueous solution or in the form of insoluble microspheres. Considering that the drug needs to be released for a long time, the optimized drug form is the poorly water-soluble drug, and the poorly water-soluble drug in the microsphere state is more optimally selected. The poorly water-soluble drug can be made into a microsphere form by spray drying and other means. Considering the effect of long-term release, the drug dosage form can be further pretreated, that is, the microsphere surface of the therapeutic drug is coated with silk fibroin, which can more effectively promote the sustained release effect of the drug during the gel degradation process. The poorly water-soluble drug is suspended in a suitable solvent in the form of microspheres, and then mixed with silk fibroin solution to induce gelation. Suitable solvents include but are not limited to water, organic solvents, solubilizers, viscosity enhancers, and penetration enhancers agent.

According to different dosages of different drugs, in the drug solution, the range of drug concentration is 0.5%-15% (w/v), wherein, the concentration of low load is 0.5-2%; medium load: 2-5%; The loading concentration is 5-15? %. The concentration of silk fibroin in the main body of the preparation is 1-30%, and the concentration of silk fibroin in the main body of the preparation is 7.5-15%.

The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases of the present invention can be applied to inner ear diseases including Meniere's disease, sudden deafness (SSNHL), Meniere's syndrome, sensorineural hearing loss , and autoimmune inner ear disease (AEID) and other inner ear diseases; correspondingly, the hormone drugs used include dexamethasone, betamethasone, prednisolone, methylprednisolone, deoxycorticosterone , 11-desoxycorticosterone, 18-H-11-desoxycorticosterone, beclomethasone, and one or more of triamcinolone acetonide and its chemically synthesized derivatives.

The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to the present invention has the drug action site in the inner ear, and the drug preparation is in a gel state, covering on and around the round window membrane in the cochlea, and can pass through the cochlea. Injection of drug preparations into the round window niche and/or injection through the tympanic cavity; there are two methods of use, one is in situ gelation and the other is pre-gelation. Injection or implantation into the inner ear, and in situ gelation is to inject the drug suspension into the entire space of the round window niche, and then form a semi-solid gel with a suitable shape to maximize the contact with the surface of the round window membrane. When the pharmaceutical dosage form is in-situ gelation, it includes but is not limited to ordinary syringes, microneedles injected into the tympanic cavity, and micro-syringes administered in a solution state.

The controlled-release and slow-release silk fibroin gel preparation for the treatment of inner ear diseases according to the present invention, if the low-molecular-weight PEG (polyethylene glycol) blending method is used and in-situ gelation is used, the drug action mechanism is as follows: after injection, The silk fibroin-PEG (polyethylene glycol) mixture fills the entire space of the round window niche, and then forms a semi-solid gel with a suitable shape, which contacts the surface of the round window membrane to the greatest extent; as the gel adheres to the On the round window membrane, the drug is continuously released and enters the inner ear to achieve the effect of treating diseases. With the release of the drug, the gel is degraded by proteases in the body, and the degradation products are polypeptides and amino acids. Degradation time It is known from experiments that it begins to degrade on the 10th day, and only a small amount of residual gel exists at the medicinal site on the 21st day. At the same time, it is found that the drug's slowdown time can reach at least 10 days.

Embodiment two

The invention provides a method for preparing a controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases, comprising the following steps:

1) Mixing: mix the hormone drugs for the treatment of inner ear diseases with the silk fibroin solution in the form of aqueous solution or in the form of insoluble microspheres to obtain a drug suspension;

2) Gelling: The drug suspension is made into the main body of the preparation by inducing gelation.

Among them, the ways to induce gelation include pH value change method, ultrasonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H ₂ O ₂ (hydrogen peroxide) blending method, and low molecular weight PEG (polyethylene glycol) ) blending method.

