A CAT / Dex-PTMC / HA multifunctional biosphere gel for the treatment of acute hair cell damage

Abstract

The present invention discloses a CAT / Dex-PTMC / HA multifunctional biological microsphere gel for treating acute hair cell damage, comprising: taking 25-35mg of component A, 35-50mg of component B, 25-35mg of component C and 50-100ml of component D respectively, and then inhaling into a medical syringe, and fully and evenly mixing the components through a double female Luer straight-through medical connector, wherein component A is CAT@CaCO3 microspheres, component B is Dex / CaO2-PTMC, component C is Dex-HA, and component D is artificial perilymph. The multifunctional biological microsphere gel can release oxygen and hormones at the same time, and integrates the multi-frequency, multi-location, and short-acting diagnosis and treatment behaviors of oxygen and hormones into one diagnosis and treatment behavior, which is easy to use, and only requires one tympanic injection to complete the treatment, which can save a lot of manpower and material resources.

Description

Technical Field

[0001] The invention relates to the technical field of biological microsphere gel, in particular to CAT / Dex-PTMC / HA multifunctional biological microsphere gel for treating acute hair cell damage. Background Art

[0002] Hair cell damage is a difficult clinical disease. Diseases caused by hair cell damage include tinnitus, sudden deafness, blast deafness, etc. At present, there are two main and most effective treatment methods: hormone therapy and hyperbaric oxygen therapy. Hormone therapy includes systemic and local medications. Systemic side effects caused by systemic medications are large. Local intratympanic injection of hormone drugs (such as dexamethasone) requires daily puncture injection in the tympanic membrane. Hyperbaric oxygen therapy requires daily treatment in a hyperbaric oxygen chamber. The two are treated in different departments (otolaryngology and hyperbaric oxygen department), the process is cumbersome, and it consumes manpower and material resources. Hospitalization is often required, and the patient has to travel between different departments every day. Local medication is highly recommended because of its high local concentration, small systemic side effects, and better effect.

[0003] However, there is currently no treatment method that can combine hormone therapy and hyperbaric oxygen therapy in one treatment process. The development of a biological compound preparation that can release glucocorticoids and oxygen to the affected ear for a long time through a single tympanic injection is an urgent need for diseases related to hair cell damage. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a CAT / Dex-PTMC / HA multifunctional biomicrosphere gel for treating acute hair cell damage to solve the traditional technical problems.

[0005] The present invention is implemented by the following technical solutions:

[0006] A CAT / Dex-PTMC / HA multifunctional biomicrosphere gel for treating acute hair cell damage comprises the following preparation steps:

[0007] Take 25-35 mg of component A, 35-50 mg of component B, 25-35 mg of component C and 50-100 ml of component D respectively, mix them, inhale them into a medical syringe, and fully and evenly mix the components through a double female Luer straight-through medical connector to obtain, wherein component A is (CAT@CaCO3) microspheres, component B is Dex / CaO2-PTMC (dexamethasone / calcium dioxide-polytrimethylene carbonate), component C is Dex-HA (dexamethasone-sodium hyaluronate), and component D is artificial perilymph.

[0008] Furthermore, the preparation steps of the A component are:

[0009] (1) Weigh 12.11 g of tris(hydroxymethyl)aminomethane (Tris) into a 100 ml beaker, add about 40 ml of deionized water, stir thoroughly to dissolve, add concentrated HCl, adjust the pH value of the solution to between 7.35 and 7.45, and then dilute the solution to 100 ml to obtain a Tris-HCl buffer;

[0010] (2) Weigh 400 mg of catalase (CAT) and dissolve it in 40 ml of Tris-HCl buffer to obtain 10 mg / mL free CAT enzyme solution;

[0011] (3) Weigh 16.52 mg of urease and dissolve it in 2 mL of Tris-HCl buffer, and shake until fully dissolved to obtain a urease solution;

[0012] (4) preparing 2 mL of a substrate solution using a Tris-HCl buffer, wherein the substrate solution contains calcium chloride and urea in a mass ratio of 4.9:4, such as 0.392 g of calcium chloride and 0.32 g of urea;

[0013] (5) Take 10 μL of free CAT enzyme solution and 170 μL of substrate solution, add them to 320 μL of urease solution, place it on a vortex oscillator, and oscillate it thoroughly at 3000 r / min for 2 min. After standing for 1.5 h, centrifuge it at 2000 r / min for 3 min. The precipitate is component A.

