Fish scale composite hydrogel for drug loading and preparation method thereof

Abstract

The invention discloses a fish scale composite hydrogel for drug loading and a preparation method thereof. The method comprises the following steps: alternately treating fish scales by adopting high-frequency and low-frequency sound waves, respectively extracting fish scale collagen and hydroxyapatite by adopting a hot-pressing method and an ionic liquid method, and then cross-linking acrylic acid modified beta-cyclodextrin with a peptide calcium chelate to prepare the fish scale composite hydrogel. According to the method, high-frequency and low-frequency sound waves are used for alternately treating the fish scales, a gluing structure of collagen and hydroxyapatite is dissociated through cooperation of the cavitation effect and mechanical vibration, and the extraction efficiency is improved while collagen denaturation is avoided; the prepared fish scale composite hydrogel has a jellyfish structure: a stable gel core layer structure is formed by coating a hydroxyapatite-chitosan cross-linked network with beta-cyclodextrin; the branched whisker structure formed by covalent linkage of the acrylic acid modified beta-cyclodextrin and the collagen realizes gradient controlled release of the drug, and the strong adhesiveness of the whisker can accurately locate the drug release part, thereby providing an innovative solution for the fields of drug delivery and tissue engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to a fish scale composite hydrogel for drug loading and a preparation method thereof. Background Art

[0002] Fish scales are layered calcified tissues derived from the epidermis of fish. They are widely found in byproducts of freshwater and marine fish processing. They are naturally renewable, abundant in resources, and low in cost. Their multi-layer composite structure consists of a dense mineralized layer on the outside and a collagen fiber network on the inside, and has excellent mechanical strength and flexibility.

[0003] Fish scales are important biomineralized tissues on the surface of fish. They are formed by the secretion of the epidermis and dermis and have a unique cross-scale composite structure. Fish scales mainly contain collagen and hydroxyapatite. Its structure consists of hydroxyapatite chips as a rigid reinforcement phase embedded in a flexible collagen fiber matrix to form an organic-inorganic hybrid plywood structure. Studies have shown that fish scales are irregular hexagons as a whole, and the surface is divided into covered areas and exposed areas, forming a microscopic corrugated topological morphology. This structure can effectively disperse external force impact and protect the fish body from external mechanical damage.

[0004] The collagen content in fish scales can reach 20-30%, and it contains active ingredients such as hydroxyapatite and glycosaminoglycans, which give it biocompatibility, degradability and cell adhesion. In recent years, with the development of the aquatic processing industry, millions of tons of fish scale waste are generated every year around the world. Traditional treatment methods are mainly landfill or incineration, which not only causes waste of resources, but also may cause environmental pollution. Therefore, the development of high-value utilization technology for fish scales, especially its conversion into biomedical materials, has become a research hotspot in the field of circular economy and green chemistry.

[0005] At present, the preparation methods of fish scale hydrogel mainly include acid / alkali extraction, enzymatic hydrolysis and physical modification. For example, patent CN201510855741.2 uses acid treatment-salting out-dialysis process to directly obtain collagen hydrogel. Although the steps are simplified, the strong acid environment easily destroys the tertiary structure of collagen, resulting in insufficient gel strength (<200g·mm). Enzymatic hydrolysis (such as trypsin hydrolysis) can improve the collagen extraction rate, but the enzyme cost is high and the product molecular weight distribution is wide, which requires ultrafiltration purification, increasing the complexity of the process. Although the physical method (such as high temperature boiling) is easy to operate, excessive thermal denaturation will reduce the biological activity of collagen, and the yield is limited by the water-scale ratio (the yield is only 3.62% at the optimal 3:1). In addition, the existing technology mostly relies on chemical cross-linking agents (such as glutaraldehyde), which may introduce cytotoxicity and limit its medical application. The existing patented technology generally ignores the reuse of hydroxyapatite inherent in fish scales, resulting in waste of mineral components and failure to achieve high value of all components. Therefore, these defects seriously restrict the industrial application of fish scale hydrogel in the fields of drug delivery, wound repair, bone regeneration, etc. Therefore, studying new preparation methods of fish scale hydrogel is conducive to the large-scale application of fish scale hydrogel in the fields of tissue engineering, drug delivery, etc., and promoting the resource utilization and high value transformation of aquatic waste. Summary of the invention