The operation steps of different induced gelation methods are as follows, and the comparison results are shown in Table 1:

a) pH value is changed to prepare silk fibroin gel

Get 10mL of 5% (w/v) silk fibroin solution with a 20mL small beaker, adjust the pH of the solution to 5.2 with NaH2PO4-Na2HPO4 buffer solution, and slowly form a gel at room temperature overnight.

b) Preparation of silk fibroin gel by ultrasonic oscillation

Use a 10mL test tube to take 5mL of 5% (w/v) silk fibroin solution, use a JYD-900 intelligent ultrasonic cell pulverizer to perform ultrasonic oscillation for 30-60sec (seconds) at a power of 500w, and then place the small beaker at room temperature A gel was formed at 25°C.

c) Preparation of silk fibroin gel by electrophoresis.

The electrode is immersed in 8% (w/v) silk fibroin aqueous solution, and energized at 25 volts direct current for 3 minutes (minutes). Due to the high water content of the silk fibroin solution, hydrolysis will occur during the electrification process to produce oxygen and hydrogen, so as the electrification proceeds, bubbles appear at the two electrodes. At the same time, gelation occurs at the anode position.

d) HRP (horseradish peroxidase)-H2O2 (hydrogen peroxide) gelation

HRP freeze-dried powder was added to deionized water to form a solution with a concentration of 1000 U/ml, and then an appropriate amount of HRP solution was added to a 3% (w/v) silk fibroin solution to prepare a final concentration of HRP in the solution of 10 U/ml. Then, 10 μl of hydrogen peroxide solution was added to each milliliter of silk fibroin solution to reach a final hydrogen peroxide concentration of 165 nM, and mixed to prepare a gel.

e) Low molecular weight PEG (polyethylene glycol) gelling

The 15% (w/v) silk fibroin solution was mixed with the same volume of 80% (w/w) PEG400 (polyethylene glycol 400) solution, and incubated at 37°C to form a gel.

Table 1 Comparison of different silk fibroin gel preparation methods

Through X-diffraction examination of the main structure of gels formed by various methods, it is known that the main structure is Silk Ⅱ. Silk Ⅱ crystal structure content is the most important factor that determines the mechanical strength, degradation rate and drug slow-release and controlled-release performance of silk fibroin biomaterials, so it can be seen that the silk fibroin gels prepared by the above different methods have no difference in material properties change. Because low-molecular-weight PEG (polyethylene glycol) gelation method is more convenient for clinical application, controllable, safe and non-toxic, so the method of inducing gelation in the following examples all adopts silk fibroin-PEG gel method, other methods No longer. In addition, it should be pointed out that when the low molecular weight PEG (polyethylene glycol) blending method is used to prepare the gel, the above-mentioned hormone drugs can also be mixed with polyethylene glycol, or mixed with silk fibroin and polyethylene glycol at the same time. Diol mix.

Embodiment Three

The invention provides a method for preparing a controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases, which is applied to the preparation of different hormone drug preparations:

a) Preparation of silk fibroin-PEG-mDEX (micronized dexamethasone, dexamethasone microspheres)

A certain amount of mDEX microspheres is suspended in 0.5ml of 15% (w/v) silk fibroin aqueous solution, and then the suspension is mixed with the same volume of 0.5ml 80% (w/w) PEG400 (polyethylene glycol 400) solution into glue.

b) Preparation of silk fibroin-PEG-DA (dexamethasone acetate) gel

Mix sterile DA (dexamethasone acetate) solution with high-concentration silk fibroin aqueous solution to achieve a certain drug load. The final concentration of silk protein is 15% (w/v), and then mix with 80% (w /w) PEG400 (polyethylene glycol 400) solution is mixed to form a gel.

c) Silk fibroin-PEG-MPS (dexamethasone sodium phosphate) gel preparation

Sterile DSP (dexamethasone sodium phosphate) solution is mixed evenly with high-concentration silk fibroin aqueous solution to achieve a certain drug load. The final concentration of silk protein is 15% (w/v), and then mixed with 80% (w/v) of the same volume ( w/w) PEG400 (polyethylene glycol 400) solution is mixed to form a gel.

Due to the different efficacy and dosage of different drugs, it is difficult for the present invention to illustrate them in detail one by one. Therefore, in the following examples, only the preparation of silk fibroin-PEG-mDEX is used as an example to describe in detail.