[0014] Furthermore, in step (5), the following post-processing steps are also included:

[0015] The precipitate was washed three times with deionized water, centrifuged again at 2000 r / min for 2 min, and stored in a refrigerator at 4°.

[0016] Furthermore, the preparation steps of the B component are:

[0017] a. Weigh 30mg Dex, 100mg PTMC, and 10mg-50mg CaO2;

[0018] b. Measure 10 ml of DCM (dichloromethane solvent) into a sample bottle, put 30 mg of Dex, 100 mg of PTMC and CaO2 into the DCM sample bottle (i.e., each DCM sample bottle contains Dex, PTMC and CaO2), and seal it; then, stir with a magnetic stirrer until it is completely dissolved; then, vacuum dry for 72 hours, grind repeatedly until it can pass through a 300-mesh sieve (pore size 48 microns), and store in a refrigerator at 4°.

[0019] Furthermore, in step b, the stirring speed of the magnetic stirrer is 800 rpm.

[0020] Furthermore, the preparation steps of the C component are: weigh 30 mg of Dex and 30 mg of sodium hyaluronate gel powder, mix the two, and store them in a refrigerator at 4°.

[0021] Further, the preparation steps of the D component are as follows: weigh 0.0191g calcium chloride, 0.0171g magnesium chloride, 0.0403g potassium chloride, 0.8006g sodium chloride, 0.0991g glucose and 0.1192g HEPES (4-hydroxyethylpiperazineethanesulfonic acid), dissolve in 100ml deionized water, stir with a magnetic stirrer until completely dissolved, and store in a refrigerator at 4°. The above solutions are prepared in a simulated inner ear environment.

[0022] Furthermore, the stirring speed of the magnetic stirrer is 800 rpm.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The multifunctional bio-microsphere gel of the present invention can release oxygen and hormones simultaneously, integrating the multi-frequency, multi-location, and short-acting treatments of oxygen and hormones into one treatment. It is easy to use and only requires one tympanic injection to complete the treatment, which can save a lot of manpower and material resources.

[0025] 2. The multifunctional bio-microsphere gel of the present invention has a wide range of uses and can be used for diseases related to acute damage to hair cells, such as sudden deafness, acute tinnitus, and blast-induced deafness. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The encapsulation efficiency of CAT at different mass concentrations in Example 1;

[0027] Figure 2 This is a graph showing the loading amount of CAT at different mass concentrations in Example 1;

[0028] Figure 3 This is an evaluation diagram of the oxygen production of the CAT / Dex-PTMC / HA multifunctional biomicrosphere gel in Example 2;

[0029] Figure 4 The real-time concentration curve and cumulative drug release curve of the drug-loaded coating in Example 3;

[0030] Figure 5 This is a graph showing the cell viability of L929 in Example 4. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0032] Example 1 Determination of the immobilization capacity of CAT in component A

[0033] 1. The preparation steps of component A are as follows:

[0034] Weigh 12.11 g of Tris into a 100 ml beaker, add 40 ml of deionized water, stir thoroughly to dissolve, add concentrated HCl, adjust the pH value of the solution to between 7.35-7.45, and dilute the solution to 100 ml to prepare Tris-HCl buffer.

[0035] Weigh 400 mg of catalase (CAT) and dissolve it in 40 ml of Tris-HCl buffer to prepare 10 mg / mL free CAT enzyme solution;

[0036] Weigh 16.52 mg of urease and dissolve it in 2 mL of Tris-HCl buffer, shake until fully dissolved to prepare a urease solution;

[0037] Prepare 2 mL of substrate solution containing 0.392 g of calcium chloride and 0.32 g of urea using Tris-HCl buffer;

[0038] Take 10 μL of free CAT enzyme solution and 170 μL of substrate solution and add them to 320 μL of urease solution. Place the vortex oscillator on a plate and oscillate at 3000 r / min for 2 min. After standing for 1.5 h, centrifuge at 2000 r / min for 3 min to precipitate CAT-immobilized calcium carbonate (CAT@CaCO3) microspheres. Wash them three times with deionized water and centrifuge them again at 2000 r / min for 2 min. Record them as (CAT@CaCO3) microspheres and mark them as component A.

