Stability-enhanced alginate-keratin-based double-coated nanoparticles and preparation method and application thereof

Abstract

The present invention discloses a method for preparing stability-enhanced alginate-keratin-based double-coated nanoparticles, which includes: keratin is subjected to controlled enzymatic hydrolysis, enzyme inactivation, centrifugation, and freeze-drying to obtain self-assembled keratin nanoparticles; they are dispersed in an acidic buffer for structural development, a bioactive molecule is added, and the pH is adjusted to neutral to cause the keratin nanoparticles to restructure and close to obtain a keratin nanoparticle solution loaded with the bioactive molecule; it is dispersed in a sodium alginate solution, and then calcium chloride solution is added dropwise for cross-linking to form a calcium alginate outer shell, and free bioactive molecules are removed by dialysis or centrifugation, and then freeze-dried to obtain the alginate-keratin-based double-coated nanoparticles loaded with the bioactive molecule. The present invention also discloses the double-coated nanoparticles and their applications. Through controlled enzymatic hydrolysis and pH regulation, the present invention improves the self-assembly of keratin nanoparticles and the encapsulation of bioactive molecules, and forms double-coated nanoparticles with regular shapes, good dispersibility, redissolubility, and environmental stability.

Description

Technical Field

[0001] The invention relates to the technical field of nano-biomaterials, and in particular to stability-enhanced alginate-keratin-based double-coated nanoparticles and a preparation method and application thereof. Background Art

[0002] In recent years, with the development of in vitro screening technology, more and more natural bioactive molecules have been found to have nutritional and disease regulation functions and have been widely used in food, health care products and pharmaceutical fields, such as protein peptides, polyphenols and flavonoids. Studies have shown that these bioactive molecules have shown application potential in anti-oxidation, anti-cancer, anti-inflammatory and other aspects. However, most bioactive molecules still have problems such as poor water solubility, easy enzymatic degradation, environmental instability (storage, heat, extreme pH, etc.) and low biomembrane permeability, which greatly limit their bioavailability and applicability. Therefore, researchers have developed a variety of delivery systems to improve the physicochemical properties and bioavailability of bioactive molecules. Among them, nanoparticles, as an effective delivery carrier, can significantly improve the solubility and stability of bioactive molecules, and achieve targeted delivery and controlled release. Among the many encapsulation wall materials, proteins, as a natural polymer, are widely used in nano-delivery systems due to their biocompatibility, biodegradability and low toxicity. However, most proteins have poor stability, resulting in low encapsulation efficiency of bioactive molecules, premature leakage and degradation. For example, the patent with application number 202110562800.2 discloses a feather keratin / sodium alginate microgel drug delivery system and its preparation method and application. Although it increases the drug loading and reduces the drug leakage rate, the maximum drug loading is only 65%, and it is easily degraded in an acidic environment and has poor stability. Therefore, how to increase the drug loading, improve the stability of the protein-based nano-delivery system, solve the problems of high aggregation, poor water solubility, and easy degradation of bioactive molecules, so as to improve the bioavailability of bioactive molecules, is still a technical bottleneck and research hotspot that needs to be solved in this field. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] Another object of the present invention is to provide a method for preparing stability-enhanced alginate-keratin-based double-coated nanoparticles. Through controllable enzymatic hydrolysis and pH regulation techniques, moderate enzymatic hydrolysis can reduce keratin aggregation and self-assemble to form nanoparticles with regular shapes and good dispersibility, with an average particle size of about 123 nm; pH regulation (from acidic to neutral) can cause the keratin nanoparticles to exhibit a "unfolding-closing" structural change, which is beneficial for the encapsulation of bioactive molecules. By further establishing a calcium alginate coating layer, spherical nanoparticles with uniform sizes can be formed, with an average particle size of about 170-173 nm and a bioactive molecule encapsulation rate of 92.0-95.6%. The preparation method of the present invention has low raw material costs and wide sources, improves production efficiency, and has no pollution from introduced chemical reagents throughout the production process.

[0005] Another object of the present invention is to provide a stability-enhanced alginate-keratin-based double-coated nanoparticle, which has a high bioactive molecule encapsulation rate, is a spherical nanoparticle with a regular shape, has an average particle size of 170-173 nm, has good redissolvability and environmental stability (such as heat treatment and low-temperature storage), and its responsiveness to gastrointestinal pH changes can significantly improve the stability of bioactive molecules.

[0006] Another object of the present invention is to provide the application of the stability-enhanced alginate-keratin-based double-coated nanoparticles in the preparation of a bioactive molecule nano-delivery carrier. The stability-enhanced alginate-keratin-based double-coated nanoparticles of the present invention have good encapsulation effects on both hydrophilic molecules (insulin) and hydrophobic molecules (resveratrol) in the preparation of a bioactive molecule nano-delivery carrier, and can be widely used in the encapsulation and delivery of bioactive molecules, including but not limited to oral nano-delivery systems.