Embodiment Four

The invention provides a method for preparing a controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases, which is used for the preparation of silk fibroin-PEG-mDEX:

a) mDEX-silk fibroin coating preparation: about 20 mg of mDEX was suspended in 1 ml of 0.1% (w/v) silk fibroin solution. The suspension was shaken slowly at room temperature for 2 min. The microsphere suspension was then sonicated for approximately 1 min to disperse aggregated microspheres. Shake again for 2 minutes and centrifuge the solution for 2 minutes (14000r.p.m) to remove the upper silk fibroin solution. The microspheres were washed twice with 1ml water, shaken for 1min, and centrifuged. The washed microspheres were dried with nitrogen gas. The above coating steps can be repeated several times until the desired coating thickness and corresponding drug release rate are obtained.

b) Preparation of silk fibroin-PEG-mDEX gel: the prepared silk fibroin-coated mDEX is suspended in 0.5ml of high-concentration silk fibroin aqueous solution, the final concentration of silk protein is 15% (w/v), and then suspended The solution was mixed with the same volume of 0.5ml 80% (w/w) PEG400 (polyethylene glycol 400) solution to form a gel.

It should be noted that: the above step b) can be used alone to prepare silk fibroin-PEG-mDEX gel without silk fibroin coating, and can also be combined with step a) to prepare silk fibroin coating. Protein-PEG-mDEX gel.

Embodiment five

The present invention provides a method for preparing a controlled-release and slow-release silk fibroin gel preparation for the treatment of inner ear diseases, which is used for the preparation of silk fibroin-PEG-mDEX. The method is consistent with that in Example 4, and the difference lies in:

The concentration of silk fibroin is adjusted to 1-30%. However, when the final concentration of silk protein is 1%, the release rate of the drug is faster due to the lower gel concentration and poor drug loading. When the final concentration of silk protein is 30% , because the gel concentration is higher, the drug loading is higher, the adsorption effect on the drug is more significant, and the release rate of the drug is slower, therefore, the concentration of silk fibroin in the main body of the preparation is preferably 7.5-15%.

Embodiment six

The present invention provides a controlled-release and slow-release silk fibroin gel preparation for the treatment of inner ear diseases, which is prepared by the preparation method in Example 4, and then SEM (scanning electron microscopy) is used to carry out the silk fibroin-PEG-mDEX gel preparation. Morphological examination. The test method is:

Silk fibroin-PEG gel (7.5% w/v) embedded mDEX (0%, 1.5% loading) was prepared according to the method in Example 4, and 200 μL of the prepared solution was dropped into a 24-well plate, and placed in a 37°C environment Incubate until the solution gels. The orifice plate was immersed in a glass beaker of 1000 ml of purified water and stirred slowly over 24 hours, during which time the purified water was replaced 4 times. The washed gel was frozen overnight at -80°C and lyophilized for 48 hours. Samples were observed by SEM.

As shown in Figure 1, PEG has been removed from the gel after water washing, resulting in a porous sponge-like appearance of the remaining gel, as observed by SEM morphology. As shown in Figure 1A-1C, they are the scanning electron microscope results of silk fibroin-PEG gel without mDEX embedding at magnifications of 20, 5 and 1 μm, respectively, and the silk fibroin-PEG gel without mDEX embedding has a porous structure , with interconnected pores of about 100 μm. As shown in Figure 1D-1F, the scanning electron microscopy results of silk fibroin-PEG gel embedded with mDEX under the magnifications of 20, 5 and 1 μm respectively, and the silk fibroin-PEG gel embedded with mDEX showed Similar to the porous lamination structure, the particulate mDEX (1-5μm) is embedded in the silk fibroin matrix. As shown in Figure 1G-1I, the scanning electron microscopy results of unembedded mDEX water-insoluble microspheres at magnifications of 20, 5 and 1 μm, respectively, mDEX microparticles measured alone and microparticles embedded in silk fibroin gel The sizes are the same, which indicates that the process of silk fibroin gel formation has no effect on the size and dispersibility of mDEX microparticles.