[0039] 2. The encapsulation efficiency and loading capacity of the immobilized enzyme were calculated according to the following formula:

[0040] Encapsulation efficiency = [1-(M sp,CAT -M sp,Urease ) / M Uinitial ]×100% (1)

[0041] Load = [M immobilized,CAT / M CaCO3 ] (2)

[0042] Where M sp,CAT is the mass of CAT in the supernatant; M sp,Urease is the mass of urease in the supernatant; M Uinitial is the total mass of CAT added; M immobilized,CAT is the mass of the CAT being loaded; M CaCO3 is the total mass of CaCO3 particles. All experimental results are the average of 3 repeated experiments.

[0043] 3. The Bradford method (Coomassie Brilliant Blue method) was used to determine the encapsulation capacity of calcium carbonate microspheres for CAT, and the enzyme encapsulation efficiency and loading capacity were measured under different enzyme concentration conditions. Figure 1 and Figure 2 shown.

[0044] The results showed that: (1) When the CAT concentration reached 0.05 mg / mL, the enzyme encapsulation efficiency and loading capacity were 63.64±1.51% and 5.65±0.53 mg / g, respectively. (2) When the CAT concentration reached 0.1 mg / mL, the enzyme encapsulation efficiency and loading capacity were 70.02±1.46% and 9.36±0.90 mg / g, respectively. (3) When the CAT concentration reached 0.15 mg / mL, the enzyme encapsulation efficiency and loading capacity were 98.59±1.04% and 25.25±0.63 mg / g, respectively. (4) When the CAT concentration reached 0.20 mg / mL, the enzyme encapsulation efficiency and loading capacity were 97.82±0.66% and 22.82±0.66 mg / g, respectively. (5) When the CAT concentration reached 0.25 mg / mL, the enzyme encapsulation efficiency and loading capacity were 92.37±1.14% and 20.71±0.71 mg / g, respectively. (6) When the CAT concentration reached 0.30 mg / mL, the enzyme encapsulation efficiency and loading capacity were 89.45±0.86% and 17.12±1.50 mg / g, respectively.

[0045] In summary, as the CAT mass concentration increases, the encapsulation rate gradually decreases, while the loading capacity gradually increases and begins to decrease after reaching the maximum value. It can be seen that when the CAT mass concentration reaches 0.15 mg / mL, the enzyme encapsulation rate reaches 98.59%, and the loading capacity reaches 25.25±0.63 mg / g, which has higher enzyme encapsulation rate and loading capacity than other CAT mass concentrations. Therefore, the mass concentration of CAT was selected as 0.15 mg / mL in subsequent experiments.

[0046] Example 2 Oxygen production test of CAT / Dex-PTMC / HA multifunctional biomicrosphere gel

[0047] In component B, 10 mg, 20 mg, 30 mg, and 50 mg of CaO2 were weighed respectively to prepare components B, and marked as B1, B2, B3, and B4 respectively (the other steps are the same as the preparation steps described in the present invention and will not be repeated here).

[0048] That is, the preparation steps of component B are:

[0049] Weigh 30mg Dex, 100mg PTMC, and mix 10mg, 20mg, 30mg, and 50mg CaO2, respectively. Take 10ml DCM in 4 sample bottles. Put 30mg Dex, 100mg PTMC, and each amount of CaO2 into each DCM sample bottle and seal. Stir with a magnetic stirrer (800rpm) until completely dissolved. After vacuum drying for 72 hours, grind repeatedly until it can pass through a 300-mesh sieve (pore size 48 microns). Record it as Dex / CaO2-PTMC, and mark it as B1, B2, B3, and B4, respectively, and store it in a refrigerator at 4°.

