Silk fibroin freeze-dried dressing and preparation method thereof

Abstract

The invention discloses a silk fibroin freeze-dried dressing which comprises silk fibroin, mannitol and sodium chloride, a preparation method comprises the steps that raw materials are mixed, stirred and freeze-dried, and the freeze-drying step comprises the steps of pre-freezing, vacuumizing and drying; the crystal network structure of mannitol shortens the redissolution time of the freeze-dried powder, a uniform solution is formed after redissolution, the use convenience is improved, and the use safety risk possibly caused by incompletely dissolved protein particles is reduced; the sodium chloride maintains the ion balance of the protein solution by adjusting the osmotic pressure; and the mannitol is not easy to absorb moisture, so that the long-term stability of the product can be kept in the packaging and storage processes.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a silk fibroin freeze-dried dressing and its preparation method. Background Technology

[0002] Silk fibroin is a natural high-molecular-weight fibrous protein extracted from silkworm silk. It has excellent biocompatibility, mechanical properties and processability, and is widely used in biomedicine, medical aesthetics and skin care and tissue engineering.

[0003] Of the two existing lyophilized silk fibroin products, the first one consists only of silk fibroin. This single-component silk fibroin formulation has limitations. Without lyophilization excipients, the structure of the dried protein sample will unfold, leading to decreased protein stability, loss of activity, aggregation, or denaturation. Morphologically, lyophilization fails to form a uniform, loose, porous structure; a thin, transparent film may only form on the tube wall or bottom, potentially misleading consumers into believing there is insufficient product. Reconstitution may result in inaccurate dosage due to uneven dispersion, and prolonged reconstitution time, coupled with incomplete dissolution, will affect ease of use. Incompletely dissolved protein particles may also pose safety risks.

[0004] In terms of storage, it exhibits poor storage stability, and is more prone to chemical degradation (such as deamidation) and physical instability (such as aggregation) during long-term storage, resulting in a shorter product shelf life and increased storage and transportation costs. Moreover, it is more sensitive to the process; phenomena such as ice crystal growth, pH changes, increased ionic strength, and phase separation during freeze-drying are more likely to cause irreversible damage to the protein, narrowing the production process window and increasing the difficulty and cost of quality control.

[0005] The second type consists of silk fibroin and trehalose, with trehalose acting as a freeze-drying protectant, but it still has shortcomings. During freeze-drying, trehalose may crystallize due to improper process control, disrupting its hydrogen bond network protection mechanism and leading to decreased protein stability. Ideally, trehalose should maintain its amorphous state (glassy state) during freeze-drying to exert its best protective effect. However, trehalose itself has a certain tendency to crystallize. Studies have found that freeze-dried products using trehalose as a protectant easily absorb moisture when exposed to a humid environment during storage. This can cause the freeze-dried cake to shrink, collapse, and potentially turn yellow, even resulting in decreased solubility and clarity, severely affecting the long-term stability and appearance of the product. For example, a study on recombinant carboxypeptidase B freeze-dried products found that samples using trehalose as a protectant exhibited the aforementioned moisture absorption problems after a period of storage.

[0006] For high-concentration protein formulations, the protective effect of trehalose may be limited. High-concentration proteins themselves face challenges such as increased viscosity and prolonged reconstitution time during the freeze-drying process. Even with the addition of protective agents such as trehalose, incomplete dissolution may still occur during the reconstitution of high-concentration freeze-dried protein products. This not only affects the accuracy of dosage, but undissolved protein aggregates may also increase the risk of immune reactions in patients.

[0007] Furthermore, neither of the above two formulations, when reconstituted, provides an isotonic solution to body fluids. Hypertonic dressings can lead to cell dehydration, cell membrane shrinkage, impaired cell function, and even cell death. Hypotonic dressings can cause cells to absorb water, swell, and even rupture, resulting in tissue edema. Osmotic imbalance disrupts the wound microenvironment, affecting cell migration, proliferation, and angiogenesis. Osmotic imbalance can also damage the skin barrier function, making it easier for bacteria to invade.