[0006] Technical problem to be solved: In view of the above technical problems, the purpose of the present invention is to disclose a fish scale composite hydrogel for drug loading and a preparation method thereof, which utilizes high-frequency and low-frequency sound waves to alternately treat fish scales to synergistically dissociate the bonding structure of collagen and hydroxyapatite through cavitation effect and mechanical vibration, thereby avoiding collagen denaturation and improving extraction efficiency; at the same time, the prepared fish scale composite hydrogel has a "jellyfish" structure: a stable gel core layer structure is formed by a β-cyclodextrin-coated hydroxyapatite-chitosan cross-linked network; a branched tentacle structure formed by covalently linking acrylic acid-modified β-cyclodextrin and collagen can achieve gradient controlled release of drugs, and the strong adhesion of the tentacles can accurately locate the drug release site, providing innovative solutions for the fields of drug delivery and tissue engineering.

[0007] Technical solution: A method for preparing a fish scale composite hydrogel, comprising the following steps: S1. The fish scales are first cleaned, dried, crushed, and sieved to obtain fish scale powder, and water is added to prepare a fish scale powder solution; S2. treating the fish scale powder solution with high-frequency-low-frequency sound waves, filtering the fish scale filtrate and the calcium ion-containing filtrate; S3. The fish scale filtrate is added with water and then subjected to hot pressing to obtain fish scale collagen and residue, wherein the residue is subjected to ionic liquid extraction, dried to obtain hydroxyapatite, and a portion of the fish scale collagen is enzymatically hydrolyzed to obtain fish scale collagen peptides; S4. treating the fish scale collagen peptide and the calcium ion-containing filtrate obtained in S2 at pH 6.5-8.0 and 45-55° C. for 30-45 min to obtain a peptide calcium chelate; S5. Add acetic acid to chitosan and hydroxyapatite obtained in S3 and ultrasonically dissolve them uniformly, add acrylic acid-modified β-cyclodextrin and peptide calcium chelate and treat them at 60-70°C for 60-90 minutes, then add fish scale collagen and treat them at 30-45°C for 3-6 hours to obtain fish scale composite hydrogel.

[0008] Furthermore, in step S1, the mass ratio of fish scale powder to water is 1:(10-15).

[0009] Furthermore, the specific operation of the high-frequency-low-frequency acoustic wave treatment in step S2 is to perform high-frequency acoustic wave treatment and low-frequency acoustic wave treatment alternately for 3-6 times; the conditions of the high-frequency acoustic wave treatment are an acoustic wave frequency of 0.5-1 MHz and a treatment time of 10-20 min; the conditions of the low-frequency acoustic wave treatment are an acoustic wave frequency of 20-50 kHz and a treatment time of 40-60 min.

[0010] Furthermore, in step S3, the mass ratio of the fish scale filtrate to water is 1:(20-30); the hot pressing temperature is 110-130°C, and the processing time is 30-60min.

[0011] Furthermore, the conditions for enzymolysis in step S3 are: an enzyme addition amount of 2-6wt%, an enzymolysis temperature of 50-55°C, and an enzymolysis time of 3-5h; and the protease used in the enzymolysis is papain, neutral protease or alkaline protease.

[0012] Furthermore, the ionic liquid in step S3 is a mixed solution of choline chloride and 1,4-butanediol; the conditions for the ionic liquid extraction are extraction temperature 50-60°C, extraction time 1.5-2.5h, and a solid-liquid ratio of residue to ionic liquid of 1:(10-15).

