Preparation method and application of controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging

Abstract

The present invention relates to a method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging and their application, belonging to the field of pharmaceutical engineering drug preparations. The present invention's preparation method produces controlled-release enzyme-loaded microspheres loaded with cathepsin D and cathepsin K, combining the advantages of microsphere controlled release with the therapeutic strategy of cathepsins. The resulting microspheres have the effect of preventing and treating skin photoaging, representing a major breakthrough in the research and development of specific drugs for preventing and treating photoaging and related skin lesions. The present invention can provide a new strategy for developing better small-molecule drugs for the prevention and treatment of photoaging and related skin diseases, and has important theoretical and practical value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical engineering drug preparations, and particularly relates to a preparation method and application of controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging. Background Art

[0002] As we all know, skin photoaging is the most important manifestation of exogenous skin aging. It is also closely related to a variety of serious skin diseases, such as light-related immune and inflammatory skin diseases, and can lead to skin tumors. For example, the incidence of various skin malignancies such as basal cell carcinoma and squamous cell carcinoma is significantly increased in photoaged areas of the human body. The onset of photodermatoses (polymorphic light eruption, chronic actinic dermatitis) and photosensitive skin diseases (lupus erythematosus, dermatomyositis) is also related to skin photoaging. Therefore, in recent years, the study of photoaging has not only become a hot topic of research in multiple disciplines at home and abroad, especially in the context of increasingly serious environmental pollution and the rising incidence of skin tumors, but has also become a popular and cutting-edge research topic in the field of dermatology.

[0003] Although great progress has been made in the research and application of intervention means and methods for photoaging, such as the development and application of retinoids, various antioxidants that scavenge reactive oxygen free radicals (ROS), chemical peels, intense pulsed light, radio frequency, fractional laser and other drugs, chemical components, and physical treatment methods, they have not been widely used due to corresponding side effects and lack of specificity.

[0004] Currently, the mechanisms underlying skin photoaging remain unclear and require further investigation. Studies have shown that the expression of cathepsin D and cathepsin K (cathepsin D and cathepsin K) is altered in chronically photodamaged skin and is closely associated with skin condition. Although cathepsin D gel has been prepared and demonstrated its effectiveness in humans, the instability and susceptibility of these enzymes to inactivation during both in vitro and in vivo application have hindered further investigation into their mechanisms in preventing and treating skin photoaging, limiting their application in this area. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and provide a preparation method and application of controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging, comprising the following steps:

[0008] (1) Add gelatin (Gel) and oxidized sodium alginate (OSA) to a buffer solution and stir evenly to obtain an OSA / Gel mixed solution; then add a CaCO3 microsphere suspension and sodium alginate (SA) to the obtained OSA / Gel mixed solution and stir evenly to obtain a gel solution;

[0009] (2) dispersing the enzyme in a buffer solution to obtain an enzyme solution, adding the obtained enzyme solution to the gel solution obtained in step (1), and shaking to obtain an enzyme-loaded gel solution; the enzyme is cathepsin D and / or cathepsin K;

[0010] (3) adding the enzyme-loaded gel solution obtained in step (2) to the oil phase, emulsifying to obtain an emulsion, and then stirring the obtained emulsion in an ice bath to form a gel to obtain microspheres;

[0011] (4) The microspheres obtained in step (3) are centrifuged and washed in an ice bath; the washed microspheres are stirred in a calcium salt solution to completely cross-link the sodium alginate.

[0012] The preparation method of the present invention produces controlled-release enzyme-loaded microspheres loaded with cathepsin D and cathepsin K, combining the advantages of microsphere controlled release with the therapeutic strategy of cathepsins. The resulting microspheres have the effect of preventing and treating photoaging of the skin. The microspheres of the present invention are first obtained by low-temperature gelation of gelatin under stirring, followed by cross-linking of alginate with calcium ions. Low-temperature gelation of gelatin under stirring makes the microspheres more spherical, while calcium ion cross-linking further stabilizes the microsphere morphology, preventing the microspheres from fusing together during centrifugal sedimentation. The microspheres prepared by the emulsion method in this application have a small particle size and a large specific surface area, which is conducive to enzyme loading and release. Furthermore, due to the low-temperature setting, the viscosity of peanut oil increases, enhancing its adsorption on the microsphere surface. Further washing is performed to remove the oil phase on the microsphere surface, ensuring that the final enzyme release effect is not affected by the peanut oil on the microsphere surface. In addition, to maximize the activity of the enzyme, the present application uses a buffer solution as the solvent for microsphere preparation. Compared with pure water, this solution component can better maintain enzyme activity.

[0013] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging of the present invention, the buffer solution in step (1) and step (2) is a solution composed of Tris and NaCl with a pH of 7.4 to 8.4.

[0014] Preferably, the buffer solution in step (1) and step (2) is a solution composed of 20 mM Tris and 150 mM NaCl with a pH of 8.0.

