A dressing and method of making the same

Abstract

The present application relates to a kind of dressings and its preparation method, belong to preparation technical field.The dressing of the present application, component safety, irritability is low, with good drug efficacy and skin care effect, can be used to treat or improve skin damage.

Description

Technical Field

[0001] This invention relates to a dressing and its preparation method, belonging to the field of pharmaceutical technology. Background Technology

[0002] The skin covers the outer surface of the body and is the largest organ in the human body. Skin-related conditions, especially those affecting clearly visible areas such as the face and arms, such as pigmentation and scarring, not only affect physical health but also severely impact the social and psychological well-being of patients and their families. Tuberous sclerosis is a genetic disorder characterized by pigmentation and tumor formation in multiple organ systems, including the skin. It is caused by gene mutations, and common symptoms include skin lesions and tumors. For example, facial angiofibroma is a prominent skin symptom, manifesting as flesh-colored to red or pink papules, mainly distributed in the nasolabial folds, cheeks, and chin, with lesions appearing in early childhood. Various surgical removal methods are available to treat this condition, but most are uncomfortable, especially for children, and often require repeated treatments at periodic intervals to prevent recurrence. Currently, facial angiofibroma can be treated with topical rapamycin, and there are literature reports that topical application of rapamycin can treat skin conditions with minimal or no systemic toxicity.

[0003] Rapamycin, also known as sirolimus, is a white to off-white powder with very low solubility in water (only 2.6 μg / mL). It is readily soluble in benzyl alcohol, chloroform, acetone, and acetonitrile. Furthermore, it is unstable to light and water. These properties have presented challenges for the development of its topical formulations. Currently, the rapamycin topical gel approved for marketing in the United States contains 51% ethanol. A high ethanol content can cause strong skin irritation and easily lead to poisoning. For individuals with ethanol allergies and children, the presence of ethanol is particularly detrimental.

[0004] Gels, transdermal patches, and dressings are all commonly used in the treatment of skin conditions. Ideally, topical medications should be effective, low in toxicity and irritation, and also provide skin care and improve skin condition. However, achieving multiple beneficial properties simultaneously is quite difficult. Therefore, it is both challenging and necessary to develop a safe and effective topical rapamycin formulation that can exert multiple effects for the treatment of facial angiofibroma or other skin conditions. Summary of the Invention

[0005] Based on the existing problems and needs, the present invention aims to provide a medicated dressing that does not contain ethanol, has low irritation, and has good efficacy and skin care effects. It can safely treat, prevent or relieve skin diseases or discomfort symptoms and the skin damage caused by them, such as angiofibroma or its rapid recurrence, skin damage, itching, and scars after angiofibroma surgery, and itching during the skin wound healing period. At the same time, it can improve the skin condition of the affected area and its surrounding area and repair postoperative or damaged skin.

[0006] Therefore, the present invention provides a dressing comprising: a hyaluronic acid-collagen membrane, a γ-cyclodextrin inclusion complex of rapamycin, and a gel of low molecular weight hyaluronic acid and low molecular weight collagen.

[0007] Sodium hyaluronate (HA) is an important polymer widely used in dressings for wounds and skin lesions. Collagen is also a commonly used substance in dressings. Furthermore, both sodium hyaluronate and collagen have multiple functions and can participate in various physiological processes, such as cell proliferation, migration, differentiation, blood clot formation, phagocytic cell activity, fibroblast proliferation, and angiogenesis. They also exhibit good biocompatibility and safety. Therefore, sodium hyaluronate and collagen play a positive and important role in skin wound healing and repair, and in improving skin health.

[0008] The hyaluronic acid salt can be one or more of hyaluronic acid, sodium hyaluronate, and zinc hyaluronate.

[0009] The hyaluronic acid-collagen membrane is a porous sponge that can be cut into shapes suitable for the application site.

[0010] In the membrane, the mass ratio of hyaluronic acid to collagen can be 1:4 to 1:0.25. In some embodiments, the mass ratio of hyaluronic acid to collagen in the membrane is 1:4 to 1:1, which is beneficial for obtaining a more functional sponge and for stimulating and promoting cell proliferation. In some embodiments, the mass ratio of hyaluronic acid to collagen in the membrane is 4:3, which is beneficial for stimulating and promoting cell proliferation and reducing adverse effects such as swelling.

[0011] In the membrane, the molecular weight of the hyaluronic acid salt can be between 2.2 million and 3 million Daltons. In some embodiments, the molecular weight of the hyaluronic acid salt in the membrane is between 2.2 million and 2.8 million Daltons. In some embodiments, the molecular weight of the hyaluronic acid salt in the membrane is between 2.2 million and 2.6 million Daltons. Hyaluronic acid salts within this molecular weight range are beneficial for forming membranes with good mechanical properties; if the molecular weight is too low, it is difficult to form a membrane or the formed membrane is easily damaged by external forces.

[0012] The hyaluronic acid content in the membrane can be 5 mg / g-20 mg / g. In some embodiments, the hyaluronic acid content in the membrane is 10 mg / g-20 mg / g, which is beneficial for forming a membrane with good mechanical properties and suitable swelling. In some embodiments, the hyaluronic acid content in the membrane is 5 mg / g-10 mg / g.

[0013] The collagen content in the membrane can be 2 mg / g-20 mg / g. In some embodiments, the collagen content in the membrane can be 3 mg / g-10 mg / g. In some embodiments, the collagen content in the membrane is 4 mg / g, 5 mg / g, 6 mg / g, 7 mg / g, 7.5 mg / g, 12.5 mg / g, 15 mg / g, or 17.5 mg / g.

[0014] Collagen, with its triple helix structure, can interact with receptors such as integrins on the cell surface, participate in the regulation of extracellular matrix and intracellular signal transduction, and affect cell proliferation, differentiation and apoptosis. It has strong biological activity and will be more conducive to restoring or improving the health of damaged skin.