[0013] Furthermore, in step S5, the mass ratio of chitosan, hydroxyapatite and acrylic acid-modified β-cyclodextrin is (0.5-1.5):(1-3):(1.5-4).

[0014] Furthermore, in step S5, the amount of peptide calcium chelate added is 2.5-5wt%; and the amount of fish scale collagen added is 2-10wt%.

[0015] A fish scale composite hydrogel prepared by any of the methods described above.

[0016] Furthermore, the fish scale composite hydrogel is used in drug delivery.

[0017] Beneficial Effects

[0018] 1. The fish scale composite hydrogel prepared by the present invention adopts alternating high-frequency and low-frequency sound waves. On the one hand, the high-frequency sound waves can cause a cavitation effect and generate micro-bubble nuclei in the fish scale powder solution through cavitation. The micro-bubble nuclei quickly gather growth energy through oscillation, growth, contraction, and collapse, and release huge energy when they break. At the same time, the violent mechanical vibration generated by the high-frequency sound waves synergistically destroys the plywood structure between the collagen fibers and hydroxyapatite in the fish scales, and promotes the collagen to break away from the constraints of the apatite lattice; on the other hand, the low-frequency sound waves can further destroy the binding force between collagen and hydroxyapatite, help loosen the connection between collagen and hydroxyapatite, promote collagen dissociation, and have little effect on the structure of collagen, thereby retaining the biological activity and functionality of collagen.

[0019] 2. The fish scale hydrogel prepared by the present invention has a unique "jellyfish" structure. On the one hand, the core layer of the gel structure that acts as an umbrella-shaped jellyfish head is composed of β-cyclodextrin-enclosed hydroxyapatite and chitosan. Under the cross-linking action of peptide calcium chelate, chitosan and hydroxyapatite form a cross-linked network structure through ionic coordination bonds and hydrogen bonds, and are enclosed in the internal cavity structure of β-cyclodextrin to form the core layer of the fish scale hydrogel structure; on the other hand, the acrylic acid group of acrylic acid-modified β-cyclodextrin forms a covalently cross-linked long chain with the amino group in collagen, which is attached to the outside of the annular cavity of β-cyclodextrin to form a branched tentacle structure of the fish scale hydrogel.

[0020] 3. The jellyfish structure gel core layer and the branched tentacle structure in the fish scale hydrogel prepared by the present invention can both be used for drug loading. The drugs carried by the gel at the branched tentacle structure are quickly released, which can be used for the acute treatment of the disease, and can alleviate the adverse reactions caused by the disease in advance. At the same time, the drugs carried by the gel in the gel core layer are slowly released, which can be used for the late treatment of the disease, and realize the gradient controlled release of the drug. In addition, the branched tentacle structure of the fish scale hydrogel can make the hydrogel adhere tightly to the drug release site, which is conducive to the precise release of the drug and its effect.

[0021] 4. The method for processing fish scales of the present invention can solve the problem of collagen denaturation caused by traditional acid-base treatment of fish scales, ensure the biological value of collagen and hydroxyapatite extracted from fish scales, and at the same time, the preparation of peptide calcium chelate can improve the raw material utilization rate of fish scales; in addition, the preparation method of fish scale hydrogel can provide good technical support for the high-value utilization of fish scales. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The collagen extraction rate, peptide calcium chelation rate and hydroxyapatite extraction rate of Examples 1-5 and Comparative Examples 1-2; Figure 2 is the drug loading of Examples 6-12 and Comparative Examples 3-7; Figure 3 is the cumulative release rate of the drug in Example 9 and Comparative Examples 3-7; Figure 4 It is the cell survival rate of Example 9 and Comparative Examples 3-7. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1