Embodiment seven

The present invention provides the controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases described in Example 4, the test and analysis results of the controlled-release and sustained-release effects in vitro, and the preparation method of silk fibroin-PEG-mDEX gel Consistent with the method provided in Example 4, the difference is that the drug loading amount of the drug is different, and the mDEX drug microspheres are not coated with silk fibroin. The in vitro release test is as follows:

Silk fibroin-PEG-mDEX gel is divided into low-dose (0.5%) and high-dose (2.5%) according to the different drug loading mDEX. For the different gelation methods, it is divided into in-situ gelation group and pre-gelation group. into glue group. Among them, for the in situ gelation group, 5 μl of silk fibroin-PEG-mDEX solution was injected into a 2-ml microcentrifuge tube through a 25G needle, and then incubated at 37°C to form a gel within 30 minutes. For the pre-gelation group, the silk fibroin-PEG-mDEX solution contained in the syringe was first incubated at 37°C for 30 minutes to form a gel, and then 5ml of the gel was injected into a 2-ml microcentrifuge tube (without a needle). ). At the beginning of the experiment, 1ml of PBS, PH=7.4 buffer solution was added to each tube, and all centrifuge tubes were shaken at 37°C. On the 1st, 6th, 24th hour, 2nd, 3rd, 4th, 5th, 6th, and 7th day, extract 0.95ml of the release medium into a blank tube, and then store it at 4°C for LC/MS/MS detection. Add 0.95ml of PBS, pH 7.4 buffer to the original centrifuge tube at constant temperature.

The results are shown in Figure 2, wherein Figure 2A is a comparison of the detection results of the DEX concentration (μg/ml) in the release medium (PBS, pH 7.4) of four silk fibroin-PEG-mDEX gels at different times, Fig. 2B is a comparison chart of the cumulative release of DEX from four silk fibroin-PEG-mDEX gels within 144 hours at different times. As shown in Figure 2A, the silk fibroin-PEG-mDEX gel with a drug loading of 0.5% showed a rapid release process at the initial stage, and reached the maximum rapidly at 2 h in both in situ gelation and pregelation modes The concentration value (approximately 4 μg/ml) decreased rapidly to the baseline value after 6 hours. When the drug loading of the gel was 2.5%, the sustained release time of the high concentration level of DEX was longer. In the in situ gelation mode, the concentration of DEX changed within 5-10μg/ml in 96h, and dropped to 1.6μg/ml, in the pregelation mode, the concentration of DEX varied from 5-10μg/ml within 72h, and dropped to 2.2μg/ml at 96h. When the drug loading of mDEX was 0.5%, the cumulative release amount of the in-situ gelation mode and the pre-gelation mode were 38% and 25%, respectively (Fig. 2B). When the mDEX drug loading was 2.5%, the in situ gelation and pregelation almost achieved zero-order release, and after 144h, the release rates were close to 44% and 30%, respectively (Fig. 2B)

Embodiment eight

The present invention provides the controlled-release and slow-release silk fibroin gel preparation described in Example 4 for treating inner ear diseases, and the experimental analysis results of the controlled-release and sustained-release effects in vivo. The silk fibroin-PEG-mDEX gel preparation method is consistent with the method provided in Example 4, the difference is that the drug loading and silk fibroin concentration are different, and the in vivo pharmacokinetic test method is as follows:

a) Ear surgery:

method:

In the silk fibroin-PEG-mDEX gel, the concentration of silk fibroin was 7.5% (w/v), and the loading of mDEX was 0.5% and 2.5% (w/v), which were used for in vivo experimental research. The 0.5% and 2.5% mDEX solutions in the control group were prepared by suspending mDEX in 10mM PBS, pH 7.4. The whole process is aseptically performed on a sterile operating table. Guinea pigs were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (35 mg/kg). During the whole operation, the animal needs to be placed on an electric heating pad (38°C) to maintain body temperature, 1% lidocaine is injected subcutaneously for local anesthesia, the auditory bulb is exposed through the operation behind the ear, and a small hole with a diameter of 3 mm is drilled on the auditory bulb , to clearly see the round window niche. Use a tuberculin syringe (27G needle) to instill 10 μl of mDEX suspension (control group) or silk fibroin-PEG-mDEX mixed suspension on the round window membrane. After injection, the guinea pigs should be fixed in this position for about 30 minutes to wait for the solution to gel , The hole is sutured with sutures and sealed with filling cement.