[0007] To achieve these and other advantages according to the present invention, a method for preparing stability-enhanced alginate-keratin-based double-coated nanoparticles is provided, which includes the following steps:

[0008] Step 1: Obtain self-assembled keratin nanoparticles after subjecting keratin to controllable enzymatic degradation, enzyme inactivation, centrifugation, and freeze-drying treatments;

[0009] Step 2: Disperse the keratin nanoparticles obtained in Step 1 in an acidic buffer solution for structural unfolding, add bioactive molecules, stir, and adjust the pH value to neutral to cause the keratin structure to reorganize and close to obtain a keratin nanoparticle solution loaded with bioactive molecules;

[0010] Step 3: Disperse the keratin nanoparticles loaded with bioactive molecules obtained in Step 2 in the sodium alginate solution. After mixing evenly, dropwise add it into the calcium chloride solution to crosslink and form a calcium alginate shell. Remove the free bioactive molecules by dialysis or centrifugation, and then freeze-dry to obtain the alginate-keratin-based double-coated nanoparticles loaded with bioactive molecules.

[0011] Preferably, Step 1 specifically includes:

[0012] S11: Disperse keratin evenly in the phosphate buffer solution, stir for 12 - 16 h for sufficient hydration, adjust the pH to 8.5, the temperature to 50 - 60 °C, the enzyme dosage to 5000 - 8000 U / g, and control the degree of hydrolysis to 5 - 15% for enzymatic degradation;

[0013] S12: After the enzymatic hydrolysis, heat at 95 °C for 3 min to inactivate the enzyme, adjust the pH to 7.0, and centrifuge at 6000 - 8000×g for 20 - 30 min, then freeze-dry to obtain the self-assembled keratin nanoparticles.

[0014] Preferably, Step 2 specifically includes:

[0015] S21: Disperse the keratin nanoparticles obtained in Step 1 in the phosphate buffer solution with pH = 1.5 - 3.0, and stir at 600 - 800 rpm for 1 - 2 h for structural development;

[0016] S22: Add the bioactive molecules, continuously stir for 1 - 2 h, adjust the pH to 7.0, and continue to stir for 1 - 2 h for structural recombination and closing to form the keratin nanoparticle solution loaded with bioactive molecules.

[0017] Preferably, Step 3 specifically includes:

[0018] S31: Dropwise add the keratin nanoparticle solution loaded with bioactive molecules obtained in Step 2 into an equal volume of sodium alginate solution with a mass-volume concentration of 1 - 3%, and continuously stir for 1 - 2 h to obtain a mixed solution;

[0019] S32: Dropwise add the mixed solution into an equal volume of 2 - 10 mM calcium chloride solution, continuously stir for 1 - 2 h, then remove the free bioactive molecules by dialysis or centrifugation, and freeze-dry to obtain the alginate-keratin-based double-coated nanoparticles loaded with bioactive molecules.

[0020] Preferably, the bioactive molecule is insulin or resveratrol.

[0021] Preferably, in S11, the temperature is 55°C and the enzyme dosage is 6000 U / g; in S12, the freeze-drying specifically includes: freeze-drying the enzymatic hydrolysate supernatant at -76°C and 0.009 mbar for 72 h.

[0022] Preferably, the mass ratio of keratin nanoparticles to bioactivity is 2:1 or 25:1, respectively.

[0023] Preferably, S32 specifically includes: dropwise adding the mixture into an equal volume of 4 mM calcium chloride solution, continuously stirring at 700 rpm for 1 h, then dialyzing with a dialysis bag with a molecular weight cut-off of 7 kDa to remove free bioactive molecules, the dialysis time ≥ 36 h, and freeze-drying at -76°C and 0.009 mbar for 72 h to obtain alginate-keratin-based double-coated nanoparticles loaded with bioactive molecules.

[0024] The object of the present invention can also be further achieved by stability-enhanced alginate-keratin-based double-coated nanoparticles.

[0025] Another object of the present invention is achieved by the application of stability-enhanced alginate-keratin-based double-coated nanoparticles in the preparation of nano-delivery carriers for bioactive molecules.

[0026] The present invention at least includes the following beneficial effects:

[0027] First, the preparation method of the stability-enhanced alginate-keratin-based double-coated nanoparticles of the present invention uses controllable enzymatic hydrolysis and pH regulation techniques. Moderate enzymatic hydrolysis can reduce keratin aggregation and self-assemble to form nanoparticles with regular shapes and good dispersibility, with an average particle size of about 123 nm; pH regulation (from acidic to neutral) can cause the keratin nanoparticles to exhibit a "unfolding - closing" structural change, which is beneficial for the encapsulation of bioactive molecules. By further establishing an alginate calcium coating layer, spherical nanoparticles with uniform sizes can be formed, with an average particle size of about 170 - 173 nm, and the bioactive molecule encapsulation efficiency reaches 92.0 - 95.6%. The raw material cost of the preparation method of the present invention is low and the source is wide, which improves production efficiency, and there is no pollution by introduced chemical reagents throughout the production process.