[0015] The collagen in the membrane, based on a mass ratio, comprises at least 75% having a triple helix structure. In some embodiments, based on a mass ratio, at least 80%, 85%, 90%, or 95% of the collagen has a triple helix structure. Collagen with a triple helix structure is beneficial for film formation and also for restoring or improving the health of damaged skin.

[0016] According to the present invention, the formation of the membrane may include freezing or other suitable methods. In some embodiments, the formation of the membrane includes freezing at -30°C to -20°C for 3 to 14 days, followed by thawing at 20°C to 25°C.

[0017] According to the present invention, the collagen in the dressing may be collagen extracted from cattle, yaks or pigs.

[0018] The collagen in the membrane has a molecular weight of not less than 200,000 Daltons. In some embodiments, the collagen in the membrane has a molecular weight of 250,000 to 500,000 Daltons. In some embodiments, the collagen in the membrane has a molecular weight of 300,000 to 400,000 Daltons.

[0019] By adjusting the ratio of hyaluronic acid salts and collagen as described above, and considering their respective properties, a membrane with suitable properties such as softness, hardness, viscoelasticity, and tensile strength can be obtained.

[0020] The use of a γ-cyclodextrin inclusion complex of rapamycin can improve the stability of rapamycin, while also facilitating its dissolution and enhancing its bioavailability. This inclusion complex, combined with the aforementioned membrane and gel, will contribute to obtaining a dressing with high dissolution and high bioavailability capable of long-term drug release. According to the present invention, the mass ratio of rapamycin to γ-cyclodextrin in the rapamycin γ-cyclodextrin inclusion complex can be 1:120-1:250, which helps to obtain a highly stable, highly dissolution, long-acting release dressing. In some embodiments, the mass ratio of rapamycin to γ-cyclodextrin is 1:120-1:150, which is beneficial for obtaining a stable dressing with good efficacy.

[0021] The molecular weight of the low molecular weight hyaluronic acid salt can be 10,000 to 300,000 Daltons. In some embodiments, the molecular weight of the low molecular weight hyaluronic acid salt can be 10,000 to 200,000 Daltons, or 10,000 to 100,000 Daltons.

[0022] The low molecular weight collagen has a molecular weight not exceeding 50,000 Daltons. Collagen with this molecular weight is easily absorbed and utilized by the skin. In some embodiments, the molecular weight of the low molecular weight collagen is between 1,000 Daltons and 50,000 Daltons. In some embodiments, the low molecular weight collagen is a combination of collagen with a molecular weight of 10,000 Daltons to 50,000 Daltons and collagen with a molecular weight of less than 10,000 Daltons, and the mass ratio of collagen with a molecular weight of 10,000 Daltons to collagen with a molecular weight of less than 10,000 Daltons is 6:1 to 2:1.

[0023] The mass ratio of the low molecular weight hyaluronic acid salt to the low molecular weight collagen can be 2:1 to 1:2. In some embodiments, the mass ratio of the low molecular weight hyaluronic acid salt to the low molecular weight collagen is 1:1 to 1:2. In some embodiments, the mass ratio of the low molecular weight hyaluronic acid salt to the low molecular weight collagen is 1:1.3 to 1:2.

[0024] In the gel, the content of the low molecular weight hyaluronic acid salt can be 5 mg / g-30 mg / g based on the total mass of the gel. In some embodiments, the content of the low molecular weight hyaluronic acid salt in the gel is 15 mg / g-30 mg / g based on the total mass of the gel. In some embodiments, the content of the low molecular weight hyaluronic acid salt in the gel is 5 mg / g-20 mg / g based on the total mass of the gel. In some embodiments, the content of the low molecular weight hyaluronic acid salt in the gel is 10 mg / g or 20 mg / g based on the total mass of the gel.

[0025] The gel with the aforementioned characteristics and properties is beneficial for repairing damaged skin, facilitating drug absorption, and reducing drug irritation.

[0026] In the dressing, the rapamycin content can be between 0.2 mg / g and 4 mg / g, based on the total mass of the dressing. Using an inclusion complex form where rapamycin is encapsulated by γ-cyclodextrin not only improves the stability of rapamycin but also enhances its dissolution and bioavailability. Therefore, the rapamycin content in the dressing can be appropriately adjusted. Lowering the rapamycin content can yield a dressing with suitable dissolution, while increasing the rapamycin content can result in a more potent dressing.

[0027] In some embodiments, the rapamycin content in the dressing is 0.5 mg / g-4 mg / g, or 0.5 mg / g-2 mg / g, or 1 mg / g-2 mg / g, based on the total mass of the dressing.

[0028] In the dressing, the gel content can be 5%-40% based on the total mass of the dressing. In some embodiments, the gel content is 10%-30% based on the total mass of the dressing. In some embodiments, the gel content is 15%, 20%, 25%, or 35% based on the total mass of the dressing. Too low a gel content is detrimental to reducing drug irritation; too high a content will make it difficult to ensure stable release of rapamycin. A gel content at this level is beneficial for the stable release of the drug in the dressing, repairing damaged skin, and reducing drug irritation.

[0029] The dressing provided by this invention uses rapamycin encapsulated by γ-cyclodextrin, which can effectively improve the stability and solubility of rapamycin, facilitating its stability, release, and absorption. Rapamycin can treat angiofibroma; therefore, the dressing can treat and prevent or slow down the rapid recurrence of angiofibroma. The membrane in the dressing is formed by collagen and hyaluronic acid, and does not contain other unsafe or irritating / damaging components, exhibiting extremely high safety and biocompatibility, making it beneficial and suitable for the care of delicate skin. Furthermore, the γ-cyclodextrin inclusion complex of rapamycin is encapsulated by collagen and hyaluronic acid. The formed membrane encapsulation makes the delivery of rapamycin safer and smoother, avoiding burst release. Simultaneously, the gel contained in the dressing is also extremely safe and biocompatible, consisting of hyaluronic acid salts and collagen, suitable for delicate skin care and easily adhered to the intended application site. All components of the dressing have extremely high safety; collagen and hyaluronic acid salts can act as a "skeleton" for tissue regeneration, absorbing fluids excreted from the skin, moisturizing the skin and tissues, and improving the ability of skin fibroblasts to promote collagen regeneration, thus benefiting the recovery of damaged skin or improving its overall health.