[0008] Existing formulations suffer from structural protein denaturation, leading to easy collapse of the appearance, poor long-term storage stability, incomplete reconstitution, and non-isotonicity. Therefore, it is urgent to provide a silk fibroin freeze-dried dressing that solves these problems. Summary of the Invention

[0009] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0010] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0011] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a silk fibroin freeze-dried dressing.

[0012] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a silk fibroin freeze-dried dressing, wherein the silk fibroin freeze-dried dressing comprises silk fibroin, mannitol and sodium chloride;

[0013] The protein content is 100-150 mg of silk fibroin, 80-100 mg of mannitol, and 20-25 mg of sodium chloride.

[0014] As a preferred embodiment of the freeze-dried dressing of the present invention, the silk fibroin is 100 mg, mannitol is 100 mg, and sodium chloride is 25 mg.

[0015] The present invention also provides the following technical solution: a method for preparing a silk fibroin freeze-dried dressing, wherein the raw materials are mixed, stirred and then freeze-dried.

[0016] As a preferred embodiment of the preparation method of the present invention, the freeze-drying step is divided into pre-freezing, vacuuming, and finally drying.

[0017] In a preferred embodiment of the preparation method described in this invention, the pre-freezing temperature is -40℃ and the time is 3.5~5h.

[0018] In a preferred embodiment of the preparation method described in this invention, the evacuation temperature is -30°C, the time is 0.5 h, and the pressure is 20 Pa.

[0019] As a preferred embodiment of the preparation method described in this invention, the drying is a gradual heating drying process, wherein the temperature is increased from -40°C to -10°C, from -10°C to 0°C, from 0°C to 10°C, from 10°C to 20°C, from 20°C to 25°C, and from 25°C to 30°C.

[0020] As a preferred embodiment of the preparation method described in this invention, the temperature is raised from -40°C to -10°C at a pressure of 10-20 Pa for a time of 16.5-18.5 h.

[0021] As a preferred embodiment of the preparation method described in this invention, the temperature is increased from 0°C to 10°C, the pressure is 5 Pa, and the time is 3.5 h.

[0022] As a preferred embodiment of the preparation method described in this invention, the temperature is increased from 10°C to 30°C, the pressure is 5 Pa, and the time is 12 h.

[0023] Beneficial effects of this invention:

[0024] This invention uses silk fibroin, mannitol, and sodium chloride as a novel formulation, followed by freeze-drying. The crystalline network structure of mannitol shortens the reconstitution time of the freeze-dried powder, resulting in a homogeneous solution after reconstitution, improving ease of use and reducing the safety risks that incompletely dissolved protein particles may pose. Sodium chloride maintains the ionic balance of the protein solution by regulating osmotic pressure. The addition of mannitol as an excipient in this formulation, along with its low hygroscopicity, helps maintain the long-term stability of the product during packaging and storage. Mannitol generally does not chemically react with proteins, making it a relatively safe excipient that does not introduce new chemical degradation pathways. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0027] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0028] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available.

[0029] Example 1

[0030] This embodiment provides a method for preparing a silk fibroin freeze-dried dressing:

[0031] Pour 5 ml of purified water into a 50 ml beaker and set aside. Weigh 100 mg of silk fibroin and add it to the beaker. Stir to dissolve, then add 100 mg of mannitol and continue stirring to dissolve. Finally, add 25 mg of sodium chloride and stir to dissolve before lyophilizing. The lyophilization parameters are shown in Table 1.

[0032] Table 1

[0033] Step number Step type Temperature (°C) Vacuum (pa) Time (h) 1 Pre-freezing -40 / 2 2 Pre-freezing -40 / 3 3 Empty -30 20 0.5 4 dry -30 10 1.5 5 dry -10 10 1.5 6 dry -10 10 10 7 dry 0 5 0.5 8 dry 0 5 1 9 dry 10 5 0.5 10 dry 10 5 1.5 11 dry 20 5 0.5 12 dry 20 5 1.5 13 dry 25 5 0.5 14 dry 25 5 4 15 dry 30 5 0.5 16 dry 30 5 5

[0034] Example 2

[0035] The difference from Example 1 is that the amount of silk fibroin is 150 mg, otherwise it is the same as Example 1.