[0028] Second, the stability-enhanced alginate-keratin-based double-coated nanoparticles of the present invention have a high bioactive molecule encapsulation efficiency, are spherical nanoparticles with regular shapes, with an average particle size of 170 - 173 nm, have good redissolubility and environmental stability (such as heat treatment and low-temperature storage), and their responsiveness to gastrointestinal pH changes can significantly improve the stability of bioactive molecules.

[0029] Thirdly, the stability-enhanced alginate-keratin-based double-coated nanoparticles of the present invention have good encapsulation effects on hydrophilic molecules (insulin) and hydrophobic molecules (resveratrol) in the preparation of bioactive molecule nano-delivery carriers, and can be widely used in the encapsulation and delivery of bioactive molecules, including but not limited to oral nano-delivery systems.

[0030] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and partially understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0031] Figure 1 It is a flowchart of the preparation method of the stability-enhanced alginate-keratin-based double-coated nanoparticles of the present invention;

[0032] Figure 2 It is a theoretical structure diagram of the stability-enhanced alginate-keratin-based double-coated nanoparticles of the present invention;

[0033] Figure 3 In it, A is the microscopic structure diagram of the keratin nanoparticles self-assembled by optimal enzymatic hydrolysis (5% degree of hydrolysis) in Example 1 of the present invention, B is the microscopic structure diagram of the keratin nanoparticles self-assembled by enzymatic hydrolysis (15% degree of hydrolysis) in Example 3, C is the microscopic structure diagram of the keratin nanoparticles not enzymatically hydrolyzed in Comparative Example 1, and D is the microscopic structure diagram of the keratin nanoparticles self-assembled by excessive enzymatic hydrolysis (20% degree of hydrolysis) in Comparative Example 2;

[0034] Figure 4 In it, A is the "unfolding" diagram of the keratin nanoparticle structure in Example 1 of the present invention under acidic conditions (pH = 2.0); B is the "closing" diagram of the keratin nanoparticle structure in Example 1 under neutral conditions (pH = 7.0);

[0035] Figure 5 It is the particle size analysis and redissolution comparison diagram of the insulin-loaded keratin nanoparticles (KNP-INS) obtained by pH regulation in Step 2 of Example 1 of the present invention and the insulin-loaded keratin nanoparticles (KNP-INS-pH7) prepared without pH regulation in Comparative Example 3;

[0036] Figure 6 It is the microscopic structure diagram of different substances in Example 1 of the present invention. Among them, A is the microscopic structure diagram of insulin (INS), B is the microscopic structure diagram of calcium alginate-insulin (ALG-INS); C is the microscopic structure diagram of keratin-insulin (KNP-INS); D is the microscopic structure diagram of calcium alginate-keratin-insulin nanoparticles (ALG-KNP-INS);

[0037] Figure 7This is the comparison chart of the redissolution results and the particle size analysis chart of nanoparticles of different substances in Example 1 of the present invention. Among them, A is the comparison chart of the redissolution results of INS, ALG-INS, KNP-INS, and ALG-KNP-INS, and B is the particle size analysis chart of INS, ALG-INS, KNP-INS, and ALG-KNP-INS;

[0038] Figure 8 This is the infrared structure analysis chart of nanoparticles of different substances in Example 1 of the present invention;

[0039] Figure 9 This is the stability result chart of nanoparticles of different substances treated at 80 °C for 0 - 60 min in Example 1 of the present invention;

[0040] Figure 10 This is the stability result chart of nanoparticles of different substances stored at 4 °C for 0 - 28 days in Example 1 of the present invention;

[0041] Figure 11 This is the apparent diagram of ALG-KNP-INS prepared in Example 1 of the present invention with the change of gastrointestinal pH;

[0042] Figure 12 This is the gastrointestinal stability result chart of INS and ALG-KNP-INS in Example 1 of the present invention;

[0043] Figure 13 In it, A is the redissolution result chart of resveratrol (RES) and calcium alginate - keratin - resveratrol (ALG-KNP-RES) nanoparticles in Example 2 of the present invention, B is the particle size analysis chart of calcium alginate - keratin - resveratrol (ALG-KNP-RES) nanoparticles, and C is the microscopic structure diagram of calcium alginate - keratin - resveratrol nanoparticles (ALG-KNP-RES);

[0044] Figure 14 In it, A is the transmembrane transport diagram of ALG-KNP-INS in Example 1 of the present invention; B is the cell absorption diagram of ALG-KNP-INS provided in Example 1 of the present invention. Detailed implementation manners

[0045] The following further elaborates on the present invention in conjunction with the attached drawings, so that those skilled in the art can implement it with reference to the text of the specification.