b) Sampling and DEX concentration detection

Method: Guinea pigs were divided into 2 groups (50): silk fibroin-PEG-mDEX gel group (sampling time: 1h, 1, 4, 7, 10, 14 days) and mDEX control group (sampling time: 1, 3, 6, 12h). For each sampling point, 5 guinea pigs (3 gel group and 2 control group) were sampled and analyzed. For the extraction of cerebrospinal fluid (CSF), a midline incision was made at the back of the head, the muscle layer was peeled off, and the cisterna magna was exposed, and 20 μl of CSF was extracted with a micro-regulation syringe. The blood sample was extracted by heart puncture, and the blood sample was transferred to a centrifuge tube added with heparin and centrifuged for 5min (3500rpm/s). The supernatant serum (approximately 100 μl each) was then transferred to an empty tube. In order to avoid cerebrospinal fluid contamination when sampling perilymph fluid, it was extracted in vitro. The cochlea was isolated from the temporal bone of the experimental animals after general anesthesia. A small hole was made in the apical ring of the cochlea, and the perilymph (5-7 μl) was aspirated with a microregulated syringe pump. All samples were stored at -80°C before testing.

result:

After injecting silk fibroin-PEG-mDEX gel and mDEX solution (control group) into the round window membrane of guinea pigs, the concentration of DEX in perilymph fluid, CSF (cerebrospinal fluid) and plasma samples of guinea pigs was regularly sampled for detection. As shown in Figure 3, it is a comparison chart of the DEX concentration released in the perilymph at different time points for the silk fibroin-PEG-mDEX gel with a drug loading of 0.5% and 2.5%, and the mDEX solution, and Figure 3A is The result map of 0-200h, Figure 3B is a locally enlarged comparison map of the 0-30h area. It can be seen from the figure that in the control group, the perilymph DEX concentration reached the peak value (13.47±6.02μg/ml) in 1 hour, dropped to 2.984±1.33μg/ml in 6 hours, and fell below the detection line in 12 hours. In the silk fibroin-PEG-mDEX gel group with DEX loading of 0.5%, the DEX concentration in the perilymph was 8.06±4.56μg/ml at 1h and 2.70±0.97μg/ml at 12h, indicating that the release time of DEX in this group was longer than that of the control group. The group should be long. When the DEX load increased to 2.5%, in the silk fibroin-PEG-mDEX group, the time and concentration of DEX in the inner ear increased significantly, and the detection concentration of DEX in the perilymph was 25.71±11.50μg/ml at 1h and 6.46±2.89 at 4d μg/ml, 10d reached 0.26±0.15μg/ml. After topical administration, the concentration of DEX in the whole body is very low. One hour after middle ear injection in the silk fibroin-PEG-mDEX (2.5%) group, the concentrations of DEX detected in CSF and plasma were 0.23±0.10 and 0.032±0.014μg, respectively /ml. Compared to the concentration in the perilymph, the reduction was about 110-fold and 800-fold. After 2h, DEX cannot be detected. In the low-load (0.5%) silk fibroin-PEG-mDEX group, DEX could not be detected in CSF and plasma even after 1 hour. These results indicated that the silk fibroin-PEG gel sustained release of DEX in the cochlea for more than 10 days.

c) Assessment of Auditory Brainstem Response (ABR)

method:

In order to evaluate the safety of silk fibroin-PEG-mDEX gel and the effect of its application in the course of auditory surgery, hearing function was evaluated by ABR test at 4 time points: before surgery, 1 day after surgery, and 4 days after surgery , and 14 days after surgery. Twelve guinea pigs were divided into two groups: operation control group (no drug, n=6) and silk fibroin-PEG-mDEX gel group (n=6). ABR was analyzed at specific frequencies (4K, 8K, 16K, 24K) using a TDT-II recording system (Alachua, FL, USA). The experimental animals were anesthetized by intraperitoneal injection of 1% pentobarbital (35 mg/kg). The experimental animals were placed on electric blankets (38°C) and tested in a soundproof room. Three needle electrodes were implanted subcutaneously to measure brainstem activity: one implanted behind the ear (reference electrode), one implanted at the apex of the skull (working electrode), and one implanted on the opposite side of the hind leg (ground electrode). Evoked potentials were band-pass filtered at 100-300 Hz, averaging 512 times. At each given frequency, the tone burst intensity starts at 90dB SPL and tapers off by 5dB until it reaches the threshold. The ABR threshold is positioned at the lowest intensity reproducible Band III that can be collected.

result:

The variation of the threshold with the sound burst intensity in the experiment is shown in Fig. 4A. In the surgical control group, hearing function was not affected at all time points (preoperative, 1, 4, 14 days) and at any frequency (Fig. 4B). In the silk fibroin-PEG-mDEX high-dose (2.5%) gel group (Figure 4C), there was no change in hearing in the 4kHz and 8kHz frequency bands, and no threshold elevation occurred. In the 16K and 24K frequency bands, compared with the preoperative threshold level, there was a small transient hearing loss after gel injection, and the threshold value increased by 5-15dB SPL within 1 day (39.2±6.6vs 30.8±5.8at 16k Hz; 45.0 ±7.1 vs 34.2±6.6 at 24k Hz, p<0.01). This phenomenon of hearing threshold elevation began to weaken on the 4th day (37.5±5.2vs 30.8±5.8at 16k Hz; 41.7±7.5vs 34.2±6.6at 24k Hz, p<0.01), and completely disappeared on the 14th day (34.2±5.8vs 30.8±5.8at 16k Hz; 35.8±5.8vs 34.2±6.6at 24k Hz, p>0.05), indicating that the effect of gel injection on hearing function is temporary and will gradually disappear with the degradation of the gel and the release of the drug.

d) Histological evaluation

method:

After the test animals were killed, the cochlea was quickly separated and perfused with PBS containing 4% paraformaldehyde, and then soaked in fixative at 4°C overnight. To obtain the entire Organ of Corti, the basement membrane and Organ of Corti were carefully dissected apart under a microscope. Rhodamine-phalloidin (Rhodamine phalloidin, 1:100) stained ear hair cells to identify cochlear hair cells. Each cochlea was examined separately under a Leica TCS SPE microscope. Three randomly selected sections of 200 μm were counted for each cochlea. The percentage of active hair cells is obtained by dividing the number of active hair cells by the total number of hair cells (the number of active cells plus inner hair cells). Cochlear slices were prepared by immersing the cochlea and middle ear in 0.1M EDTA for 14 days to decalcify, and sliced along the longitudinal axis of the cochlea with a thickness of 7 μm, stained with H&E, and observed with an optical microscope.

result:

Histological evaluation of the middle and inner ear began on day 10 after surgery. The analysis focused on possible inflammation in the tympanum, round window niche, round window membrane, and scala tympani. The effects of silk fibroin-PEG-mDEX gel on the production of the organ of Corti and the spiral ganglion were also evaluated. From the results, no obvious severe inflammatory reaction (effusion, edema, fibrosis, etc.) appeared in all groups. A small amount of inflammatory cells and blood cells appeared in the scala tympani adjacent to the round window membrane, but from the comparison of the number of inflammatory cells in the experimental group (silk fibroin-PEG-mDEX gel, Figure 5B) and the control group (untreated ear, Figure 5A), There is also no significant difference in their values. Compared with the control group (mDEX solution group, FIG. 5C ), the gel administration group ( FIG. 5D ) did not produce any changes in the size, morphology and number of ganglion cells.