[0050] 35 mg of component A, 30 mg of component C, 100 ml of component D were mixed with 50 mg of components B1, B2, B3, and B4 to prepare microsphere gel No. 1, No. 2, No. 3, and No. 4 for oxygen production test (the preparation steps of component A, component C, and component D are the same as those described in the present invention and will not be repeated here). The test results are as follows: Figure 3 The initial dissolved oxygen content in water was between 63±2μM and 66.67±3.21μM.

[0051] The results showed that: (1) When the action time was 1 hour, the dissolved oxygen content of microsphere gel No. 1, No. 2, No. 3, and No. 4 was measured to be 83±4.36μM, 81.33±5.51μM, 84.33±4.16μM, and 83.33±3.51μM, respectively. (2) When the action time was 4 hours, the dissolved oxygen content of microsphere gel No. 1, No. 2, No. 3, and No. 4 was measured to be 93.67±3.21μM, 91.67±3.79μM, 88.33±7.51μM, and 89±6.56μM. That is, within 4 hours, the dissolved oxygen content produced was close to 30μM. (3) At 24 hours, the dissolved oxygen content of microsphere gel No. 1, No. 2, No. 3, and No. 4 was measured to be 105.67±4.04145μM, 99.33±2.314μM, 104.33±14.36μM, and 103±7.81μM. (4) At two weeks, the dissolved oxygen content of microsphere gel No. 1, No. 2, No. 3, and No. 4 was 100.67±3.79μM, 107±2.65μM, 106±10.58μM, and 105.67±4.041μM. (5) At 4 weeks, the dissolved oxygen content of microsphere gel No. 1, No. 2, No. 3, and No. 4 was 96.33±1.53μM, 94.33±4.041μM, 98.67±3.06μM, and 103.67±5.13μM.

[0052] The 2019 Guidelines for Sudden Deafness in the United States emphasize the importance of oxygen therapy. The guidelines point out two points: (1) If hyperbaric oxygen therapy is used as initial treatment, hyperbaric oxygen combined with glucocorticoid therapy should be used within 2 weeks after the onset of sudden deafness; (2) If hyperbaric oxygen is used as rescue treatment: clinicians can provide hyperbaric oxygen combined with glucocorticoid therapy as rescue treatment within 1 month after the onset of sudden deafness.

[0053] From Figure 3 It can be seen that from 24 hours to nearly one month, the dissolved oxygen content was stably maintained between 94.33±4.041μM and 106±10.58μM. Microsphere gels No. 1, 2, 3, and 4 can fully meet the dissolved oxygen content requirements in the above-mentioned 2019 American Guidelines for Sudden Deafness.

[0054] Example 3 Drug sustained release performance

[0055] Weigh 30 mg of Dex, 100 mg of PTMC, 30 mg of CaO2, and 10 ml of DCM into a sample bottle. Seal the bottle and stir with a magnetic stirrer (800 rpm) until it is completely dissolved. After vacuum drying for 72 hours, grind repeatedly until it can pass through a 300-mesh sieve (pore size 48 microns). Record it as Dex / CaO2-PTMC, mark it as component B, and store it in a refrigerator at 4°.

[0056] The preparation steps of component A, component C and component D are the same as those described in the present invention and will not be repeated here.

[0057] After mixing 25 mg of component A, 50 mg of component B, 25 mg of component C and 100 ml of component D, 100 g was taken and marked as CAT / Dex-PTMC / HA multifunctional biomicrosphere gel No. I. The four components were respectively sucked into medical syringes and fully and evenly mixed through double female Luer straight-through medical connectors.

[0058] After mixing 30 mg of component A, 50 mg of component B, 30 mg of component C and 100 ml of component D, take 100 g and mark it as CAT / Dex-PTMC / HA multifunctional biomicrosphere gel II. Suck them into medical syringes respectively and mix the four components thoroughly and evenly through double female Luer straight-through medical connectors.

[0059] After mixing 35 mg of component A, 50 mg of component B, 35 mg of component C and 100 ml of component D, take 100 g and mark it as CAT / Dex-PTMC / HA multifunctional biomicrosphere gel III. Suck them into medical syringes respectively and mix the four components thoroughly and evenly through double female Luer straight-through medical connectors.