[0046] It should be noted that the experimental methods described in the following implementation schemes are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0047] Keratin is the third most abundant natural polymer after cellulose and chitin. It has good biocompatibility, biodegradability and low toxicity, and has been widely used in the field of biomedical materials. Keratin is rich in cystine (>7 mol%), and its abundant disulfide bonds provide a highly stable structure for keratin. Studies have shown that keratin has good aqueous solution stability and resistance to degradation by traditional proteases. At the same time, it can penetrate kidney and liver cells, showing good cell adhesion and proliferation ability and penetrability, and demonstrating great application potential in the field of nano-delivery systems. Alginate, as a natural polysaccharide, has good gel-forming property, pH sensitivity and mucoadhesion, and is an excellent carrier excipient, which has been applied to the preparation of carriers such as nanoparticles, liposomes, capsules, tablets, hydrogels and microspheres. Alginate coating can further protect the loaded bioactive molecules from gastrointestinal pH and enzyme degradation. At present, nanoparticle preparation technologies are mainly divided into three categories: physical methods (such as spray drying method and anti-solvent method), chemical methods (such as covalent cross-linking) and enzymatic hydrolysis methods. However, most processes have disadvantages such as chemical reagent residues, long process time, complex operation, high cost and uneven product quality. For example, the anti-solvent precipitation method will cause chemical reagent residues; the covalent cross-linking method takes a long time, has large particle size and involves chemical reagent residues; the thermal aggregation method has high energy consumption. Therefore, how to provide a green and efficient technology for preparing stability-enhanced nanoparticles for the encapsulation and delivery of bioactive molecules has become an urgent problem in this field.

[0048] In order to improve the disadvantages of poor solubility, poor stability and low bioavailability of the bioactive molecule delivery system, the present invention is based on keratin materials, and combines controllable enzymatic hydrolysis modification and pH regulation technology to regulate the self-assembly behavior of keratin molecules, so as to improve the dispersibility of keratin nanoparticles and the encapsulation rate of bioactive molecules. Further, a calcium alginate coating layer is formed through a gel technology to construct a double-coated nanoparticle structure, thereby effectively enhancing the processing / storage stability of the bioactive molecule delivery system, and realizing gastrointestinal pH-responsive structural changes to enhance its delivery efficiency and bioavailability.

[0049] The present invention uses controllable enzymatic hydrolysis and pH regulation technology to improve the microstructure and self-assembly behavior of keratin molecules, prepares keratin nanoparticles with regular shape and good dispersibility to encapsulate bioactive molecules, and then establishes a calcium alginate coating layer through a gel technology to form an alginate-keratin-based double-coated nanoparticle with enhanced bioactive molecule encapsulation ability and stability. The technical process of the present invention is as Figure 1As shown. Through a controllable enzymatic hydrolysis technology, the present invention moderately opens the disulfide bonds and peptide bonds of keratin molecules by using keratinase, reduces the degree of cross-linking of keratin molecules, reduces the aggregation behavior of keratin molecules, and self-assembles to form keratin nanoparticles with good dispersion and regular shape. By using the pH regulation technology of the present invention, the intermolecular forces of keratin molecules (such as electrostatic interaction and hydrogen bond) are broken in an acidic environment, and the keratin nanoparticles present a state of expanded structure; with the adjustment to a neutral environment, the keratin molecules achieve structural recombination and closure. This "expansion-closure" process can promote the encapsulation of bioactive molecules. By using the sodium alginate gel technology of the present invention, a calcium alginate shell is formed outside the nanoparticles through the cross-linking of sodium alginate and calcium ions to construct a sodium alginate-keratin-based double-coated nanoparticle system, improving the stability of bioactive molecules; based on the pH sensitivity of sodium alginate (structural contraction in an acidic environment), the "aggregation-redispersion" structural responsive change of the double-coated nanoparticles in the gastrointestinal environment is realized, improving their digestive stability. The theoretical structure diagram of the stability-enhanced sodium alginate-keratin-based double-coated nanoparticles prepared by the present invention is as shown in Figure 2 shown.

[0050] In the present invention, ALG represents calcium alginate, KNP represents keratin nanoparticles, INS represents insulin, and RES represents resveratrol.

[0051] Example 1

[0052] A preparation method of stability-enhanced sodium alginate-keratin-based double-coated nanoparticles, which comprises the following steps:

[0053] Step 1: Uniformly disperse keratin at a concentration of 5% (w / v) in phosphate buffer (pH = 7.0, 10 mM), stir at 700 rpm for 12 h for sufficient hydration, then adjust the pH of the buffer to 8.5 with NaOH solution (1 M) for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 55°C, the enzyme dosage is 6000 U / g, and the degree of keratin hydrolysis is controlled at 5%. After the enzymatic hydrolysis is completed, heat the keratin hydrolysate at 95°C for 3 min to inactivate the enzyme, then adjust the pH to 7.0 with HCl solution (1 M), and centrifuge at 8000 × g for 20 min to obtain self-assembled keratin nanoparticles (KNP), which are freeze-dried for use at -76°C and 0.009 mbar, and the freeze-drying time is 72 h. Among them, the average particle size of the obtained keratin nanoparticles is 123 ± 7.0 nm, and the microscopic morphology is as shown in Figure 3 A in;