The possibility of loss of inner and outer hair cells after injection of silk fibroin-PEG-mDEX gel was investigated by microscopically observing the overall surface histology of the cochlea. Quantitative counting analysis in Table 2 showed that both the treatment group and the blank group lost some outer hair cells, but there was no significant difference (p<0.01). As shown in Figure 6, there was no change in the inner hair cells of all groups (A: control group; B: treatment group).

Table 2 Percentage of inner hair living cells and outer hair living cells on the 10th day after injection of silk fibroin-PEG-mDEX gel (n=3)

e) Degradation of silk fibroin-PEG-mDEX gel in vivo

method:

Animals were sacrificed 1h, 4, 7, 10, 14, 21 days after gel injection, and auditory vesicles were obtained. The auditory bulb was opened to expose the cochlea, and the silk fibroin-PEG-mDEX gel remaining on the round window niche was observed through a microscope.

result:

After isolating the vesicles, we monitored the biodegradation properties of the silk fibroin-PEG-mDEX gels by time-lapse microscopy. As shown in Figure 7, it represents the degradation of silk fibroin-PEG-mDEX gel 1 hour, 4 days, 7 days, 10 days, 14 days and 21 days after gel injection, and the red arrow indicates the position of the round window niche and residual gel. It can be found that a white stable gel state can be observed in the round window niche 1 hour after the injection of the gel. On the 10th day, the volume of the gel changes, and the gel begins to degrade. The gel volume decreases until the 14th day. At 21 days, almost no gel residue was visible in the round window niche.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (10)

1. A controlled-release and slow-release silk fibroin gel preparation for the treatment of inner ear diseases, characterized in that: it includes a preparation body, the preparation body includes a carrier in a gel state and a drug dispersed or adsorbed in the carrier, the The medicine is hormone medicine for treating inner ear diseases, and the carrier is silk fibroin gel. 2. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 1, characterized in that: the silk fibroin gel is made of silk fibroin solution by inducing gelation. 3. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 2, characterized in that: the method of inducing gelation includes pH value change method, ultrasonic oscillation method, electrophoresis method, horseradish treatment, etc. Oxidase-hydrogen peroxide blending method, and low molecular weight polyethylene glycol blending method. 4. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 3, characterized in that: the way of inducing gelation is low molecular weight polyethylene glycol blending method. 5. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 2, characterized in that: the main body of the preparation is composed of the drug in the form of an aqueous solution or in the form of insoluble microspheres and silk fibroin. The protein solution is made by inducing gelation after mixing. 6. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 1, characterized in that: the concentration of silk fibroin in the main body of the preparation is 1-30%. 7. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 6, characterized in that: the concentration of silk fibroin in the main body of the preparation is 7.5-15%. 8. The controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to claim 1, characterized in that: the hormone drugs include dexamethasone, betamethasone, prednisolone, methylprednisolone One or more of pine dragon, deoxycorticosterone, 11-deoxycorticosterone, 18-H-11-desoxycorticosterone, beclomethasone, triamcinolone acetonide and their chemically synthesized derivatives. 9. A method for preparing a controlled-release and slow-release silk fibroin gel preparation for treating inner ear diseases according to any one of claims 1-6, characterized in that: comprising the following steps: 1) Mixing: mix the hormone drugs for the treatment of inner ear diseases with the silk fibroin solution in the form of aqueous solution or in the form of insoluble microspheres to obtain a drug suspension; 2) Gelling: The drug suspension is made into the main body of the preparation by inducing gelation. 10. The preparation method according to claim 9, characterized in that: in the mixing step, the hormone drug is uniformly mixed with the silk fibroin solution in the form of insoluble microspheres before coating treatment, and the coating treatment includes the following step: a) suspending the hormone drug microspheres in a low-concentration silk fibroin solution, mixing them uniformly, and ultrasonically treating them for a period of time to disperse the agglomerated microspheres; b) The solution obtained in step a) is vibrated, centrifuged, supernatant removed, washed with water and dried to obtain hormone drug microspheres coated with silk fibroin.