[0015] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (1), the CaCO3 microspheres are prepared by stirring and reacting carbonate, casein and calcium salt solution; and the CaCO3 microsphere suspension is obtained by dispersing the CaCO3 microspheres in a solvent.

[0016] Preferably, the solvent of the CaCO3 microsphere suspension is water.

[0017] Preferably, the preparation method of the CaCO3 microspheres of the present invention is as follows: Na2CO3 and casein are dissolved in deionized water to prepare a Na2CO3 / casein solution (filter sterilized); CaCl2 is dissolved in deionized water to prepare a CaCl2 solution (filter sterilized); a 50mM Na2CO3 solution (containing 8mg / mL casein) is added to a beaker and stirred, and an equal volume of an equal concentration of CaCl2 solution is uniformly added and stirred to obtain a CaCO3 suspension; after centrifugation and cleaning, the mixture is added to a buffer solution, ultrasonically resuspended, and frozen for later use. The CaCO3 microspheres are dispersed in water to obtain a CaCO3 microsphere suspension.

[0018] Preferably, the gel solution obtained in step (1) is allowed to stand overnight in a refrigerator at 4°C to ensure that the gel and OSA fully react; and then dissolved in a water bath at 37°C to obtain the gel solution used in step (2).

[0019] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (2), in the enzyme-loaded gel solution, the concentration of Gel is 0.01 g / mL-0.03 g / mL; the mass volume concentration of SA is 0.005 g / mL-0.01 g / mL; the mass volume concentration of OSA is 0-0.01 g / mL; and the concentration of CaCO3 microspheres is 2 mg / mL-4 mg / mL.

[0020] Preferably, in step (2), in the enzyme-loaded gel solution, the mass volume concentration of the Gel is 0.01 g / mL; the mass volume concentration of the SA is 0.005 g / mL; the mass volume concentration of the OSA is 0.005 g / mL-0.01 g / mL; more preferably, the mass volume concentration of the OSA is 0.005 g / mL; and the concentration of the CaCO3 microspheres is 3 mg / mL.

[0021] As a preferred embodiment of the method for preparing the controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging of the present invention, in step (2), the concentration of the enzyme in the enzyme solution is 0.05 mg / mL-0.015 mg / mL. Preferably, the concentration of the enzyme is 0.1 mg / mL.

[0022] Preferably, in step (4), the washing is first performed by washing away the oil phase with an organic solvent A, and then by washing with an organic solvent B miscible with water; the organic solvent A comprises at least one of dichloromethane, pentane, hexane, and cyclohexane; and the organic solvent B comprises at least one of ethanol, n-propanol, and ethylene glycol.

[0023] Preferably, in step (4), the washed microspheres can be filtered through a 70-90 mesh (preferably 80 mesh) filter to remove larger microspheres, and then the filtered microsphere ethanol solution is passed through a 40 μm cell strainer to collect the microspheres; that is, the particle size of the washed microspheres is between 40 μm and 212 μm (70 mesh). In the present invention, the microspheres are first washed with cold dichloromethane to fully remove the peanut oil, and then the dichloromethane is washed away with cold anhydrous ethanol to ensure that the release of the enzyme in the finally obtained enzyme-loaded microspheres will not be affected by the peanut oil and dichloromethane. The temperature of the cold dichloromethane or cold anhydrous ethanol is -4°C to 4°C.

[0024] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (4), the solvent of the calcium salt solution is a solution composed of 20 mM Tris and 150 mM NaCl at a pH of 8.0; the concentration of calcium ions in the calcium salt solution is 0.2 mol / L-0.3 mol / L; preferably, the concentration of calcium ions in the calcium salt solution is 0.25 mol / L. Preferably, the calcium salt is calcium chloride.

[0025] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (1), the concentration of CaCO3 microspheres in the CaCO3 microsphere suspension is 22.2 mg / mL-44.4 mg / mL; preferably, the concentration of CaCO3 microspheres in the CaCO3 microsphere suspension is 33.3 mg / mL.

[0026] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging of the present invention, in step (1), the temperature during stirring is 37°C-43°C; preferably, the temperature during stirring is 40°C.

[0027] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (2), the shaking time is 5-10 minutes; preferably, the shaking time is 10 minutes. After adding the enzyme, the solution system needs to be shaken to ensure uniform dispersion of the enzyme in the solution, and stirring should not be used, as stirring will affect the gel state and enzyme dispersion.

[0028] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (3), the stirring time is 30 minutes to 60 minutes; and in step (3), sufficient stirring in an ice bath ensures that the emulsion is completely gelled and the microspheres are fixed. The ice bath conditions are conventional experimental operations, and the ice bath temperature is 0°C.

[0029] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to the present invention, in step (3), the oil phase is an edible oil having a viscosity greater than 35 mPa·s at 37° C.; preferably, the oil phase is peanut oil; preferably, the method for adding the enzyme-loaded gel solution to the peanut oil in step (3) is: adding the obtained enzyme-loaded gel solution to the peanut oil via injection, ensuring that the needle is always below the peanut oil liquid surface during addition.