[0024] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1000g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was treated with high-frequency - low-frequency sonic waves for 4 cycles, wherein the high-frequency sonic wave treatment frequency was 0.5 MHz and the treatment time was 10 min, and the low-frequency sonic wave treatment conditions were a sonic wave frequency of 30 kHz and a treatment time of 45 min; and then filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate; S3.50g of fish scale filtrate was added with 1250g of water, and the mixture was hot-pressed at 110°C for 40min, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue was added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55°C for 2h, filtered, washed and purified to obtain hydroxyapatite; S4.10g fish scale collagen was evenly dissolved in 100g water, and then 2g papain was added for enzymatic hydrolysis at 50°C for 4h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the calcium ion filtrate in S2, treated at pH 8.0 and 50°C for 30min, centrifuged, washed and dried to prepare peptide calcium chelate.

[0025] Example 2

[0026] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1500g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was treated with high-frequency - low-frequency sonic waves for 4 cycles, wherein the high-frequency sonic wave treatment frequency was 0.5 MHz and the treatment time was 10 min, and the low-frequency sonic wave treatment conditions were a sonic wave frequency of 30 kHz and a treatment time of 45 min; and then filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate; S3.50g of fish scale filtrate was added with 1250g of water, and the mixture was hot-pressed at 110°C for 30min, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue was added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55°C for 1.5h, filtered, washed and purified to obtain hydroxyapatite; S4.10g fish scale collagen was evenly dissolved in 100g water, and then 3g papain was added for enzymatic hydrolysis at 50°C for 3h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the calcium ion filtrate in S2, treated at pH 8.0 and 50°C for 30min, centrifuged, washed and dried to prepare peptide calcium chelate.

[0027] Example 3

[0028] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1000g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was treated with high-frequency - low-frequency sonic waves for 4 cycles, wherein the high-frequency sonic wave treatment frequency was 0.75 MHz and the treatment time was 10 min, and the low-frequency sonic wave treatment conditions were a sonic wave frequency of 30 kHz and a treatment time of 45 min; and then filtered to obtain the fish scale filtrate and the calcium ion-containing filtrate; S3.50g of fish scale filtrate was added with 1250g of water, and the mixture was hot-pressed at 110°C for 40min, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue was added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55°C for 2h, filtered, washed and purified to obtain hydroxyapatite; S4.10g fish scale collagen was evenly dissolved in 100g water, and then 2g papain was added for enzymatic hydrolysis at 50℃ for 4h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the calcium ion filtrate in S2, treated at pH 8.0 and 50℃ for 45min, and centrifuged, washed and dried to prepare peptide calcium chelate.

[0029] Example 4

[0030] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1000g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was treated with a high-frequency - low-frequency sonic wave cycle 4 times, wherein the high-frequency sonic wave treatment frequency was 0.5 MHz and the treatment time was 10 min, and the low-frequency sonic wave treatment conditions were a sonic wave frequency of 50 kHz and a treatment time of 45 min; and then filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate; S3.50g of fish scale filtrate was added with 1250g of water, and the mixture was hot-pressed at 110°C for 40min, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue was added with 12.5 times the volume of choline chloride + 1,4-butanediol solution, and treated at 60°C for 2h, and filtered, washed and purified to obtain hydroxyapatite; S4.10g fish scale collagen was added with 100g water and dissolved evenly, and then 4g papain was added for enzymatic hydrolysis at 50℃ for 4h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the calcium ion filtrate in S2, treated at pH 8.0 and 55℃ for 30min, centrifuged, washed and dried to prepare peptide calcium chelate.