[0060] Weigh 100g of CAT / Dex-PTMC / HA multifunctional biomicrosphere gel No. I, No. II, and No. III respectively and put them into dialysis bags MD44MM (molecular weight cutoff 2000, Viskase, USA). The volumetric flask contains the release medium - 250ml artificial perilymph (APL). Each volumetric flask is placed in a constant temperature shaker at a temperature of T = 37 ± 1 ° C, a speed of n = 100rpm, and is kept in the dark for 12 weeks. Take out 50ml of the predetermined volume of solution at a fixed time point every day and add fresh APL. Sampling is performed at 7 time points, including 8h, 1d (24h), 3d, 7d, 2w, 6w, 8w, and 12w. The extracted samples are filtered through a PVDF filter with a pore size of 0.22μm and analyzed using HPLC (manufacturer Agilent, model 1260). Mobile phase: acetonitrile-water 72:28 (v / v), flow rate 1.0mL / min, UV detection wavelength 240nm, column temperature maintained at 25°C, injection volume 20μl, analysis run time for each sample 30min. Dex standard was prepared as 0.1mg / ml solution with mobile phase, and then diluted into 5 standard solutions: 5mg / L, 10mg / L, 25mg / L, 50mg / L, 100mg / L. The area under the peak was used to calculate the concentration of DXM in the sample.

[0061] Results and Discussion:

[0062] Dexamethasone standard solution was injected into the chromatograph, the peak area was recorded, and the drug concentration was subjected to linear regression analysis to obtain the regression equation: C = 0.035A + 0.194 (r2 = 0.9987). Dex had a good linear relationship in the concentration range of 5 to 100 mg / L. Figure 4 The real-time concentration curve and cumulative drug release curve of the drug-loaded coating show the drug release behavior of the CAT / Dex-PTMC / HA multifunctional biomicrosphere gel. It is known that its in vitro drug sustained release curve consists of a rapid rise and fall period (rapid drug release stage) and a relatively stable period (sustained sustained release stage). The drug release showed a peak drug concentration within 400 hours. The peak drug concentrations of CAT / Dex-PTMC / HA multifunctional biomicrosphere gels I, II, and III were as high as 4.565±0.076, 7.091±0.060, and 9.382±0.209 mg / L. And the sustained drug release time is as long as 3 months. It can fully meet the acute and chronic repair needs after hair cell damage.

[0063] Example 4 In vitro cytotoxicity test

[0064] Weigh 30 mg of Dex, 100 mg of PTMC, 30 mg of CaO2, and 10 ml of DCM into a sample bottle. Seal it and stir it with a magnetic stirrer (800 rpm) until it is completely dissolved. After vacuum drying for 72 hours, grind it repeatedly until it can pass through a 300-mesh sieve (pore size 48 microns). Record it as Dex / CaO2-PTMC, mark it as component B, and store it in a refrigerator at 4°. The preparation of the remaining components A, C, and D is the same as above.

[0065] After mixing 35 mg of component A, 50 mg of component B, and 35 mg of component C with 100 ml of component D, 100 g was taken and marked as CAT / Dex-PTMC / HA multifunctional biomicrosphere gel. The four components were fully and evenly mixed through a double female Luer straight-through medical connector, and the preparation was successful to obtain CAT / Dex-PTMC / HA multifunctional biomicrosphere gel No. III. 100 g of CAT / Dex-PTMC / HA multifunctional biomicrosphere gel No. I of Example 3, No. II of Example 3, and No. III of Example 4 were weighed respectively and loaded into a dialysis bag MD44MM (molecular weight cutoff of 2000, Viskase, USA). After 24 hours, 10 ml of the release medium was extracted and filtered through a PVDF filter with a pore size of 0.22 μm for standby use.