[0030] As a preferred embodiment of the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging of the present invention, in step (4), the stirring time is 3 min-7 min; preferably, the stirring time is 5 min; and the stirring speed is 200 rpm.

[0031] Preferably, the prepared microspheres can be stored in a calcium salt solution or freeze-dried.

[0032] Another object of the present invention is to provide controlled-release enzyme-loaded microspheres prepared by the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging.

[0033] The microspheres produced by the preparation method of the present invention can successfully load enzymes and achieve controlled release of the enzymes, slowly releasing the protease and maintaining a stable protease concentration over a period of time. This ensures the enzymatic activity of cathepsin D and cathepsin K, providing a foundation for in vivo and in vitro studies of cathepsin D and cathepsin K in preventing and treating skin photoaging. The microspheres produced by the present invention are mostly less than 100 μm in size and can be used for subcutaneous injection.

[0034] Another object of the present invention is to provide the use of the controlled-release enzyme-loaded microspheres in preventing and treating skin photoaging.

[0035] Preferably, the application is the application of the controlled-release enzyme-loaded microspheres in preventing and treating chronic photodamage of the skin.

[0036] Preferably, the application is in the preparation of a drug for preventing and treating skin photoaging.

[0037] Preferably, in the application, the dosage of the controlled-release enzyme-loaded microspheres is 1 μg / mL-10 μg / mL.

[0038] Through research, the present invention found that two types of controlled-release enzyme-loaded microspheres have certain effects in resisting chronic photodamage in cell and mouse experiments. Among them, cathepsin D-loaded microspheres may reduce the effects of UV radiation on chronic photodamage to the skin through antioxidant effects; cathepsin K-loaded microspheres may reduce the effects of UV radiation on chronic photodamage to the skin by participating in the production and repair of extracellular matrix.

[0039] The present invention has the following beneficial effects: sodium alginate composite biomicrospheres capable of controlled release of active cathepsin D and cathepsin K are prepared, the role of the controlled-release cathepsin D and cathepsin K sodium alginate microspheres in preventing and treating skin photoaging is verified, and the mechanism thereof in preventing and treating skin photoaging is further studied, which is an important breakthrough in the research and development of specific drugs for preventing and treating photoaging and related skin lesions. This research can provide a new strategy for developing better small molecule drugs for the prevention and treatment of photoaging and related skin diseases, and has important theoretical and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is an optical microscope image of the microspheres prepared in Example 1;

[0041] Figure 2 Scanning electron micrograph of the microspheres prepared in Example 1;

[0042] Figure 3 This is the particle size distribution diagram of the enzyme-loaded microspheres in Example 1;

[0043] Figure 4 This is the result diagram of the cell experiment to detect the appropriate concentration of microspheres;

[0044] Figure 5 Figure 1 is a diagram of the status of mice injected subcutaneously with 50 μg / mL of the microspheres of Example 1 (CTSD group) and Example 2 (CTSK group);

[0045] Figure 6 To establish a chronic light-damaged mouse model, skin wrinkles were observed in mice 7 days after subcutaneous injection of medium-dose (10 μg / mL) and low-dose (1 μg / mL) microspheres.

[0046] Figure 7 To establish a chronic light-damaged mouse model, the effect of subcutaneous injection of 10 μg / mL microspheres from Example 1 on paraffin sections 7 days later was observed;

[0047] Figure 8 The results of HYP content test in the medium-dose group after UV irradiation and microsphere injection for 8 weeks;

[0048] Figure 9 This is the enzyme content release curve when the CTSD-loaded microspheres in Example 1 are co-cultured with cells;

[0049] Figure 10 This is the enzyme content release curve when the CTSK-loaded microspheres in Example 2 are co-cultured with cells;

[0050] Figure 11 Results of flow cytometric cell cycle analysis performed simultaneously with UVA irradiation and co-culture of microspheres and HDFs;

[0051] Figure 12 The ROS (reactive oxygen species) content test results of each group were performed simultaneously with UVA irradiation and co-culture of microspheres and HDFs;

[0052] Figure 13 UVA irradiation and co-culture of microspheres and HDFs were performed simultaneously. SA-β-Gal flow cytometry was used to determine the senescence of cells in each group.

[0053] Figure 14 Fluorescence microscopy images of cells during simultaneous UVA irradiation and co-culture of microspheres and HDFs;

[0054] Figure 15 The results of flow cytometry analysis of cell cycle when HDFs were co-cultured with microspheres 14 days after UVA irradiation.

[0055] Figure 16 The results of ROS (reactive oxygen species) content test in each group after UVA irradiation of HDF for 14 days and then co-cultured with microspheres;

[0056] Figure 17 SA-β-Gal flow cytometry was used to determine cell senescence in each group when HDFs were irradiated with UVA for 14 days and then co-cultured with microspheres.