[0030] The dressing provided by this invention can be used to treat angiofibroma, prevent or slow the recurrence of angiofibroma, prevent or maintain treatment after surgical treatment of angiofibroma, or care for, improve or repair the skin at the surgical site.

[0031] On the other hand, the present invention also provides a method for preparing the dressing.

[0032] A method for preparing the aforementioned dressing includes the following steps:

[0033] a) Prepare a hyaluronic acid-collagen membrane;

[0034] b) Mix the γ-cyclodextrin inclusion complex of rapamycin with water, optionally removing some of the water to obtain an aqueous mixture of rapamycin;

[0035] c) The hyaluronic acid-collagen membrane was contacted with an aqueous mixture of rapamycin, sonicated, soaked, and freeze-dried under vacuum to obtain a drug-containing membrane;

[0036] d) Prepare a gel containing low molecular weight hyaluronic acid and low molecular weight collagen; and

[0037] e) Mix the medicated film with the gel, and optionally sterilize to obtain the dressing.

[0038] In step b, the mass ratio of the γ-cyclodextrin inclusion complex of rapamycin to water in the resulting aqueous rapamycin mixture can be 1:0.5 to 1:1.5. In some embodiments, the mass ratio of the γ-cyclodextrin inclusion complex of rapamycin to water in the resulting aqueous rapamycin mixture is 1:0.9 to 1:1.2. In some embodiments, the mass ratio of the γ-cyclodextrin inclusion complex of rapamycin to water in the resulting aqueous rapamycin mixture is 1:1, which is more conducive to drug release and absorption.

[0039] In step c, the mass ratio of the hyaluronic acid-collagen membrane to the aqueous rapamycin mixture can be 15:1 to 0.2:1. In some embodiments, the mass ratio of the hyaluronic acid-collagen membrane to the aqueous rapamycin mixture in step c is 14:1 to 1:1. In some embodiments, the mass ratio of the hyaluronic acid-collagen membrane to the aqueous rapamycin mixture in step c is 14:1 to 9:1, or 0.9:1 to 0.2:1, or 7:1 to 1:1. In some embodiments, the mass ratio of the hyaluronic acid-collagen membrane to the aqueous rapamycin mixture in step c is 12:1, 11:1, 10:1, 9:1, or 5:1.

[0040] In step c, the frequency of the ultrasound can be 10Hz-30Hz.

[0041] In step c, the ultrasound duration can be 10 to 60 minutes.

[0042] In some implementations, step c involves ultrasonication for 10 to 60 minutes at 0°C-25°C and 10Hz-30Hz.

[0043] In some embodiments, step c involves soaking at 0°C-25°C for 1-12 hours. In some embodiments, step c involves soaking at 0°C-10°C for 4-12 hours. In some embodiments, step c involves soaking at 0°C-10°C for 6-12 hours. In some embodiments, step c involves soaking at 10°C-25°C for 4-8 hours.

[0044] In step c, the drug-containing film can be obtained by vacuum freeze-drying at -50℃ to -40℃ and 10Pa to 30Pa, and then kept at 0℃ to 5℃ for 2h to 10h.

[0045] The preparation of the hyaluronic acid-collagen membrane may include the following steps:

[0046] 1) Obtain aqueous solutions of hyaluronic acid and collagen, respectively;

[0047] 2) Adjust the pH of the aqueous solution of hyaluronic acid salt to 1.2-1.5, optionally remove the air bubbles, and then mix it with the aqueous solution of collagen at 0℃~10℃. Adjust the pH to 1.2-1.5 again to obtain an acidic mixture.

[0048] 3) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then melt it at 20℃ to 30℃ to obtain hyaluronic acid salt-collagen sponge;

[0049] 4) Transfer the obtained sponge to a dialysis bag and place it in water or an aqueous solution for dialysis until its pH is 6.8-7.6; or soak the obtained sponge in water or an aqueous solution until its pH is 6.8-7.6; remove the sponge and dehydrate it until the water content does not exceed 10 wt% (mass fraction) of the total water content to obtain a neutral sponge; wherein the aqueous solution is a phosphate buffer solution with a pH of 6.9-7.4;

[0050] 5) Optionally, the neutral sponge is sterilized to obtain a hyaluronic acid-collagen membrane.

[0051] The preparation of the gel containing low molecular weight hyaluronic acid and low molecular weight collagen may include the following steps:

[0052] 6) Obtain aqueous solutions of low molecular weight hyaluronic acid and low molecular weight collagen, respectively.

[0053] 7) Adjust the pH of the aqueous solution of low molecular weight hyaluronic acid salt to 0.7-1.1, optionally remove the air bubbles, and then mix it with the aqueous solution of low molecular weight collagen at 0℃~10℃. Adjust the pH to 0.7-1.1 again to obtain an acidic mixture.

[0054] 8) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then melt it at 20℃ to 30℃ to obtain hyaluronic acid salt-collagen gel;

[0055] 9) Soak the obtained gel in water or an aqueous solution until its pH is 6.8-7.6; remove it to obtain a neutral hyaluronic acid salt-collagen gel; wherein the aqueous solution is a phosphate buffer with a pH of 6.9-7.6;

[0056] 10) Optionally, the neutral hyaluronic acid salt-collagen gel is sterilized to obtain a gel of low molecular weight hyaluronic acid salt and low molecular weight collagen.

[0057] In steps 1 and 6, the collagen used can be in solid form or in solution form.