[0031] Example 5

[0032] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1000g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was treated with high-frequency - low-frequency sonic waves for 4 cycles, wherein the high-frequency sonic wave treatment frequency was 0.5 MHz and the treatment time was 10 min, and the low-frequency sonic wave treatment conditions were a sonic wave frequency of 30 kHz and a treatment time of 45 min; and then filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate; S3.50g of fish scale filtrate was added with 1250g of water, and the mixture was hot-pressed at 120°C for 40min, filtered, concentrated and dried to obtain fish scale collagen; 15 times the volume of choline chloride + 1,4-butanediol solution was added to the remaining residue, and the mixture was treated at 55°C for 2h, and filtered, washed and purified to obtain hydroxyapatite; S4.10g fish scale collagen was evenly dissolved in 100g water, and then 2g papain was added for enzymatic hydrolysis at 50°C for 4h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the calcium ion filtrate in S2, treated at pH 8.0 and 50°C for 30min, centrifuged, washed and dried to prepare peptide calcium chelate.

[0033] Comparative Example 1

[0034] The difference between this comparative example and Example 3 is that high-frequency sonication is not used.

[0035] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1000g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was treated with low-frequency sonic waves for 4 cycles, wherein the low-frequency sonic wave treatment conditions were a sonic wave frequency of 30 kHz and a treatment time of 45 min; and then filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate; S3.50g of fish scale filtrate was added with 1250g of water, and the mixture was hot-pressed at 110°C for 40min, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue was added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55°C for 2h, filtered, washed and purified to obtain hydroxyapatite; S4.10g fish scale collagen was evenly dissolved in 100g water, and then 2g papain was added for enzymatic hydrolysis at 50℃ for 4h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the calcium ion filtrate in S2, treated at pH 8.0 and 50℃ for 45min, and centrifuged, washed and dried to prepare peptide calcium chelate.

[0036] Comparative Example 2

[0037] The difference between this comparative example and Example 3 is that high-frequency-low-frequency sonic wave treatment is not used.

[0038] A method for extracting fish scale collagen and hydroxyapatite from fish scales comprises the following steps: S1. The fish scales were first cleaned and dried, crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100g of fish scale powder was added with 1000g of water to prepare a fish scale powder solution; S2. The fish scale powder solution was heat-pressed at 110°C for 40 minutes, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue was added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55°C for 2 hours, filtered, washed and purified to obtain hydroxyapatite; S3.10g of fish scale collagen was evenly dissolved in 100g of water, and then 2g of papain was added for enzymatic hydrolysis at 50°C for 4h to obtain fish scale collagen peptide; the fish scale collagen peptide was evenly mixed with the filtrate in S2, treated at pH 8.0 and 50°C for 45min, centrifuged, washed and dried to prepare peptide calcium chelate.

[0039] Index determination

[0040] (1) Collagen extraction rate The collagen extraction rates of Examples 1-5, Comparative Example 1 and Comparative Example 2 were analyzed according to the hydroxyproline content determination method in the national standard GB / T9695.23-2008.

[0041] (2) Hydroxyapatite extraction rate The weight of the residue (denoted as M1) and the weight of hydroxyapatite (denoted as M2) were weighed respectively, and the hydroxyapatite extraction rate was calculated by the following formula.

[0042] Hydroxyapatite extraction rate (%) = M1 / M2 × 100% (3) Peptide calcium chelation rate The calcium contents of Examples 1-5, Comparative Example 1 and Comparative Example 2 were measured according to the calcium content determination method of national standard GB 5009.92-2016, and the peptide calcium chelation rate was calculated by the following formula.

[0043] Peptide calcium chelation rate (%) = calcium content in peptide calcium chelate / calcium content in filtrate × 100% Depend on Figure 1 It can be seen that the collagen extraction rate, peptide calcium chelation rate and hydroxyapatite extraction rate of Examples 1-5 are all higher than those of Comparative Examples 1 and 2. Comparative Example 1 only uses low-frequency sound wave treatment. Since the destructive effect of low-frequency sound waves in the fish scale pretreatment stage is smaller than that of high-frequency sound waves, the plywood structure between collagen and hydroxyapatite in the fish scales cannot be fully destroyed, resulting in collagen not completely dissociated from hydroxyapatite, thereby reducing the collagen extraction rate and hydroxyapatite extraction rate; in addition, Comparative Example 2 does not use high-frequency-low-frequency sound wave treatment, which can not effectively destroy the plywood structure between collagen and hydroxyapatite in the fish scales in the fish scale pretreatment step, and collagen fails to dissociate, which subsequently affects the extraction of hydroxyapatite, so the collagen extraction rate and hydroxyapatite extraction rate are reduced. Therefore, the preparation method of the following fish scale composite hydrogel uses the fish scale collagen, hydroxyapatite and peptide calcium chelate extracted in Example 3.