[0057] Figure 18 This is a fluorescence microscopy image of cells after UVA irradiation of HDF for 14 days and then co-cultured with microspheres;

[0058] Figure 19 To establish a chronic light-damaged mouse model and then inject microspheres, paraffin sections of the mouse back skin tissue were obtained and stained with HE, Masson, and resorcinol elastic fiber, as well as ROS fluorescence staining of frozen sections.

[0059] Figure 20 This is the result of HYP content detection in the experiment of establishing a chronic light-damaged mouse model and then injecting microspheres;

[0060] Figure 21 To construct a chronic light damage model, the back skin of mice was subcutaneously injected with microspheres, and photos were taken for wrinkle scoring, paraffin sections of mouse tissues, and immunofluorescence detection results. DETAILED DESCRIPTION

[0061] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0062] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0063] Example 1

[0064] A method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging comprises the following steps:

[0065] (1) Add 111 mg of sterilized Gel and 55.5 mg of OSA to 9.6 mL of buffer solution and dissolve at 600 rpm in a 40°C water bath to obtain 9.6 mL of OSA / Gel solution; then add 55.5 mg of sterilized SA and 1 mL of 33.3 mg / mL CaCO3 microsphere suspension to the obtained OSA / Gel solution and dissolve at 600 rpm to obtain 10.6 mL of SA / OSA / Gel / CaCO3 gel solution. The solution is then placed in a 4°C refrigerator overnight to ensure that the Gel and OSA react fully.

[0066] (2) Disperse CTSD (cathepsin D) in a buffer solution to obtain an enzyme solution with an enzyme concentration of 0.1 mg / mL; add 0.5 mL of the obtained enzyme solution to a SA / OSA / Gel / CaCO3 solution dissolved in a 37°C water bath and shake for 10 minutes to ensure that the enzyme is evenly dispersed in the solution to obtain an enzyme-loaded gel solution;

[0067] (3) Take 30 mL of peanut oil in a 100 mL beaker, place a 3 cm long magnetic rod, place it in a 37 ° C water bath environment, and perform magnetic stirring at a speed of 600 rpm. Slowly add 3 mL of the enzyme-loaded gel solution obtained in step (2) to the peanut oil through a syringe, and ensure that the needle is always below the oil surface during the addition. After the addition is completed, emulsify for 15 minutes; then place the emulsion system in an ice bath and continue to stir at a speed of 600 rpm for 30 minutes to ensure that the emulsion droplets are completely gelled to obtain microspheres;

[0068] (4) The microspheres obtained in step (3) were transferred to a 50 mL centrifuge tube and centrifuged at 1000 rpm for 5 min at 10°C to remove the upper oil phase; then, the microspheres were washed three times with excess cold DCM in an ice bath to fully remove the peanut oil; 40 mL of cold anhydrous ethanol was then added to the microspheres containing DCM, stirred for 2 min, and filtered through an 80-mesh filter to remove larger microspheres. The filtered microsphere ethanol solution was then passed through a 40 μm cell strainer to collect the microspheres; then, the microspheres were rinsed six times with 1 mL of cold anhydrous ethanol solution to ensure that the DCM was completely removed; the washed microspheres were placed in 10 mL of 0.25 M CaCl2 buffer and slowly stirred at 200 rpm for 5 min to ensure that the sodium alginate in the microspheres was completely cross-linked, thereby obtaining CTSD-loaded microspheres; the microspheres were stored in 5 mL of 0.25 M CaCl2 buffer.

[0069] The buffer solution in step (1) and step (2) is a solution composed of 20 mM Tris and 150 mM NaCl with a pH of 8.0.

[0070] In step (1), the preparation method of CaCO3 microspheres is as follows: 0.106g Na2CO3 and 160mg casein are dissolved in 20mL deionized water to prepare a 50mM / 8mg / mL Na2CO3 / casein solution (filter sterilized); 0.111g CaCl2 is dissolved in 20mL deionized water to prepare a 50mM CaCl2 solution (filter sterilized); 20mL of a 50mM Na2CO3 solution (containing 8mg / mL casein) is added to a 100mL beaker, stirred at 600rpm, and 20mL of a 50mM CaCl2 solution is evenly added. After stirring for 20min, 40mL of a 2.5mg / mL CaCO3 suspension is obtained; after centrifugation at 4000rpm for 5min, the supernatant is removed. After ultrasonic cleaning with pure water (autoclaved), the suspension is centrifuged at 4000rpm for 5min, the supernatant is removed, and this is repeated twice. Finally, add 3 mL of buffer, resuspend by ultrasonication, and freeze at 4°C until use. Disperse the CaCO3 microspheres in water to obtain a CaCO3 microsphere suspension.

[0071] Example 2

[0072] The preparation method of enzyme-loaded microspheres in this example differs from that in Example 1 only in that, in step (2), an equal amount of CTSK (cathepsin K) is used to replace CTSD. The rest is the same as in Example 1 to prepare CTSK-loaded microspheres in this example.