[0054] Step 2: Disperse 20 mg of keratin nanoparticles in 2 mL of phosphate buffer (pH = 2.0, 10 mM), stir for 1 h at 700 rpm for structural unfolding, and gradually add 2 mL of insulin solution (5 mg / mL, dissolved in 0.01 M HCl) and continue to stir at 700 rpm for 1 h. Subsequently, adjust the pH to 7.0 using NaOH solution (1 M) and continue to stir for 2 h for structural recombination and closure to form a keratin nanoparticle solution loaded with insulin (KNP-INS). Among them, the average particle size of the obtained KNP-INS is 141.2 ± 12.5 nm, and the "unfolding-closure" process of the keratin nanoparticle structure is as Figure 4 shown, Figure 4 where A in Figure 4 is the unfolding diagram of the keratin nanoparticle structure under acidic conditions (pH = 2),

[0055] and B in

[0056] is the closure diagram of the keratin nanoparticle structure under neutral conditions (pH = 7); Figure 6 The microscopic structure diagram of insulin (INS) is as shown in A in Figure 6 ; the microscopic structure diagram of calcium alginate-insulin (ALG-INS) is as shown in B in Figure 6 ; the microscopic structure of keratin-insulin (KNP-INS) is as shown in C in

[0057] ; the microscopic structure of calcium alginate-keratin-insulin nanoparticles (ALG-KNP-INS) is as shown in D in

[0058] Example 2

[0059] A preparation method of a stability-enhanced calcium alginate-keratin-based double-coated nanoparticle, which comprises the following steps:

[0060] Step 1: Keratin was uniformly dispersed in phosphate buffer (pH 7.0, 10 mM) at a concentration of 5% (w / v), and stirred at 700 rpm for 12 h for sufficient hydration. Subsequently, the pH of the buffer was adjusted to 8.5 with NaOH solution (1 M) for enzymatic hydrolysis. The enzymatic hydrolysis temperature was 55 °C, the enzyme dosage was 6000 U / g, and the degree of keratin hydrolysis was controlled at 5%. After the enzymatic hydrolysis was completed, the keratin hydrolysate was heated at 95 °C for 3 min to inactivate the enzyme. Subsequently, the pH was adjusted to 7.0 with HCl solution (1 M), and centrifuged at 8000 ×g for 20 min to obtain self-assembled keratin nanoparticles (KNP), which were freeze-dried at -76 °C and 0.009 mbar for use. The freeze-drying time was 72 h;

[0061] Step 2: 20 mg of keratin nanoparticles were dispersed in 2 mL of phosphate buffer (pH 2.0, 10 mM), and stirred at 700 rpm for 1 h for structure development. 80 μL of resveratrol (RES) solution (10 mg / mL, dissolved in ethanol) was added dropwise and continuously stirred at 700 rpm for 1 h. Subsequently, the pH was adjusted to 7.0 with NaOH solution (1 M), and stirring was continued for 2 h for structure recombination and closure to form a keratin nanoparticle solution loaded with resveratrol (KNP-RES);

[0062] Step 3: KNP-RES was added dropwise to an equal volume of sodium alginate solution (1%, w / v), and stirred at 700 rpm for 1 h. Subsequently, the mixture was added dropwise to an equal volume of CaCl2 solution (4 mM), and continuously stirred for 1 h to form alginate - keratin - based double - coated nanoparticles loaded with resveratrol: calcium alginate - keratin - resveratrol nanoparticles (ALG - KNP - RES). The free resveratrol was removed by centrifugation under the conditions of 8000 ×g and 20 min.

[0063] Among them, the microstructure of calcium alginate - keratin - resveratrol nanoparticles (ALG - KNP - RES) is as shown in Figure 13 C, the particle size analysis diagram of calcium alginate - keratin - resveratrol nanoparticles (ALG - KNP - RES) is as shown in Figure 13 B, the redissolution result diagram of calcium alginate - keratin - resveratrol nanoparticles (ALG - KNP - RES) is as shown in Figure 13 A, and the encapsulation efficiency of resveratrol is 95.61 ± 1.44%.

[0064] Example 3

[0065] The difference between this example and Example 1 lies in that: in the enzymatic hydrolysis in Step 1, the degree of keratin hydrolysis is controlled at 15%.

[0066] Comparative Example 1

[0067] The difference between this example and Example 1 lies in that: enzymatic hydrolysis was not carried out in Step 1.