[0058] In steps 3 and 8, the freezing time is preferably 3 to 12 days. In steps 3 and 8, the thawing time can be 4 to 12 hours, or 5 to 8 hours. Using a single freeze-thaw cycle helps to obtain a membrane or gel with suitable viscoelasticity.

[0059] In steps 4 and 9, dialysis can be performed using a membrane with a molecular weight cutoff value between 1,000 Daltons and approximately 10,000 Daltons.

[0060] In steps 4 and 9, the pH can be 7.0-7.4. In some embodiments, the pH in steps 4 and 9 is 7.0 or 7.2.

[0061] In some implementations, in step 4, the sponge is dehydrated until the moisture content does not exceed 5 wt% or 2 wt% of the total moisture content.

[0062] In steps 5 and 10, the sterilization can be performed by: dry heat sterilization, steam heat sterilization, moist heat sterilization, irradiation (β-ray, γ-ray or X-ray sterilization, or electromagnetic (electron beam) sterilization).

[0063] In some embodiments, the sterilization includes irradiation sterilization.

[0064] In some embodiments, the sterilization includes irradiating the resulting membrane or gel with cobalt-60 at a dose not exceeding 15 kGy at -30°C to 0°C.

[0065] In some embodiments, the dressing is prepared using sterile raw materials under aseptic production conditions.

[0066] In some embodiments, a method for preparing the aforementioned dressing includes the following steps:

[0067] a) Preparing a hyaluronic acid-collagen membrane, comprising the following steps:

[0068] 1) Obtain aqueous solutions of hyaluronic acid and collagen, respectively;

[0069] 2) Adjust the pH of the aqueous solution of hyaluronic acid salt to 1.2-1.5, optionally remove the air bubbles, and then mix it with the aqueous solution of collagen at 0℃~10℃. Adjust the pH to 1.2-1.5 again to obtain an acidic mixture.

[0070] 3) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then melt it at 20℃ to 30℃ for 4 to 12 hours to obtain hyaluronic acid salt-collagen sponge;

[0071] 4) Transfer the obtained sponge to a dialysis bag and place it in water or an aqueous solution for dialysis until its pH is 6.8-7.6; or soak the obtained sponge in water or an aqueous solution until its pH is 6.8-7.6; remove the sponge and dehydrate it until the water content does not exceed 10 wt% of the total water content to obtain a neutral sponge; wherein the aqueous solution is a phosphate buffer solution with a pH of 6.9-7.6;

[0072] 5) Optionally, sterilize the obtained neutral sponge to obtain a hyaluronic acid-collagen membrane;

[0073] b) Mix the γ-cyclodextrin inclusion complex of rapamycin with water, optionally removing a portion of the water to obtain an aqueous mixture of rapamycin; in the obtained aqueous mixture of rapamycin, the mass ratio of the γ-cyclodextrin inclusion complex of rapamycin to water is 1:0.5-1:1.5.

[0074] c) The hyaluronic acid-collagen membrane is contacted with an aqueous mixture of rapamycin, and sonicated for 10-60 minutes at 0℃-25℃ and 10Hz-30Hz. It is then soaked at 0℃-25℃ for 1-12 hours, freeze-dried under vacuum at -50℃ to -40℃ and 10Pa-30Pa, and finally kept at 0℃-10℃ for 2-10 hours to obtain a drug-containing membrane. The mass ratio of the hyaluronic acid-collagen membrane to the aqueous mixture of rapamycin is 15:1 to 0.2:1.

[0075] d) The preparation of a gel containing low molecular weight hyaluronic acid and low molecular weight collagen includes the following steps:

[0076] 6) Obtain aqueous solutions of low molecular weight hyaluronic acid and low molecular weight collagen, respectively.

[0077] 7) Adjust the pH of the aqueous solution of low molecular weight hyaluronic acid salt to 0.7-1.1, optionally remove the air bubbles, and then mix it with the aqueous solution of low molecular weight collagen at 0℃~10℃. Adjust the pH to 0.7-1.1 again to obtain an acidic mixture.

[0078] 8) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then melt it at 20℃ to 30℃ for 4 to 12 hours to obtain hyaluronic acid salt-collagen gel.

[0079] 9) Soak the obtained gel in water or an aqueous solution until its pH is 6.8-7.6; remove it to obtain a neutral hyaluronic acid salt-collagen gel; wherein the aqueous solution is a phosphate buffer with a pH of 6.9-7.6;

[0080] 10) Optionally, the resulting neutral hyaluronic acid salt-collagen gel is sterilized to obtain a gel of low molecular weight hyaluronic acid salt and low molecular weight collagen; and

[0081] e) Mix the medicated film with the gel, and optionally sterilize to obtain the dressing.

[0082] The method provided by this invention, by controlling each step and its related conditions, forms cross-links between hyaluronic acid salts and between collagen and hyaluronic acid salts, thereby forming a complex cross-linked network and system, resulting in a membrane with relatively superior performance; the method is operable and controllable and can be used for production.

[0083] According to the present invention, in order to obtain a membrane of a suitable size, the membrane can be cut or otherwise processed to make it a membrane of a suitable size for use.

[0084] Through the above steps, a gel with good moisturizing and water-retaining properties can be obtained, thus creating a dressing with good performance, which is beneficial for the repair of damaged skin and the retention and absorption of drugs. Detailed Implementation

[0085] The terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0086] In this invention, "composed of the following components" or "consistently composed of the following components" means that it may also contain unavoidable impurities and / or water.

[0087] In this invention, "optional" means that it may or may not be present.

[0088] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "some implementations," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0089] In this invention, room temperature refers to ambient temperature, which is between 20°C and 30°C, or between 22°C and 28°C, or 25°C.

[0090] In this invention, the molecular weight of hyaluronic acid salts is expressed as viscosity-average molecular weight.