[0066] Fibroblasts (L929) were cultured in DMEM containing 10% v / v fetal bovine serum and 1% v / v penicillin in a 37C cell culture incubator with 5% carbon dioxide. Fibroblasts (50k / ml) were inoculated in 96-well plates and 24-well plates. After 12 hours of culture, CAT / Dex-PTMC / HA multifunctional biomicrosphere gel extract was added to the culture medium. Co-incubated with fibroblasts for 12, 24, and 48 hours, 10μl CCK-8 test was added, and incubated for 2 hours. The absorbance was measured at a wavelength of 450nm using a microplate reader (Biotek, USA).

[0067] Mouse fibroblast L929 is the main cell type of the inner ear and is commonly used in the study of inner ear materials. Therefore, we chose L929 cells to evaluate the biocompatibility of electrode coating materials.

[0068] The cell viability was detected by CCK-8 method (standard error is shown (n=3), ***p<0.001). The test results are shown in Figure 5The cell viability of L929 shown in the figure is greater than 95% after incubation in the CAT / Dex-PTMC / HA multifunctional biomicrosphere gel extract for 12, 24, and 48 hours. Compared with the positive control (cisplatin solution), the difference is statistically significant (p<0.05). This shows that the CAT / Dex-PTMC / HA multifunctional biomicrosphere gel has good biocompatibility and great potential for clinical application.

[0069] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A CAT / Dex-PTMC / HA multifunctional biomicrosphere gel for treating acute hair cell damage, characterized in that: The method comprises the following preparation steps: 25-35 mg of component A, 35-50 mg of component B, 25-35 mg of component C and 50-100 ml of component D are respectively taken and mixed, and then sucked into a medical syringe, and the components are fully and evenly mixed through a double female Luer straight-through medical connector to obtain, wherein component A is CAT@CaCO3 microspheres, component B is Dex / CaO2-PTMC, component C is Dex-HA, and component D is artificial perilymph; The preparation steps of the A component are: (1) Weigh 12.11 g of Tris into a 100 ml beaker, add 40 ml of deionized water, stir thoroughly to dissolve, add concentrated HCl, adjust the pH value of the solution to between 7.35 and 7.45, and then make the solution to 100 ml to obtain a Tris-HCl buffer; (2) Weigh 400 mg of CAT and dissolve it in 40 ml of Tris-HCl buffer to obtain 10 mg / mL free CAT enzyme solution; (3) Weigh 16.52 mg of urease and dissolve it in 2 mL of Tris-HCl buffer, and shake until fully dissolved to obtain a urease solution; (4) preparing 2 mL of a substrate solution using a Tris-HCl buffer, wherein the substrate solution contains calcium chloride and urea in a mass ratio of 4.9:4; (5) Take 10 μL of free CAT enzyme solution and 170 μL of substrate solution, add them to 320 μL of urease solution, place them on a vortex oscillator, oscillate them at 3000 rpm for 2 min, let them stand for 1.5 h, and centrifuge them at 2000 rpm for 3 min. The precipitate is component A. The preparation steps of the B component are: a. Weigh 30mg Dex, 100mg PTMC, and 10mg-50mg CaO2; b. Take 10 ml of DCM and put it into a sample bottle. Put 30 mg of Dex, 100 mg of PTMC and CaO2 into the DCM sample bottle and seal it. Then, stir with a magnetic stirrer until it is completely dissolved. The stirring speed of the magnetic stirrer is 800 rpm. Then, vacuum dry for 72 hours and grind repeatedly until it can pass through a 300-mesh sieve. The preparation steps of the C component are as follows: weigh 30 mg of Dex and 30 mg of sodium hyaluronate gel powder, and mix the two to obtain; The preparation steps of the D component are as follows: weigh 0.0191g calcium chloride, 0.0171g magnesium chloride, 0.0403g potassium chloride, 0.8006g sodium chloride, 0.0991g glucose and 0.1192g HEPES, dissolve them in 100ml deionized water, and stir them with a magnetic stirrer until they are completely dissolved. The stirring speed of the magnetic stirrer is 800rpm.

2. The CAT / Dex-PTMC / HA multifunctional biomicrosphere gel for treating acute hair cell damage according to claim 1, characterized in that: In step (5), the following post-processing steps are also included: The precipitate was washed three times with deionized water, centrifuged again at 2000 r / min for 2 min, and stored in a refrigerator at 4°.

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