[0044] Example 6

[0045] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0046] Example 7

[0047] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 20 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 30° C. for 4 h to obtain fish scale composite hydrogel.

[0048] Example 8

[0049] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 10 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0050] Example 9

[0051] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 30 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 30° C. for 4 h to obtain fish scale composite hydrogel.

[0052] Example 10

[0053] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 5 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 30 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0054] Embodiment 11

[0055] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 2.5 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0056] Example 12

[0057] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 10 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0058] Comparative Example 3

[0059] The difference between this comparative example and Example 6 is that the peptide calcium chelate is replaced by genipin.

[0060] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of genipin and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0061] Comparative Example 4

[0062] The difference between this comparative example and Example 6 is that the peptide calcium chelate is replaced by glutaraldehyde.

[0063] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of glutaraldehyde and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0064] Comparative Example 5

[0065] The difference between this comparative example and Example 6 is that no peptide calcium chelate is added.

[0066] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0067] Comparative Example 6

[0068] The difference between this comparative example and Example 6 is that hydroxyapatite is not added.

[0069] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan to 100 mL acetic acid solution and dissolve evenly by ultrasonication; Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60° C. for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0070] Comparative Example 7

[0071] The difference between this comparative example and Example 6 is that acrylic acid-modified β-cyclodextrin is not added.

[0072] A method for preparing a fish scale composite hydrogel comprises the following steps: Step 1. Add 5 g chitosan and 10 g hydroxyapatite to 100 mL acetic acid solution and dissolve them evenly by ultrasonication; Step 2. Add 3 g of peptide calcium chelate and treat at 60°C for 60 min; Step 3. Add 5 g of fish scale collagen and treat at 40° C. for 6 h to obtain fish scale composite hydrogel.

[0073] Performance measurement

[0074] (1) Drug loading Taking curcumin as a model drug, 50 mg of fish scale composite hydrogel was weighed and placed in 10 mL of 1.0 mg / mL ethanol solution of curcumin and mixed at a constant speed for 24 h. 3 The mixture was centrifuged at 400 rpm for 12 min, and the supernatant was separated and washed with anhydrous ethanol for 3 times. 4 mL of anhydrous ethanol was added each time, and the supernatant was separated and obtained. The total amount of supernatant collected was 20 mL. The absorbance of the supernatant mixture was measured, and the drug loading of the fish scale composite hydrogel was calculated by the curcumin standard curve. The drug loading (%) = (m1-m2) / 50×100%, wherein m1 is the mass of the drug administered, and m2 is the mass of the drug in the supernatant. A blank control was set and placed in anhydrous ethanol and the same steps were followed to eliminate the influence of the extract on the test process.

[0075] Depend on Figure 2It can be seen that the drug loading of Examples 6-12 is higher than that of Comparative Examples 3-7, among which Example 9 has the highest drug loading, indicating that the core layer of the fish scale hydrogel structure is prepared by acrylic acid-modified β-cyclodextrin, chitosan, hydroxyapatite and peptide calcium chelate, and chitosan and peptide calcium chelate are supported by ionic coordination bonds and hydrogen bonds, while hydroxyapatite is filled into the cross-linked network to support the stability of the network, and is encapsulated in the internal cavity structure of β-cyclodextrin to form the core layer of the fish scale hydrogel structure; in addition, the acrylic acid group and the amino group in the collagen form a covalently cross-linked long chain attached to the outside of the annular cavity of β-cyclodextrin to form a branched tentacle structure of the fish scale hydrogel, and the two can maximally encapsulate curcumin, increase the loading amount of curcumin, and thus increase the drug loading amount of the fish scale composite hydrogel.