[0091] In the following examples, bovine collagen with a triple helix structure was used; if dialysis was used, the dialysis solution was changed every 4-5 hours; if soaking was used, the soaking solution was changed every 2-3 hours; the dialysis bag used for dialysis had a molecular weight cutoff of 10,000; the phosphate buffer or PBS buffer was 0.01 mol / L, prepared using disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride, with a pH of 7.0, 7.2, or 7.4.

[0092] Preliminary experimental example:

[0093] At room temperature, 1072 mg of sodium hyaluronate (HA, molecular weight 2.4 million Daltons) was dissolved in 80 g of ultrapure water with mechanical stirring to obtain a sodium hyaluronate solution; at 4 °C, 125 mg of collagen (molecular weight 300,000 Da) was dissolved in 12.5 g of ultrapure water with magnetic stirring to obtain a collagen solution.

[0094] Take 7.5g of the obtained sodium hyaluronate solution, adjust the pH to 3.5 with 0.1mol / L hydrochloric acid solution, then cool to 4℃, add 2.5g of the obtained collagen solution, stir, and a small amount of white filamentous substance is found. Continue stirring for 2 hours, and the white filamentous substance is still present.

[0095] Take 7.5g of the obtained sodium hyaluronate solution, adjust the pH to 1.5 with 0.1mol / L hydrochloric acid solution, then cool to 4℃, add 2.5g of the obtained collagen solution, stir, and no filamentous or other precipitates appear. Continue stirring for 2 hours, and no precipitates appear.

[0096] Example 1

[0097] A) Preparation of sodium hyaluronate-collagen membrane:

[0098] Formula Table 1:

[0099] Sodium hyaluronate 10mg / g 10mg / g 10mg / g 20mg / g 20mg / g 20mg / g 20mg / g Collagen 20mg / g 30mg / g 40mg / g 5mg / g 10mg / g 15mg / g 20mg / g

[0100] The raw materials are: sodium hyaluronate with a molecular weight of 2.4 million Da; and collagen (bovine source) with a molecular weight of 300,000 Da and a triple helix structure.

[0101] Formula Table 2:

[0102]

[0103] According to the formula, dissolve sodium hyaluronate in water for injection and stir at room temperature until completely dissolved. Then, slowly add 1M (mol / L) HCl (hydrochloric acid) to adjust the pH of the solution to 1.5. Centrifuge to remove air bubbles and obtain an acidic sodium hyaluronate solution (formula content). Dissolve collagen in water for injection and stir at room temperature until completely dissolved to obtain a collagen solution (formula content). At 0℃, add 100g of the obtained collagen solution to 100g of the obtained sodium hyaluronate solution, stir evenly, and adjust the pH of the mixed solution to 1.5 with 1M HCl to obtain an acidic mixed solution.

[0104] The obtained acidic mixed solution was poured into a mold and frozen at -20°C for 3 days. Then it was taken out and thawed at 25°C for 5-6 hours to obtain sodium hyaluronate-collagen sponge (if it is not frozen, the sponge cannot be obtained, only the solution is obtained; if the freezing time is less than 3 days, the obtained sponge is easy to break and not easy to form a film).

[0105] The obtained sodium hyaluronate-collagen sponge was transferred to a dialysis bag (molecular weight cutoff of 10,000) and dialyzed in phosphate buffer solution with a pH of 7.2 until the pH of the sponge reached 7.0. The sponge was then removed to obtain a neutral sodium hyaluronate-collagen sponge. Most of the water was squeezed out at room temperature, and then freeze-dried at -20°C until dry to obtain a sodium hyaluronate-collagen membrane with a water content of no more than 1 wt%.

[0106] Sterile membranes can be obtained by irradiating cobalt-60 at 0°C with an irradiation dose of 10 kGy.

[0107] Results: Formulations A1-A7 yielded intact membranes that were not easily torn; the membrane obtained from formulation A8 was more easily torn than the membranes obtained from formulations A9 and A1; formulations A10 and A11 were difficult to form membranes; and the membranes obtained from formulations A12-A14 were more easily torn than the membrane obtained from A1.

[0108] B) Preparation of a gel of low molecular weight sodium hyaluronate and low molecular weight collagen:

[0109] Formula table:

[0110]

[0111] Sodium hyaluronate molecular weight: 100,000 Daltons; Collagen (bovine source): a combination of collagen with a mass ratio of 50,000 Daltons and collagen with a mass ratio of 8,000 Daltons, 4:1.

[0112] According to the formulation table, prepare each group of gels as follows: Dissolve sodium hyaluronate in water for injection, stir at room temperature until completely dissolved, then slowly add 1M HCl to adjust the pH of the solution to 0.9, centrifuge to remove air bubbles, and obtain an acidic sodium hyaluronate solution (formula content); dissolve collagen in water for injection, stir at room temperature until completely dissolved, and obtain a collagen solution (formula content); at 4℃, add 100g of collagen solution to 100g of sodium hyaluronate solution, stir evenly, and adjust the pH of the mixed solution to 1.0 with 1M HCl to obtain an acidic mixed solution; freeze the obtained acidic mixed solution at -20℃ for 3 days, then remove it and thaw it at 25℃ for 5-6 hours, then soak it in phosphate buffer with pH 7.2 until the pH of the gel is 7.0, obtaining sodium hyaluronate-collagen gel. Among them, the gel obtained from formulation group B6 is slightly harder than that from formulation groups B1-B5, but it is still usable.

[0113] C) Preparation of an aqueous solution of rapamycin:

[0114] Formula table:

[0115] Rapamycin 0.20g 0.10g 0.05g 0.05g γ-Cyclodextrin 24.00g 15.00g 10.00g 12.50g water 25.8.00g 15.00g 10.00g 15.00g

[0116] Preparation method 1: According to the formula, add γ-cyclodextrin to purified water, heat to 40°C, stir and mix well, then add rapamycin, stir for 2-3 hours, cool to room temperature, and obtain an aqueous mixture of rapamycin (the obtained mixture can be freeze-dried or spray-dried to obtain a dry product of the γ-cyclodextrin inclusion complex of rapamycin).