[0073] Example 3

[0074] The preparation method of the enzyme-loaded microspheres of this embodiment differs from that of Example 1 only in that the buffer solution used in steps (1) and (2) is a solution of Tris and NaCl with a pH of 7.4. The rest is the same as in Example 1 to prepare the CTSD-loaded microspheres of this embodiment.

[0075] Example 4

[0076] The preparation method of the enzyme-loaded microspheres in this example differs from that in Example 2 only in that the buffer solution used in the preparation is a solution composed of Tris and NaCl with a pH of 8.4. The rest is the same as in Example 2 to prepare the CTSK-loaded microspheres in this example.

[0077] Example 5

[0078] The preparation method of enzyme-loaded microspheres in this embodiment differs from that in Example 1 only in that, in step (1), the amount of Gel added is 333 mg, the amount of OSA added is 111 mg, the amount of SA added is 111 mg, and the concentration of CaCO3 microspheres in the CaCO3 microsphere suspension is 44.4 mg / mL; the rest is the same as in Example 1, and the CTSD-loaded microspheres in this embodiment are prepared.

[0079] Example 6

[0080] The preparation method of enzyme-loaded microspheres in this example differs from that in Example 5 only in that, in step (2), an equal amount of CTSK (cathepsin K) is used to replace CTSD. The rest is the same as in Example 5 to prepare CTSK-loaded microspheres in this example.

[0081] Example 7

[0082] The preparation method of the enzyme-loaded microspheres of this embodiment differs from that of Example 1 only in that, in step (1), the concentration of the CaCO3 microspheres in the CaCO3 microsphere suspension is 22.2 mg / mL; in step (2), the enzyme concentration in the enzyme solution is 0.05 mg / mL; the rest is the same as in Example 1, and the CTSD-loaded microspheres of this embodiment are prepared.

[0083] Example 8

[0084] The preparation method of enzyme-loaded microspheres in this example differs from that in Example 7 only in that, in step (2), an equal amount of CTSK (cathepsin K) is used to replace CTSD. The rest is the same as in Example 7 to prepare CTSK-loaded microspheres in this example.

[0085] Example 9

[0086] The preparation method of enzyme-loaded microspheres in this embodiment differs from that in Example 1 only in that, in step (1), the concentration of CaCO3 microspheres in the CaCO3 microsphere suspension is 44.4 mg / mL; in step (2), the enzyme concentration of the enzyme solution is 0.015 mg / mL; the rest is the same as in Example 1, and the CTSD-loaded microspheres in this embodiment are prepared.

[0087] Example 10

[0088] The preparation method of enzyme-loaded microspheres in this example differs from that in Example 9 only in that, in step (2), an equal amount of CTSK (cathepsin K) is used to replace CTSD. The rest is the same as in Example 9 to prepare CTSK-loaded microspheres in this example.

[0089] Comparative Example 1

[0090] The preparation method of the microspheres of this comparative example differs from that of Example 1 only in that, in step (1), the amount of the buffer solution is 10.1 mL, and in step (2), CTSD is not added. The rest is the same as in Example 1, and blank microspheres of this comparative example are prepared.

[0091] Comparative Example 2

[0092] The preparation method of the microspheres in this comparative example differs from that in Example 1 only in that deionized water is used to replace the buffer solution during preparation. All other steps are the same as in Example 1 to prepare the CTSD-loaded microspheres in this comparative example. The enzyme-loaded microspheres prepared in this comparative example have reduced enzyme activity and poor sustained-release effect.

[0093] Comparative Example 3

[0094] The preparation method of the microspheres in this comparative example differs from that in Example 1 only in that the buffer solution used in the preparation is 1×PBS solution, and the rest is the same as in Example 1 to prepare the CTSD-loaded microspheres in this comparative example. The enzyme-loaded microspheres prepared in this comparative example have reduced enzyme activity.

[0095] Comparative Example 4

[0096] The preparation method of the microspheres in this comparative example differed from that in Example 1 only in that, in step (3), the emulsion system was not placed in an ice bath. All other steps were the same as in Example 1, resulting in the CTSD-loaded microspheres of this comparative example. The enzyme-loaded microspheres prepared in this comparative example had poor encapsulation efficiency and low enzyme loading.

[0097] Comparative Example 5

[0098] The preparation method of the microspheres in this comparative example differs from that in Example 1 only in that, in step (1), OSA is not added and an equal amount of SA is used to replace OSA. All other steps are the same as in Example 1 to prepare the CTSD-loaded microspheres in this comparative example. The enzyme-loaded microspheres prepared in this comparative example have poor degradation performance.

[0099] Comparative Example 6

[0100] The preparation method of the microspheres in this comparative example differs from that in Example 1 only in that, in step (1), no calcium carbonate microsphere suspension is added. All other steps are the same as in Example 1, resulting in the CTSD-loaded microspheres of this comparative example. The enzyme-loaded microspheres prepared in this comparative example exhibit poor encapsulation and sustained-release properties, and exhibit low enzyme loading.