[0036] Comparative Example 1

[0037] The difference from Example 1 is that the amount of silk fibroin is 200 mg, but otherwise it is the same as Example 1.

[0038] Comparative Example 2

[0039] The difference from Example 1 is that the amount of silk fibroin is 225 mg, otherwise it is the same as Example 1.

[0040] Remelting and osmotic pressure measurements were performed on Examples 1-2 and Comparative Examples 1-2, and the data are shown in Tables 2 and 3.

[0041] Table 2

[0042] Example 1 Example 2 Comparative Example 1 Comparative Example 2 Remelting time (s) 41 45 46 48 Remelting state Clarity and transparency Clarity and transparency Clarification of trace precipitation Clarification and a small amount of precipitation

[0043] Table 3

[0044] Example 1 Example 2 Example 3 Example 4 osmotic pressure 285 287 288 290

[0045] As shown in Tables 2-3, both Examples 1 and 2 are clear and transparent with an osmotic pressure between 285 and 290. However, Example 1 has a shorter reconstitution time. Therefore, Example 1 is the best embodiment of our invention.

[0046] Example 3

[0047] The difference from Example 1 is that the amount of mannitol is 80 mg, but otherwise it is the same as Example 1.

[0048] Comparative Example 3

[0049] The difference from Example 1 is that the mannitol content is 120 mg, otherwise it is the same as Example 1.

[0050] Comparative Example 4

[0051] The difference from Example 1 is that mannitol is replaced with trehalose, otherwise it is the same as Example 1.

[0052] Comparative Example 5

[0053] The difference from Example 1 is that mannitol is replaced with sucrose, otherwise it is the same as Example 1.

[0054] Comparative Example 6

[0055] The difference from Example 1 is that mannitol is not added; otherwise, it is the same as Example 1.

[0056] Remelting and osmotic pressure measurements were performed on Examples 3 and Comparative Examples 3-6, and the data are shown in Tables 4 and 5.

[0057] Table 4

[0058] Example 3 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Remelting time (s) 50 44 52 56 40 Remelting state Clarity and transparency Clarity and transparency Clarity and transparency Clarity and transparency Clarity and transparency

[0059] Table 5

[0060] Example 3 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 osmotic pressure 263 310 227 229 172

[0061] Tables 4 and 5 show that mannitol is a suitable excipient, resulting in intact appearance and faster resolution.

[0062] Example 4

[0063] The difference from Example 1 is that the sodium chloride content is 20 mg, but otherwise it is the same as Example 1.

[0064] Comparative Example 7

[0065] The difference from Example 1 is that the sodium chloride content is 30 mg, otherwise it is the same as Example 1.

[0066] Comparative Example 8

[0067] The difference from Example 1 is that sodium chloride is not added; otherwise, it is the same as Example 1.

[0068] Remelting and osmotic pressure measurements were performed on Example 4 and Comparative Example 6, and the data are shown in Tables 6 and 7.

[0069] Table 6

[0070] Example 4 Comparative Example 7 Comparative Example 8 Remelting time (s) 42 44 42 Remelting state Clarity and transparency Clarity and transparency Clarity and transparency

[0071] Table 7

[0072] Example 4 Comparative Example 7 Comparative Example 8 osmotic pressure 252 290 108

[0073] Tables 6 and 7 show that sodium glycerol chloride at a dosage of 25 mg has an intact appearance, good reconstitution properties, and an osmotic pressure is isotonic with that of the human body.

[0074] Example 5

[0075] The difference from Example 1 is that the freeze-drying parameters are shown in Table 8, while the rest are the same as in Example 1.