[0117] Preparation method 2: According to the formula, add γ-cyclodextrin to purified water, heat to 40℃, and stir to mix well; dissolve rapamycin in a small amount of ethanol (about 1 mL), then add it to the aqueous solution of γ-cyclodextrin, stir for 2-3 hours, cool to room temperature, freeze-dry or spray-dry the resulting mixture to obtain the γ-cyclodextrin inclusion complex of rapamycin; mix the inclusion complex with water at a mass ratio of 1:1.2 to obtain an aqueous mixture.

[0118] D) Preparation of dressing:

[0119] Formula table:

[0120]

[0121] The mass of the aqueous mixture of rapamycin is based on the total mass of rapamycin and γ-cyclodextrin (excluding the mass of water).

[0122] According to the formula, at room temperature, the sodium hyaluronate-collagen membrane prepared according to the aforementioned method is mixed with an aqueous solution of rapamycin, and sonicated for 15 minutes at 15℃-20℃ and 20Hz, and then soaked at 15℃-20℃ for 4-6 hours; then freeze-dried to dryness at -50℃ and 20Pa, and kept at 0℃-5℃ for 2-10 hours to obtain a drug-containing membrane; then add a gel of low molecular weight sodium hyaluronate and low molecular weight collagen, mix on a shaker for 2 hours to obtain a dressing; the dressing can be sterilized by irradiation at 0℃ with a cobalt-60 irradiation dose of 10KGy to obtain a sterile dressing.

[0123] Example 2: Tests and Results

[0124] 1) Liquid absorption rate

[0125] The test was conducted using absorbable PBS buffer (pH = 7.4), and the absorbance rate was calculated using the following formula: Absorption rate = (W 后 -W 前 ) / W 前 *100%; where “W” 后 "W" refers to the weight of the dressing soaked in PBS buffer. 前 "This refers to the weight of the dressing before absorption."

[0126] Results: The absorbance rates of formulation groups D1-D7 in Example 1 were measured to be between 40% and 70%, which showed good absorbance and was beneficial for absorbing exudate.

[0127] 2) Water vapor transmission rate

[0128] The test was conducted in accordance with the method specified in YY / T 0471.2-2004 Test Methods for Contact Wound Dressings Part 2: Water Vapor Transmission Rate of Breathable Membrane Dressings.

[0129] Results: The water vapor permeability of dressings D1-D7 in Example 1 was relatively stable, around 1200 g / (m²). 2 ·h)-1500g / (m 2 Between ·h). Therefore, the dressing can maintain the moisture permeability of the wound area, promote gas exchange and avoid the accumulation of exudate, and can also "lock" moisture in the skin surface to prevent evaporation, thereby better promoting skin repair.

[0130] 3) Skin irritation

[0131] Negative control group: 0.9 wt% physiological saline;

[0132] Positive control group: 0.5 wt% sodium dodecyl sulfate aqueous solution;

[0133] Control group 1: The formula group D6 and its preparation method were followed, but the gel was replaced with PBS buffer (pH=7.4);

[0134] Following the method requirements of GB / T 16886.5-2017, mice were used to test the potential skin irritation of the test samples, with three mice for each sample. Equal weights of sample were applied directly to the same area of ​​skin on the back of the same region (hair was shaved 24 hours prior to application), then secured with a bandage and left on for at least 4 hours, after which the bandage was removed. Reactions to the application site and surrounding skin tissue, including erythema, edema, and necrosis, were observed at 1 hour, 24 hours, and 48 hours.

[0135] Results: No abnormalities such as erythema, edema, or necrosis occurred in the negative control group and samples D1-D7 of the formulation in Example 1; significant redness and swelling occurred in the positive control group, and mild redness and swelling occurred in control group 1. Therefore, the dressing provided by this invention is non-irritating or has low irritation to the skin.

[0136] 4) Moisturizing properties

[0137] Sample group: Formulations obtained from D1-D7;

[0138] Control group 2: According to the ratio of formulation group D7, the aqueous mixture of rapamycin in formulation group C2 was mixed with the mixture of collagen, sodium hyaluronate and water, as well as the aqueous solution of low molecular weight collagen and low molecular weight sodium hyaluronate to obtain a mixture solution (without the process of freezing to form a film and freezing to obtain a gel).

[0139] Experimental Methods: Forty healthy volunteers aged 20-40 years (excluding pregnant or lactating women) were randomly divided into eight groups of five. Each group used one test product. Subjects were prohibited from using any products (cosmetics or topical medications) on the test site for three days prior to the test. During the test, the test area was first cleaned with water, then the test sample was applied (using the same weight of sample and the same test area). The test was conducted at an ambient temperature of 20℃-25℃ and humidity of 40%-60%. A Corneometer CM825 was used to measure the skin moisture content of the test area before sample application, 4 hours after application, and 8 hours after application. The inner forearm of each subject was selected as the test area. Each person underwent three parallel measurements, each measured by the same instrument and the same person, with the measuring probe cleaned. The average value for each person was calculated, and then the average value for each group was calculated. The rate of increase in skin moisture content was then determined.

[0140] Skin moisture content growth rate = (MMV) t -MMV0) / MMV0×100%;

[0141] MMV0: Skin moisture content before use;

[0142] MMV t Skin moisture content after t hours of use.

[0143] Results: During use, no one reported any irritation (stinging, redness, itching, or other discomfort). The increase in water content is shown in the table below.

[0144] 4-hour water content growth rate 48％ 45％ 44％ 45％ 48％ 47％ 47％ 47％ 8-hour water content growth rate 40％ 38％ 38％ 38％ 41％ 39％ 40％ 31％

[0145] Therefore, the dressing provided by the present invention has good moisturizing properties.