[0101] Comparative Example 7

[0102] The preparation method of the microspheres in this comparative example differs from that in Example 1 only in that, in step (2), the mixture is stirred for 10 min instead of being shaken. The remaining steps are the same as in Example 1 to prepare the CTSD-loaded microspheres in this comparative example. The enzyme-loaded microspheres prepared in this comparative example have a significantly lower enzyme loading.

[0103] Comparative Example 8

[0104] The preparation method of the microspheres in this comparative example differed from that in Example 1 only in that, in step (3), soybean oil, having a viscosity of 31.3 mPa·s at 40°C, was used instead of peanut oil as the oil phase. All other steps were the same as in Example 1 to produce the CTSD-loaded microspheres of this comparative example. The enzyme-loaded microspheres prepared in this comparative example had an enlarged particle size and could not be injected subcutaneously.

[0105] Comparative Example 9

[0106] The preparation method of the microspheres in this comparative example differed from that in Example 1 only in that, in step (4), the washing was not performed under ice bath conditions. All other steps were the same as in Example 1, and the CTSD-loaded microspheres of this comparative example were obtained. The enzyme-loaded microspheres prepared in this comparative example had poor encapsulation effect and low enzyme loading.

[0107] Comparative Example 10

[0108] The preparation method of the microspheres in this comparative example differed from that in Example 1 only in that, in step (4), pure water was used instead of dichloromethane for washing, and then pure water was used instead of anhydrous ethanol for washing. All other steps were the same as in Example 1, and the CTSD-loaded microspheres of this comparative example were prepared. The enzyme-loaded microspheres prepared in this comparative example had a high level of residual oil phase on the surface, and the sustained-release effect was reduced.

[0109] Test Example 1

[0110] (1) Characterization of microsphere morphology: The microspheres prepared in Example 1 were observed using an optical microscope and a scanning electron microscope. Figure 1 and Figure 2 As shown in the figure, the microspheres are spherical and have a moderate particle size. Figure 3 As shown, the particle size is concentrated in the range of 40 μm to 100 μm. The morphology of the enzyme-loaded microspheres of Examples 2-10 is similar to that of Example 1, with good encapsulation effect and most of the sizes are below 100 μm.

[0111] (2) Detection of appropriate microsphere concentration

[0112] 1) Construction of in vitro and in vivo chronic light damage models:

[0113] In vitro experiments (cell experiments): Circumcision foreskins of children aged 3-9 years were collected, fibroblasts were isolated, and fibroblasts (HDF) were cultured in DMEM medium. The 3-5 passage cells were frozen. After cell recovery, human skin fibroblasts were irradiated with long-wave ultraviolet (UVA) once a day at a dose of approximately 10 J / cm 2 , a total of 14 days; during the irradiation period, cells were routinely passaged every 5-7 days.

[0114] In vivo experiment (mouse experiment): 5-week-old female Balb / c-nude mice were selected and irradiated with UVA+UVB for 8 weeks, 5 days per week (UVA: 1000mJ / cm 2 / d, UVB: 1 MED / d in the first week, then increase by one MED per week until 4 MED and maintain until 8 weeks).

[0115] 2) Detection of appropriate concentrations of microspheres for in vitro and in vivo applications:

[0116] 1. In vitro experiment (cell experiment): DMEM medium was used to prepare different concentration gradient microsphere solutions (100, 10, 1 ng / mL), and co-cultured with UVA irradiated HDF (human dermal fibroblasts) for 24h and 48h. The cytotoxicity (cell activity) was detected by CCK8. The results showed that Figure 4 middle.

[0117] Figure 4 In the experiment, group C was the blank control group, and the UV group was the control group irradiated with ultraviolet light but without adding microspheres. From the results, it can be concluded that the cell proliferation ability of the 10 μg / mL group was stronger than that of the other two groups.

[0118] 2. In vivo experiment (mouse experiment): Since the effective concentration of HDF requires 10 μg / mL, PBS was used to prepare the microsphere solution. It was prepared and used immediately. Ten points with similar distances on the back of the mouse were selected and the concentrations of 1, 10, 50, and 100 μg / mL were used for subcutaneous injection. The skin of the mouse back was taken, photographed for wrinkle scoring, paraffin sections of the skin tissue were stained with HE, and the HYP (hydroxyproline) content was detected to evaluate the chronic light damage of the mouse skin. The test results are as follows: Figure 5-8 shown.

[0119] The results showed that at 50 and 100 μg / mL, the mice's skin became red, swollen, and ulcerated. Figure 5 The results showed that subcutaneous injection of 50 μg / mL of the microspheres of Example 1 (CTSD group) and Example 2 (CTSK group) caused redness, swelling and ulceration of the mouse skin, followed by skin infection and death of the mice. Figure 6 To establish a chronic light-damaged mouse model, the skin wrinkles of mice were observed after subcutaneous injection of medium-dose (10 μg / mL) and low-dose (1 μg / mL) microspheres for 7 days. The Blank group was the blank microsphere group in comparative example 1. Figure 7 To establish a chronic light-damaged mouse model, the effect of subcutaneous injection of 10 μg / mL microspheres from Example 1 on paraffin sections 7 days later was observed. Figure 8 Figure 3 shows the HYP content in the medium-dose group after 8 weeks of UV irradiation and microsphere injection. Since no significant changes were observed in the 1 and 10 μg / mL groups, the 1 and 10 μg / mL groups were selected for subsequent experiments.