[0076] Comparative Examples 3 and 4 were cross-linked with genipin and glutaraldehyde, respectively, and the cross-linking effects between them and chitosan and hydroxyapatite were weaker than those of peptide calcium chelates. Comparative Examples 5 and 6 did not add peptide calcium chelates or hydroxyapatite, respectively, which would affect the stability of the core structure of the fish scale composite hydrogel. In addition, Comparative Example 7 did not use acrylic acid-modified β-cyclodextrin, and the prepared fish scale composite hydrogel did not form a branched tentacle structure, thus reducing the drug loading of the fish scale composite hydrogel.

[0077] (2) Cumulative drug release rate According to the provisions of the 2015 edition of the Chinese Pharmacopoeia, the curcumin-loaded fish scale composite hydrogel was subjected to an in vitro release experiment. The specific method was as follows: a dilute hydrochloric acid solution (pH = 1.4) containing 20% ​​ethanol by mass was used as a simulated artificial gastric juice for in vitro drug release testing. At the same time, a molecule with a relative molecular mass of 8×10 3 -10×10 3 dialysis bag, weigh 50 mg of drug-loaded sample and put it into the dialysis bag, then put it into the sustained-release solution, stir it in a 37.5°C water bath, start the in vitro release test of curcumin, take out 3 mL of sustained-release solution at each time point set in advance, and immediately add 3 mL of corresponding sustained-release solution when the sustained-release solution is taken out, use an ultraviolet spectrophotometer to measure the absorbance of the sample solution at 427 nm, and calculate the cumulative release rate of the drug according to the following formula; Cumulative drug release rate (%) = (V 0 c n +V ) / m×100%, where V 0 is the total volume of the solution; V is the volume of the solution taken out each time; i = 1, 2...n is the number of times the solution is drawn; ci is the drug concentration measured when the solution is obtained for the i-th time; m is the total mass of curcumin encapsulated in the drug-loaded hydrogel used for testing.

[0078] Depend on Figure 3It can be seen that the cumulative drug release rate of curcumin loaded on the fish scale composite hydrogel prepared in Example 9 is lower than that of Comparative Examples 3 to 7 when acting for a long time, and the curcumin loaded on the branched tentacle structure of the fish scale composite hydrogel is released suddenly in a short time, which can achieve the maximum relief effect in the initial stage of actual drug treatment of the disease; in addition, the curcumin on the surface of the fish scale composite hydrogel synthetic structure needs to be released through the chitosan-hydroxyapatite network first, and then enter the gastric juice through the pores on the surface of the β-cyclodextrin inclusion structure to release, which is equivalent to constructing a double-layer controlled release structure, prolonging the sustained release time of the drug, thereby achieving the effect of gradient controlled release of curcumin.

[0079] (3) Biocompatibility In vitro cytotoxicity experiments were conducted on the raw materials of the medium with reference to the biological test rules of ISO10993.1-2018 for medical devices, and the CCK-8 method was used to test the cytotoxicity. Weigh 0.9g of the test sample and soak it in industrial ethanol for 24h, filter and dry it, and then sterilize it with ultraviolet light; the test sample was soaked in 20mL of complete culture medium, cultured at 37℃ for 24h, filtered with a microporous filter membrane, and the leaching solution was recycled, and the complete culture solution was used as the control experiment; in vitro cell experiments used Chinese hamster uterine cells to be injected into 96-well plates, with 10 wells in each group; according to the classification method, 200µL of the leaching solution or complete culture solution (control) of different samples was added to each group, and after culturing for 24h, 48h, and 72h, CCK-8 experimental reagent was added to each well, and after gently shaking, it was placed in a temperature-controlled cell culture incubator for further culture. After 2-3h, the absorbance of each well sample at a wavelength of 450nm was measured using an instant enzyme-linked immunosorbent assay to calculate the relative cell proliferation rate.