[0146] 5) In vitro release

[0147] Sample group: Samples obtained from formulation group D6;

[0148] Control group 3: The aqueous mixture obtained from formulation group C1 was prepared with gel from formulation group B2 to obtain a rapamycin content of 2 mg / g;

[0149] Release medium: PBS buffer, pH = 7.4;

[0150] Method: 2g of each sample was placed in a dialysis bag with a molecular weight cutoff of 5000, and then placed in PBS buffer at 37℃ and pH 7.4. At set time points (1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 60h, 72h), 0.5mL of sample was precisely transferred, and 0.5mL of PBS buffer (pH=7.4) was added to maintain a constant volume. Six parallel groups were set up. After filtering the release medium through a 0.2μm syringe filter, the absorbance at a wavelength of 277nm was measured using a UV spectrophotometer. The drug concentration was determined according to the drug standard curve, and the cumulative drug release was calculated.

[0151] Results: The cumulative release of rapamycin in the sample group was 18% at 4h, 36% at 12h, 56% at 24h, 72% at 36h, 80% at 48h, and 93% at 72h, showing stable and continuous release of rapamycin within 72h. In the control group 3, the cumulative release was 56% at 4h and 90% at 12h.

[0152] The results show that the dressing provided by the present invention can release rapamycin for a long time and achieve a long-lasting effect, while the gel mixture containing only the compound releases quickly and cannot achieve a long-lasting effect.

[0153] 6) In vitro percutaneous infiltration

[0154] Sample group: Samples obtained from formulation group D6;

[0155] Control group 3: The aqueous mixture obtained from formulation group C1 was prepared with gel from formulation group B2 to obtain a rapamycin content of 2 mg / g;

[0156] Methods: Small pigskin was prepared by removing subcutaneous fat and washing with physiological saline. 2g of the sample was taken and placed in a Franz vertical diffusion cell with PBS buffer (pH=7.4, air bubbles removed) as the receiving solution. A magnetic stir bar was placed in the diffusion cell, and the cell was filled with absorbent (approximately 10mL). Pigskin (soaked in PBS buffer for 30min before use) was cut to a suitable size and placed between the supply and receiving cells. The magnetic stirring speed was 300 rpm, and the water bath temperature was 32℃±0.5℃. At set time points (1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 60h, 72h), 0.5mL of sample was precisely transferred, and 0.5mL of PBS buffer (pH=7.4) was added to maintain a constant volume. Three parallel studies were performed. The absorbance at 277nm was measured using a UV spectrophotometer. The drug concentration was determined according to the drug standard curve, and the cumulative drug penetration was calculated.

[0157] Results: The cumulative permeation of the sample in formulation group D6 was 18% at 12h, 32% at 24h, 56% at 48h, and reached 80% at 72h; the cumulative permeation of control group 3 exceeded 50% at 12h.

[0158] Based on the above test results, the dressing provided by this invention can provide a stable and sustained release of rapamycin over a long period of time, while being essentially non-irritating. It can keep the application site moist, absorb exudate, and has good breathability. It can be used to treat and prevent angiofibromas or their recurrence, as well as to prevent recurrence after surgical treatment of angiofibromas, and to care for, improve, and repair damaged skin. Compared with topical gels, it can be applied only once over a longer period of time, without the need for multiple applications, thus improving ease of use.

[0159] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the solutions or applications described herein within the scope and content of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve the conditions / parameters to implement and / or apply the technology of this invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within this invention.

Claims

1. A dressing, comprising: A hyaluronic acid-collagen membrane, a rapamycin-γ-cyclodextrin inclusion complex, and a gel of low molecular weight hyaluronic acid and low molecular weight collagen; wherein, based on the total mass of the dressing, the content of rapamycin is 0.2 mg / g-4 mg / g, the mass ratio of rapamycin to γ-cyclodextrin is 1:120-1:150, and the content of the gel of low molecular weight hyaluronic acid and low molecular weight collagen is 5%-40%; in the membrane, the molecular weight of collagen is 250,000 Daltons-500,000 Daltons, and based on the mass ratio, at least 95% of the collagen has a triple helix structure; the mass ratio of hyaluronic acid to collagen is 1:4-1:0.25, the molecular weight of hyaluronic acid is 2.2 million Daltons-2.8 million Daltons, and the content of hyaluronic acid is 10 mg / g-2 mg / g. The content of collagen is 3mg / g-10mg / g, and the hyaluronic acid salt is one or more of sodium hyaluronate and zinc hyaluronate. The molecular weight of the low molecular weight hyaluronic acid salt is 100,000 Daltons-300,000 Daltons. The low molecular weight collagen is a combination of collagen with a molecular weight of 10,000 Daltons-50,000 Daltons and collagen with a molecular weight of less than 10,000 Daltons, and the mass ratio of collagen with a molecular weight of 10,000 Daltons-50,000 Daltons to collagen with a molecular weight of less than 10,000 Daltons is 6:1-2:

1. In the gel, based on the total mass of the gel, the content of the low molecular weight hyaluronic acid salt is 5mg / g-20mg / g, and the mass ratio of the low molecular weight hyaluronic acid salt to the low molecular weight collagen is 2:1-1:

2.

2. The dressing according to claim 1, comprising the following conditions: In the membrane, the mass ratio of hyaluronic acid salt to collagen is 4:3; In the membrane, the molecular weight of the hyaluronic acid salt is 2.2 million to 2.6 million Daltons; The hyaluronic acid content in the membrane is 10 mg / g-20 mg / g; The membrane contains 5 mg / g to 10 mg / g of collagen; the dressing can continuously release rapamycin for 24 to 72 hours. In the gel, based on the total mass of the gel, the content of the low molecular weight hyaluronic acid salt is 10 mg / g-20 mg / g; and The mass ratio of the low molecular weight hyaluronic acid salt to the low molecular weight collagen is 1:1 to 1:

2.

3. The dressing according to claim 1, wherein the content of rapamycin is 0.5 mg / g to 2 mg / g based on the total mass of the dressing.