[0120] Test Example 2

[0121] In vitro cell metabolism experiments with enzyme-loaded microspheres:

[0122] HDFs irradiated with UVA were co-cultured with 10 μg / mL microspheres of Example 1 and Example 2 for 1 week, and irradiated with UVA. 100 μL of supernatant was collected every day, and the CTSD and CTSK levels released by the microspheres were detected using an ELISA kit within one week. The results are as follows: Figure 9 (CTSD-loaded microspheres of Example 1) and Figure 10 (CTSF-loaded microspheres of Example 2) This indicates that the enzyme-loaded microspheres of the present invention have a certain sustained-release effect. In addition, the enzyme-loaded microspheres of Examples 3-10 have comparable sustained-release effects to those of Examples 1 and 2.

[0123] Figure 11-14 These are the test results of cell experiments when UVA irradiation and microspheres co-cultured with HDF were performed simultaneously. Figure 11 The results of flow cytometry cell cycle analysis were obtained from Figure 11 It can be seen that co-culture of CTSD-loaded microspheres (Example 1) with HDFs can alleviate the inhibition of cell cycle by UVA, while the CTSK-loaded microspheres (Example 2) and the CTSD-loaded microspheres and CTSK microspheres co-culture group have no obvious effect on alleviating the cell cycle inhibition caused by UVA. Figure 12 The test results of ROS (reactive oxygen species) content in each group are shown in Figure 2. Figure 13 In order to determine the senescence of cells in each group using SA-β-Gal flow cytometry, Figure 12 It was found that co-culture of CTSD-loaded microspheres with HDFs could reduce the production of ROS after UVA irradiation. Figure 13 It can be seen that co-culture of CTSD-loaded microspheres, CTSK-loaded microspheres and HDFs can alleviate cell senescence caused by UVA. Figure 14 Fluorescence microscopy images of cells during simultaneous UVA irradiation and co-culture of microspheres and HDFs.

[0124] Figure 15-18These are the results of a cell experiment where HDFs were irradiated with UVA for 14 days and then co-cultured with microspheres. Figure 15 The results of flow cytometry cell cycle analysis were obtained from Figure 15 It can be seen that co-culture of CTSD-loaded microspheres (Example 1) with HDFs can alleviate the inhibition of cell cycle by UVA, while the CTSK-loaded microspheres (Example 2) and the CTSD-loaded microspheres and CTSK microspheres co-culture group have no obvious effect on alleviating the cell cycle inhibition caused by UVA. Figure 16 The test results of ROS (reactive oxygen species) content in each group are shown in Figure 2. Figure 17 In order to determine the senescence of cells in each group using SA-β-Gal flow cytometry, Figure 16 It was found that co-culture of CTSD-loaded microspheres with HDFs could reduce the production of ROS after UVA irradiation. Figure 17 It can be seen that co-culture of CTSD-loaded microspheres, CTSK-loaded microspheres and HDFs can alleviate cell senescence caused by UVA. Figure 18 Fluorescence microscopy images of HDF cells irradiated with UVA for 14 days and then co-cultured with microspheres.

[0125] Test Example 3

[0126] In vivo subcutaneous injection experiment of enzyme-loaded microspheres in mice:

[0127] 10 μg / mL enzyme-loaded microspheres were injected subcutaneously into mice at a symmetrical position on the back every 1 day. Mouse tissues at the injection site were collected on the 7th day, and paraffin-embedded sections were stained.

[0128] Figure 19-20 To establish a chronic light-damaged mouse model and then inject microspheres, the skin of the mouse back was taken and paraffin sections of the skin tissue were stained with HE, Masson, and resorcinol elastic fiber, and HYP content was detected to evaluate the chronic light-damaged mouse skin. Figure 19 As shown; HYP content test results are as follows Figure 20 shown.

[0129] from Figure 19-20Comprehensive analysis of mouse tissue paraffin sections using HE staining, Masson staining, resorcinol elastic fiber staining, and HYP content analysis revealed that the control group exhibited normal, structurally intact epidermis, with orderly and evenly distributed dermal collagen and elastic fibers, and normal blood vessels. However, after UV irradiation, the epidermis of mice exhibited hyperkeratosis, with thickening of the stratum granulosum and stratum spinosum. Dermal collagen fibers were disorganized and unevenly distributed, capillaries were dilated, and elastic fibers were curled and reduced. In the CTSD and CTSK groups, collagen fiber alignment was restored, with an increase in elastic fibers, particularly in the cathepsin K group. Furthermore, ROS immunofluorescence analysis revealed that mice treated with modeling followed by subcutaneous injection of CTSD microspheres produced less ROS.