[0068] Comparative Example 2

[0069] The difference between this example and Example 1 lies in that: in the enzymatic hydrolysis in Step 1, the degree of keratin hydrolysis is controlled at 20%. The microscopic morphologies of the self-assembled keratin nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2 are as Figure 3 shown, Figure 3 where A in Figure 3 is the microscopic morphology of the keratin nanoparticles prepared by controlling enzymatic hydrolysis (5% hydrolysis degree) in Example 1, Figure 3 B in Figure 3 is the microscopic morphology of the keratin nanoparticles prepared by controlling enzymatic hydrolysis (15% hydrolysis degree) in Example 3, Figure 3 C in

[0070] is the microscopic morphology diagram of the keratin nanoparticles prepared without enzymatic hydrolysis,

[0071] and D in

[0072] is the microscopic morphology of the keratin nanoparticles prepared by excessive enzymatic hydrolysis (20%). It can be seen from

[0073] that the average particle size of the keratin nanoparticles formed without enzymatic hydrolysis is in the micron range, with a compact structure and an irregular shape; the keratin particles formed by excessive enzymatic hydrolysis have an irregular shape and are prone to tight aggregation; the average particle size of the keratin nanoparticles formed by controlling the optimal enzymatic hydrolysis at 5% is 123 ± 7.0 nm, presenting a uniformly distributed hollow spherical shape. Controllable enzymatic hydrolysis can effectively prepare spherical keratin nanoparticles with regular shapes and uniform distributions. Figure 5 Comparative Example 3 Figure 6 Figure 5 shown. Through particle size analysis and redissolution analysis of the comparison between Example 1 and Comparative Example 3, the average particle size of KNP-INS prepared by pH adjustment in Step 2 of Example 1 of the present invention is 141.2 ± 12.5 nm, and the redissolved solution is clear and transparent. From Figure 6As can be seen from Figure C, KNP-INS presents a tightly granular structure with good dispersion, effectively improving insulin (INS) aggregation, reducing particle size, and enhancing dispersibility. The average particle size of KNP-INS-pH7 prepared without pH regulation is 316.0 ± 53.1 nm, and the reconstitution solution is relatively turbid with poor reconstitution ability. pH regulation can effectively reduce the particle size of keratin nanoparticles loaded with insulin and improve the reconstitution ability of KNP-INS. In addition, as Figure 4 shown, through pH regulation, the structure of keratin nanoparticles is completely opened at pH = 2.0, presenting a network structure; when the pH is adjusted back to 7.0, keratin recombines and closes to form tightly structured nanoparticles, and this "unfolding - closing" process can promote the encapsulation of bioactive molecules.

[0074] Performance Experiments

[0075] I. Microscopic Morphology of Nanoparticles:

[0076] The microscopic morphology of nanoparticles was observed using a transmission electron microscope (TEM). The microscopic morphology of calcium alginate - keratin - insulin nanoparticles (ALG - KNP - INS) prepared in Example 1 is as shown in Figure 6 Figure D. As can be seen from Figure 6 Figure D, ALG - KNP - INS presents a regular spherical shape with good dispersion. The microscopic structure of calcium alginate - keratin - resveratrol nanoparticles (ALG - KNP - RES) prepared in Example 2 is as shown in Figure 13 Figure C. As can be seen from Figure 13 Figure C, ALG - KNP - RES presents a regular spherical shape with good dispersion.

[0077] II. Particle Size Analysis and Reconstitution Ability of Nanoparticles:

[0078] The particle size of nanoparticles was analyzed using a Malvern ZetaSizer; the reconstitution ability was judged based on the dispersion clarity of the nanoparticle aqueous dispersion (1 mg / mL).

[0079] The particle size analysis results of the alginate - keratin - based double - coated nanoparticles loaded with insulin (ALG - KNP - INS) prepared in Example 1, and the particle size comparison results with INS, ALG - INS, and KNP - INS are as shown in Figure 7 Figure B. As can be seen from Figure 7 Figure B, the average particle size of ALG - KNP - INS is 173.0 ± 14.4 nm; the reconstitution ability results of calcium alginate - keratin - insulin nanoparticles (ALG - KNP - INS) prepared in Example 1, and the reconstitution ability comparison results with INS, ALG - INS, and KNP - INS are as shown in Figure 7 Figure A. As can be seen fromFigure 7 It can be obtained from A that the ALG-KNP-INS complex solution is clear and transparent, and has good redissolution property. The particle size analysis diagram of the calcium alginate-keratin-resveratrol nanoparticles (ALG-KNP-RES) prepared in Example 2 is as Figure 13 shown in B, and the redissolution result diagram is as Figure 13 shown in A. From Figure 13 it can be obtained that the average particle size of the calcium alginate-keratin-resveratrol nanoparticles (ALG-KNP-RES) is 170.5 ± 11.3 nm, the complex solution is clear and transparent, and has good redissolution property. The encapsulation rate of resveratrol is 95.61 ± 1.44%. The alginate-keratin-based double coating layer can also efficiently encapsulate the hydrophobic molecule resveratrol, effectively reduce the particle size of resveratrol, and improve its aqueous solution dispersibility and solubility.

[0080] III. Chemical structure analysis of nanoparticles:

[0081] The Fourier transform infrared spectroscopy (FTIR) spectrometer was used to analyze the chemical structure of the nanoparticles. The infrared structure analysis diagram of the calcium alginate-keratin-insulin nanoparticles (ALG-KNP-INS) prepared in Example 1 is as Figure 8 shown. From Figure 8 it can be seen that through FTIR analysis, the results show that the ALG-KNP-INS infrared spectrum has characteristic peaks of alginate, indicating the presence of an effective calcium alginate coating layer.