4. The dressing according to claim 1, wherein, The membrane is formed by the following steps: 1) Obtain aqueous solutions of hyaluronic acid and collagen, respectively; 2) Adjust the pH of the aqueous solution of hyaluronic acid salt to 1.2-1.5, optionally remove the air bubbles, and then mix it with the aqueous solution of collagen at 0℃~10℃. Adjust the pH to 1.2-1.5 again to obtain an acidic mixture. 3) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then thaw it at 20℃ to 30℃ for 4 to 12 hours to obtain hyaluronic acid salt-collagen sponge; 4) Transfer the obtained sponge to a dialysis bag and place it in water or an aqueous solution for dialysis until its pH is 6.8-7.6; or soak the obtained sponge in water or an aqueous solution until its pH is 6.8-7.6; remove the sponge and dehydrate it until the water content does not exceed 10 wt% of the total water content to obtain a neutral sponge; wherein the aqueous solution is a phosphate buffer solution with a pH of 6.9-7.6; 5) Optionally, the neutral sponge is sterilized to obtain a hyaluronic acid-collagen membrane; The gel of the aforementioned low molecular weight hyaluronic acid and low molecular weight collagen is formed by the following steps: 6) Obtain aqueous solutions of low molecular weight hyaluronic acid and low molecular weight collagen, respectively; 7) Adjust the pH of the aqueous solution of low molecular weight hyaluronic acid salt to 0.7-1.1, optionally remove the air bubbles, and then mix it with the aqueous solution of low molecular weight collagen at 0℃~10℃. Adjust the pH to 0.7-1.1 again to obtain an acidic mixture. 8) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then thaw it at 20℃ to 30℃ for 4 to 12 hours to obtain hyaluronic acid salt-collagen gel. 9) Soak the obtained gel in water or an aqueous solution until its pH reaches 6.8-7.6; remove it to obtain a neutral hyaluronic acid-collagen gel; wherein the aqueous solution is a phosphate buffer with a pH of 6.9-7.6; 10) Optionally, the neutral hyaluronic acid salt-collagen gel is sterilized to obtain a gel of low molecular weight hyaluronic acid salt and low molecular weight collagen.

5. A method for preparing the dressing according to any one of claims 1-3, comprising the following steps: a) Prepare a hyaluronic acid-collagen membrane; b) Mix the γ-cyclodextrin inclusion complex of rapamycin with water, optionally removing a portion of the water to obtain an aqueous mixture of rapamycin; in the obtained aqueous mixture of rapamycin, the mass ratio of the γ-cyclodextrin inclusion complex of rapamycin to water is 1:0.5-1:1.

5. c) The hyaluronic acid-collagen membrane is contacted with an aqueous mixture of rapamycin, sonicated, soaked at 0℃-25℃ for 1h-12h, and then freeze-dried under vacuum at -50℃ to -40℃ and 10Pa-30Pa until dry. The membrane is then kept at 0℃-5℃ for 2h-10h to obtain a drug-containing membrane. The mass ratio of the hyaluronic acid-collagen membrane to the aqueous mixture of rapamycin is 15:1-0.2:

1. The sonication frequency is 10Hz-30Hz, the sonication time is 10-60 minutes, and the sonication temperature is 0℃-25℃. d) A gel containing low molecular weight hyaluronic acid and low molecular weight collagen was prepared; and e) Mix the medicated film with the gel, and optionally sterilize to obtain the dressing.

6. The method according to claim 5, wherein, The preparation of the hyaluronic acid-collagen membrane includes the following steps: 1) Obtain aqueous solutions of hyaluronic acid and collagen, respectively; 2) Adjust the pH of the aqueous solution of hyaluronic acid salt to 1.2-1.5, optionally remove the air bubbles, and then mix it with the aqueous solution of collagen at 0℃~10℃. Adjust the pH to 1.2-1.5 again to obtain an acidic mixture. 3) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then melt it at 20℃ to 30℃ to obtain hyaluronic acid salt-collagen sponge; 4) Transfer the obtained sponge to a dialysis bag and place it in water or an aqueous solution for dialysis until its pH is 6.8-7.6; or soak the obtained sponge in water or an aqueous solution until its pH is 6.8-7.6; remove the sponge and dehydrate it until the water content does not exceed 10 wt% of the total water content to obtain a neutral sponge; wherein the aqueous solution is a phosphate buffer solution with a pH of 6.9-7.6; 5) Optionally, the neutral sponge is sterilized to obtain a hyaluronic acid-collagen membrane; The preparation of the gel containing low molecular weight hyaluronic acid and low molecular weight collagen includes the following steps: 6) Obtain aqueous solutions of low molecular weight hyaluronic acid and low molecular weight collagen, respectively; 7) Adjust the pH of the aqueous solution of low molecular weight hyaluronic acid salt to 0.7-1.1, optionally remove the air bubbles, and then mix it with the aqueous solution of low molecular weight collagen at 0℃~10℃. Adjust the pH to 0.7-1.1 again to obtain an acidic mixture. 8) Freeze the obtained acidic mixture at -30℃ to -20℃ for 3 to 14 days, and then melt it at 20℃ to 30℃ to obtain hyaluronic acid salt-collagen gel; 9) Soak the obtained gel in water or an aqueous solution until its pH reaches 6.8-7.6; remove it to obtain a neutral hyaluronic acid-collagen gel; wherein the aqueous solution is a phosphate buffer with a pH of 6.9-7.6; 10) Optionally, the neutral hyaluronic acid salt-collagen gel is sterilized to obtain a gel of low molecular weight hyaluronic acid salt and low molecular weight collagen.

7. The method according to claim 6, wherein, In step 4, the obtained sponge is dehydrated until the moisture content does not exceed 5 wt% or 2 wt% of the total moisture content; in step 3 or step 8, the dissolution time is 4 h-12 h; the sterilization includes sterilization using cobalt 60 at a dose not exceeding 15 KGy at -30℃ to 0℃.

Images