[0076] Table 8

[0077] Step number Step type Temperature (°C) Vacuum (pa) Time (h) 1 Pre-freezing -40 / 0.5 2 Pre-freezing -40 / 3 3 Empty -30 20 0.5 4 dry -30 10 1.5 5 dry -10 10 1.5 6 dry -10 10 10 7 dry 0 5 0.5 8 dry 0 5 1 9 dry 10 5 0.5 10 dry 10 5 1.5 11 dry 20 5 0.5 12 dry 20 5 1.5 13 dry 25 5 0.5 14 dry 25 5 4 15 dry 30 5 0.5 16 dry 30 5 5

[0078] Comparative Example 9

[0079] The difference from Example 1 is that the freeze-drying parameters are shown in Table 9, while the rest are the same as in Example 1.

[0080] Table 9

[0081] Step number Step type Temperature (°C) Vacuum (pa) Time (h) 1 Pre-freezing -40 / 2 2 Pre-freezing -40 / 3 3 Empty -30 20 0.5 4 dry -30 10 1.5 5 dry -10 10 1.5 6 dry -10 10 8 7 dry 0 5 0.5 8 dry 0 5 1 9 dry 10 5 0.5 10 dry 10 5 1.5 11 dry 20 5 0.5 12 dry 20 5 1.5 13 dry 25 5 0.5 14 dry 25 5 4 15 dry 30 5 0.5 16 dry 30 5 5

[0082] Remelting and osmotic pressure measurements were performed on Example 5 and Comparative Example 9, and the data are shown in Tables 10 and 11.

[0083] Table 10

[0084] Example 5 Comparative Example 9 Remelting time (s) 89 94 Remelting state Some remained undissolved. Some remained undissolved.

[0085] Table 11

[0086] Example 5 Comparative Example 9 osmotic pressure 285 285

[0087] All embodiments and comparative examples were evaluated for their appearance, as shown in Table 12.

[0088] Table 12

[0089] Example 1 Complete loose round cake Example 2 Complete loose round cake Example 3 The bottom is wrinkled and cracked. Example 4 Complete loose round cake Example 5 The bottom is wrinkled, and some products are not completely freeze-dried. Comparative Example 1 Complete loose round cake Comparative Example 2 Complete loose round cake Comparative Example 3 Complete loose round cake Comparative Example 4 A complete, loose, round cake with crystals forming. Comparative Example 5 A complete, loose, round cake with crystals forming. Comparative Example 6 Irregular shape, not freeze-dried Comparative Example 7 The bottom is slightly wrinkled Comparative Example 8 Complete loose round cake Comparative Example 9 The bottom is wrinkled, and some products are not completely freeze-dried.

[0090] 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 it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A silk fibroin lyophilized dressing, characterized by: The silk fibroin lyophilized dressing comprises silk fibroin, mannitol and sodium chloride. The silk fibroin is 100-150 mg, the mannitol is 80-100 mg, and the sodium chloride is 20-25 mg.

2. The silk fibroin lyophilized dressing of claim 1, wherein: The silk fibroin is 100 mg, the mannitol is 100 mg, and the sodium chloride is 25 mg.

3. The method of claim 1 or 2, wherein the silk fibroin lyophilized dressing is prepared by the steps of: The raw materials of claims 1-2 are mixed, stirred and then lyophilized.

4. The production method according to claim 3, characterized by: The lyophilization step is divided into pre-freezing, vacuumizing and drying.

5. The production method according to claim 4, characterized by: The pre-freezing temperature is -40℃, and the time is 3.5-5 h.

6. The production method according to claim 4, characterized by: The vacuumizing temperature is -30℃, the time is 0.5 h, and the pressure is 20 pa.

7. The production method according to claim 4, wherein: The drying is stepwise temperature rising drying, from -40℃ to -10℃, -10℃ to 0℃, 0℃ to 10℃, 10℃ to 20℃, 20℃ to 25℃, and 25℃ to 30℃.

8. The production method according to claim 4, wherein: From -40℃ to -10℃, the pressure is 10-20 pa, and the time is 16.5-18.5 h.

9. The production method according to claim 8, characterized by: From 0℃ to 10℃, the pressure is 5 pa, and the time is 3.5 h.

10. The production method according to claim 4, wherein: From 10℃ to 30℃, the pressure is 5 pa, and the time is 12 h.