[0080] Depend on Figure 4 It can be seen that with the extension of the culture time, the relative cell proliferation rate of Example 9 is greater than 90%, indicating that the fish scale composite hydrogel prepared in Example 9 has almost no toxicity to cell proliferation, while Comparative Examples 3 and 4 respectively use genipin and glutaraldehyde cross-linking to prepare fish scale composite hydrogels, wherein both genipin and glutaraldehyde have certain cytotoxicity, and glutaraldehyde has stronger cytotoxicity, which inhibits cell proliferation during cell culture proliferation and reduces the relative cell proliferation rate; Comparative Examples 5-7 have little effect on the relative cell proliferation rate.

[0081] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a fish scale composite hydrogel, characterized in that: The following steps are involved: S1. The fish scales are first cleaned, dried, crushed, and sieved to obtain fish scale powder, and water is added to prepare a fish scale powder solution; S2. treating the fish scale powder solution with high-frequency-low-frequency sound waves, filtering the fish scale filtrate and the calcium ion-containing filtrate; S3. The fish scale filtrate is added with water and then subjected to hot pressing to obtain fish scale collagen and residue, wherein the residue is subjected to ionic liquid extraction, dried to obtain hydroxyapatite, and a portion of the fish scale collagen is enzymatically hydrolyzed to obtain fish scale collagen peptides; S4. treating the fish scale collagen peptide and the calcium ion-containing filtrate obtained in S2 at pH 6.5-8.0 and 45-55° C. for 30-45 min to obtain a peptide calcium chelate; S5. Add acetic acid to chitosan and hydroxyapatite obtained in S3 and ultrasonically dissolve them uniformly, add acrylic acid-modified β-cyclodextrin and peptide calcium chelate and treat them at 60-70°C for 60-90 minutes, then add fish scale collagen and treat them at 30-45°C for 3-6 hours to obtain fish scale composite hydrogel.

2. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The mass ratio of the fish scale powder to water in step S1 is 1:(10-15).

3. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The specific operation of the high-frequency-low-frequency sonic treatment in step S2 is to perform high-frequency sonic treatment and low-frequency sonic treatment alternately for 3-6 times; the conditions for the high-frequency sonic treatment are a sonic wave frequency of 0.5-1 MHz and a treatment time of 10-20 min; the conditions for the low-frequency sonic treatment are a sonic wave frequency of 20-50 kHz and a treatment time of 40-60 min.

4. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: In step S3, the mass ratio of the fish scale filtrate to water is 1:(20-30); the hot pressing treatment temperature is 110-130° C. and the treatment time is 30-60 min.

5. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The conditions for enzymolysis in step S3 are: an enzyme addition amount of 2-6wt%, an enzymolysis temperature of 50-55°C, and an enzymolysis time of 3-5h; and the protease used in the enzymolysis is papain, neutral protease or alkaline protease.

6. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The ionic liquid in step S3 is a mixed solution of choline chloride and 1,4-butanediol; the conditions for the ionic liquid extraction are extraction temperature of 50-60°C, extraction time of 1.5-2.5h, and a solid-liquid ratio of residue to ionic liquid of 1:(10-15).

7. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: In the step S5, the mass ratio of chitosan, hydroxyapatite and acrylic acid-modified β-cyclodextrin is (0.5-1.5):(1-3):(1.5-4).

8. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: In the step S5, the amount of peptide calcium chelate added is 2.5-5wt%; the amount of fish scale collagen added is 2-10wt%.

9. The fish scale composite hydrogel prepared according to the method according to any one of claims 1 to 8.

10. Use of the fish scale composite hydrogel according to claim 9 in drug delivery.

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