[0130] Figure 21 To establish a chronic light damage model, the back skin of mice was subcutaneously injected with microspheres, and photos were taken for wrinkle scoring, paraffin sections of mouse tissues, and immunofluorescence detection results. Figure 21 Hematoxylin and eosin (HE), Masson's, and resorcinol-elastic fiber staining of paraffin sections of mouse tissue revealed that the control group exhibited normal epidermis with intact structure, orderly and evenly distributed dermal collagen and elastic fibers, and normal blood vessels. However, after UV irradiation, the epidermis of mice showed hyperkeratosis, thickening of the stratum granulosum and spinosum, and disorganized and unevenly distributed dermal collagen fibers. Capillaries were dilated, and elastic fibers were curled and reduced. In the CTSD and CTSK groups, collagen fiber arrangement was restored, and elastic fibers increased, with a more pronounced effect in the cathepsin K group.

[0131] The results of Experimental Examples 2 and 3 show that the CTSD-loaded microspheres of the present invention may reduce the effects of chronic photodamage to the skin caused by UV irradiation through antioxidant effects; the CTSK-loaded microspheres may reduce the effects of chronic photodamage to the skin caused by UV irradiation by participating in the production and repair of the extracellular matrix; the effect of using CTSD- and CTSK-loaded microspheres simultaneously is not as good as using them separately, and it is speculated that the two proteins interact with each other; the two protein-loaded microspheres have certain effects on anti-chronic photodamage in cell and mouse experiments, and their related mechanisms and prevention and treatment rules can be further studied by collecting cells and mouse tissues to extract RNA, and detecting the expression of related genes and proteins through qPCR and WB.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging, characterized in that: The following steps are involved: (1) adding gelatin and oxidized sodium alginate to a buffer solution and stirring uniformly to obtain a mixed solution; then adding a CaCO3 microsphere suspension and sodium alginate to the obtained mixed solution and stirring uniformly to obtain a gel solution; (2) dispersing the enzyme in a buffer solution to obtain an enzyme solution, adding the obtained enzyme solution to the gel solution obtained in step (1), and shaking to obtain an enzyme-loaded gel solution; the enzyme is cathepsin D and / or cathepsin K; (3) adding the enzyme-loaded gel solution obtained in step (2) to the oil phase, emulsifying to obtain an emulsion, and then stirring the obtained emulsion in an ice bath to form a gel to obtain microspheres; (4) centrifuging the microspheres obtained in step (3) and washing them in an ice bath; stirring the washed microspheres in a calcium salt solution to completely crosslink the sodium alginate; In the step (1), the CaCO3 microspheres are prepared by stirring a carbonate, casein and a calcium salt solution; and the CaCO3 microsphere suspension is obtained by dispersing the CaCO3 microspheres in a solvent.

2. The method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to claim 1, characterized in that: The buffer solution in step (1) and step (2) is a solution composed of Tris and NaCl with a pH of 7.4 to 8.

4.

3. The method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to claim 1, characterized in that: In the step (2), in the enzyme-loaded gel solution, the concentration of gelatin is 0.01 g / mL-0.03 g / mL; the concentration of sodium alginate is 0.005 g / mL-0.01 g / mL; the concentration of oxidized sodium alginate is 0-0.01 g / mL; and the concentration of CaCO3 microspheres is 2 mg / mL-4 mg / mL.

4. The method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to claim 3, characterized in that: In the step (2), in the enzyme-loaded gel solution, the concentration of gelatin is 0.01 g / mL; the concentration of sodium alginate is 0.005 g / mL; the concentration of oxidized sodium alginate is 0.005 g / mL; and the concentration of CaCO3 microspheres is 3 mg / mL.

5. The method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to claim 1, characterized in that: In the step (2), the concentration of the enzyme in the enzyme solution is 0.05 mg / mL-0.015 mg / mL.

6. The method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to claim 1, characterized in that: In step (4), the solvent of the calcium salt solution is a solution composed of 20 mM Tris and 150 mM NaCl with a pH of 8.0; the concentration of calcium ions in the calcium salt solution is 0.2 mol / L-0.3 mol / L.

7. The method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to claim 1, characterized in that: At least one of the following is met: (1) In step (1), the concentration of CaCO3 microspheres in the CaCO3 microsphere suspension is 22.2 mg / mL-44.4 mg / mL; (2) In step (1), the temperature during stirring is 37°C-43°C; (3) In step (2), the shaking time is 5 min to 10 min; (4) In step (3), the stirring time is 30 min to 60 min; (5) In step (3), the oil phase is an edible oil having a viscosity greater than 35 mPa·s at 37°C; (6) In step (4), the stirring time is 3 min to 7 min.

8. Controlled-release enzyme-loaded microspheres prepared by the method for preparing controlled-release enzyme-loaded microspheres for preventing and treating skin photoaging according to any one of claims 1 to 7.

9. Use of the controlled-release enzyme-loaded microspheres according to claim 8 in the preparation of a drug for preventing and treating skin photoaging.

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