[0082] IV. Thermal stability and storage stability:

[0083] Thermal stability: Taking the calcium alginate-keratin-insulin nanoparticles (ALG-KNP-INS) prepared in Example 1 as an example: The aqueous dispersion of ALG-KNP-INS (1 mg / mL) was heated in a water bath at 80 °C for 0 - 60 minutes, and then cooled to room temperature with running water. Insulin was quantitatively analyzed by HPLC. The thermal stability of the nanoparticles was evaluated according to the insulin retention rate after heat treatment for a certain period of time. The analysis results are as Figure 9 shown.

[0084] Storage stability: Taking the calcium alginate-keratin-insulin nanoparticles (ALG-KNP-INS) prepared in Example 1 as an example: The aqueous dispersion of ALG-KNP-INS (1 mg / mL) was stored at 4 °C for 0 - 28 days, and then insulin was quantitatively analyzed by HPLC. The storage stability of the nanoparticles was evaluated according to the insulin retention rate after low-temperature storage for a certain period of time. The analysis results are as Figure 10 shown.

[0085] From Figure 9 and Figure 10It can be seen that through the analysis of thermal stability and storage stability, the results show that during heat treatment (80 °C, 0 - 60 min) and low-temperature storage (4 °C, 0 - 28 days), the protective effect of ALG-KNP-INS on insulin is significantly enhanced, and its insulin retention rate is significantly higher than that of INS, KNP-INS, and ALG-INS. After 30 min of heat treatment and 3 weeks of refrigeration, the insulin retention rate is still above 90%.

[0086] V. Gastrointestinal digestion stability:

[0087] Taking the calcium alginate-keratin-insulin nanoparticles (ALG-KNP-INS) prepared in Example 1 as an example:

[0088] Gastric digestion: The sample was dispersed in HCl solution with a pH of 2.0 to make the final insulin concentration 1 mg / mL, 1.6 mg / mL of pepsin was added, and digestion was simulated at a stirring speed of 250 rpm in a 37 °C water bath for 90 min. After gastric digestion, the pH was adjusted to 6.8 using 1M NaOH and quickly cooled in an ice bath.

[0089] Intestinal digestion: After gastric digestion, the pH of the sample was adjusted to 6.8 with NaOH (1M) and mixed with an equal volume of trypsin solution (10 mg / mL, dissolved in phosphate buffer with a pH of 6.8 and 10 mM). Subsequently, digestion was continued in a 37 °C water bath for 120 minutes at a stirring rate of 250 rpm. Digested samples were collected at predetermined time points (30, 60, and 120 min) and quickly cooled. The particle size change of the digested sample without added enzyme was observed, and the results are as Figure 11 shown. The insulin stability of the enzymatically digested sample was analyzed using the SDS-PAGE method, and the results are as Figure 12 shown.

[0090] The results of gastrointestinal simulated digestion show that ALG-KNP-INS has a pH-responsive structural change in the gastrointestinal environment ( Figure 11 ), and ALG-KNP-INS first aggregates and then disperses as the pH changes from 2.0 to 6.8, potentially blocking the degradation of the loaded insulin by proteases. Figure 12 It is shown that ALG-KNP-INS can effectively alleviate the degradation of insulin by gastrointestinal proteases. Compared with other forms, the double coating layer of calcium alginate-keratin (ALG-KNP) can effectively improve insulin aggregation, reduce particle size, improve its dispersibility and redissolubility, and significantly improve the stability of insulin during heating, storage, and digestion.

[0091] VI. Transmembrane transport and cellular uptake:

[0092] Taking the calcium alginate - keratin - insulin nanoparticles (ALG - KNP - INS) prepared in Example 1 as an example:

[0093] A monolayer cell membrane was constructed using Caco2 cells to simulate small intestinal epithelial cells, and the transmembrane transport ability of the nanoparticles was analyzed (the results are shown in Figure 14 A as shown below); by co - culturing Caco2 cells and the nanoparticles, the cell delivery ability of the nanoparticles to insulin (INS) was evaluated. Among them, insulin (INS) was labeled with fluorescein, and its cell uptake was observed using a confocal laser scanning microscope (CLSM) (the results are shown in Figure 14 B as shown below).

[0094] Through the Caco2 cell experiment, it was found that ALG - KNP - INS could cross the Caco2 monolayer cell membrane ( Figure 14 shown in A below); the cell uptake experiment showed that ALG - KNP - INS had the cell delivery ability to INS ( Figure 14 shown in B below). The ALG - KNP double - coating layer has certain application potential for improving the aggregation, water solubility, stability of bioactive molecules and achieving transmembrane delivery.

[0095] Traditional bioactive molecules such as resveratrol and insulin have disadvantages such as poor water solubility, easy environmental degradation, poor intestinal absorption, and poor transmembrane penetration, resulting in poor storage stability and low bioavailability. In the present invention, through controllable enzymatic hydrolysis and pH regulation techniques, by regulating the self - assembly behavior of keratin molecules, the problems of poor water solubility and easy aggregation of keratin are effectively solved, and the encapsulation ability of bioactive molecules is improved. The average particle size of the keratin nanoparticles loaded with bioactive molecules prepared is 131 - 141 nm. Through the alginate - keratin - based double - coating technology, spherical double - coated nanoparticles with regular shapes can be prepared, with an average particle size of 170 - 173 nm, having improved water solubility and environmental stability (such as heat treatment and low - temperature storage). Its responsive change to gastrointestinal pH can significantly improve the stability of bioactive molecules and can achieve cell delivery of bioactive molecules. The alginate - keratin - based double - coating layer prepared by the invention has an encapsulation effect on both hydrophilic molecules (insulin) and hydrophobic molecules (resveratrol), and can be widely applied to the encapsulation and delivery of bioactive molecules, including but not limited to oral nano - delivery systems. The preparation method of the present invention simplifies the preparation process of nanoparticles loaded with bioactive molecules, improves production efficiency, and does not involve chemical reagent pollution.

[0096] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated examples herein.

Claims

1. A method for preparing stability-enhanced alginate-keratin-based double-coated nanoparticles, characterized in that: The steps include: Step 1, keratin is evenly dispersed in a phosphate buffer, stirred for 12 to 16 hours for full hydration, the pH is adjusted to 8.5, the temperature is 50 to 60°C, the enzyme dosage is 5000-8000 U / g, and the degree of hydrolysis is controlled to be 5 to 15% for enzymatic degradation; after the enzymatic hydrolysis is completed, the enzyme activity is inactivated by heating at 95°C for 3 minutes, the pH is adjusted to 7.0, and the mixture is centrifuged at 6000 to 8000 × g for 20 to 30 minutes, and lyophilized to obtain self-assembled keratin nanoparticles; Step 2: dispersing the keratin nanoparticles obtained in step 1 in an acidic buffer solution with a pH of 1.5 to 3.0 for structural development, adding bioactive molecules, stirring, and adjusting the pH value to neutral to allow the keratin structure to be reorganized and closed to obtain a keratin nanoparticle solution loaded with bioactive molecules; Step 3: Disperse the keratin nanoparticle solution loaded with bioactive molecules obtained in step 2 in a sodium alginate solution, add a calcium chloride solution dropwise to cross-link and form a calcium alginate shell after mixing evenly, dialyze or centrifuge to remove free bioactive molecules, and freeze-dry to obtain alginate-keratin double-coated nanoparticles loaded with bioactive molecules.

2. The preparation method according to claim 1, characterized in that Step 2 specifically includes: S21, dispersing the keratin nanoparticles obtained in step 1 in a phosphate buffer solution with a pH of 1.5 to 3.0, and stirring at 600 to 800 rpm for 1 to 2 h to develop the structure; S22, adding bioactive molecules, continuing stirring for 1 to 2 hours, adjusting the pH to 7.0, and continuing stirring for 1 to 2 hours to reorganize and close the structure to form a keratin nanoparticle solution loaded with bioactive molecules.

3. The preparation method according to claim 1, characterized in that: Step three specifically includes: S31, adding the keratin nanoparticle solution loaded with bioactive molecules obtained in step 2 dropwise into an equal volume of a sodium alginate solution having a mass volume concentration of 1 to 3%, and continuously stirring for 1 to 2 hours to obtain a mixed solution; S32. Add the mixed solution dropwise into an equal volume of 2-10 mM calcium chloride solution, continue stirring for 1-2 h, remove free bioactive molecules by dialysis or centrifugation, and freeze-dry to obtain alginate-keratin-based double-coated nanoparticles loaded with bioactive molecules.

4. The preparation method according to claim 1, characterized in that: The bioactive molecule is insulin or resveratrol.

5. The preparation method according to claim 1, characterized in that: In step 1, the enzymatic hydrolysis temperature is 55° C., and the enzyme dosage is 6000 U / g; freeze-drying specifically includes: freeze-drying the enzymatic hydrolysis supernatant at -76° C. and 0.009 mbar for 72 h.

6. The preparation method according to claim 4, characterized in that: When the bioactive molecule is insulin, the mass ratio of keratin nanoparticles to the bioactive molecule is 2:1; when the bioactive molecule is resveratrol, the mass ratio of keratin nanoparticles to the bioactive molecule is 25:

1.

7. The preparation method according to claim 3, characterized in that: S32 specifically includes: adding the mixed solution dropwise into an equal volume of 4 mM calcium chloride solution, stirring continuously at 700 rpm for 1 h, dialyzing with a dialysis bag with a molecular weight cutoff of 7 kDa to remove free bioactive molecules, the dialysis time is ≥36 h, and freeze-drying at -76°C and 0.009 mbar for 72 h to obtain alginate-keratin-based double-coated nanoparticles loaded with bioactive molecules.

8. Stability-enhanced alginate-keratin-based double-coated nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the stability-enhanced alginate-keratin-based double-coated nanoparticles according to claim 8 in preparing nano-delivery carriers for bioactive molecules.

Images