A disulfide cross-linked starch-based micelle and a preparation method and application thereof

Abstract

The application discloses a kind of disulfide crosslinked starch-based micelles and its preparation method and application.The preparation method includes: using pullulanase to carry out branch removal treatment to waxy corn starch, and prepare dextrin;Using periodate on the dextrin is oxidized, and prepare dialdehyde dextrin;Make cysteamine and the dialdehyde dextrin grafting reaction, and prepare cysteamine-dextrin;The cysteamine-dextrin is dissolved in water and self-assembled to form micelles, then ultrasonic sulfhydryl oxidation is carried out, and prepare disulfide crosslinked starch-based micelles.The disulfide crosslinked starch-based micelles provided by the application with hydrophobic functional factor has higher hydrophobic functional factor loading and environmental stress stability, in vitro simulation release presents colon targeting, and has wide application prospect in the field of oral delivery of hydrophobic functional factor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanomaterials, and particularly relates to a disulfide cross-linked starch-based micelle as well as a preparation method and application thereof. BACKGROUND

[0002] Many hydrophobic functional factors beneficial to human health are limited in oral absorption efficiency due to low water solubility, sensitivity to environment (digestive enzymes, temperature, pH, etc.), difficulty in passing through the intestinal barrier, and easy interaction with food.

[0003] Micelles, as a new encapsulation and delivery system of hydrophobic functional factors, have attracted the attention of many researchers in recent decades. Micelles are nanocarriers with core-shell structure composed of amphiphilic compounds. The internal hydrophobic core can encapsulate hydrophobic molecules, and the external hydrophilic shell is in contact with the water environment, significantly improving the solubility of hydrophobic functional factors in water. The micelles have the characteristics of small size, strong controllability, and high loading capacity, which can enhance the penetration of nanocarriers through the intestinal mucosa, achieve controlled release and targeted delivery in the gastrointestinal tract, and greatly improve the oral bioavailability of drugs. Compared with chemically synthesized materials, micelles made of polysaccharides have the advantages of low toxicity, environmental friendliness, and biocompatibility, which are beneficial to reduce potential adverse effects.

[0004] Starch is a polysaccharide widely existing in nature, which has the advantages of low cost, easy availability, safety, etc. The rich hydroxyl structure of starch is an ideal group for chemical reaction, which can be introduced into other structures through simple chemical modification, thereby endowing starch with new properties and functions, and is considered as an ideal material for preparing micelles.

[0005] The modification methods of starch-based micelles have also been widely explored, such as using cross-linking agents (epichlorohydrin or aldehyde compounds) to react with the hydroxyl groups on the starch molecules to form a cross-linked network; introducing inorganic or organic acids to convert the hydroxyl groups into ester groups, thereby changing the hydrophilic or hydrophobic properties of starch; introducing acetylated groups to increase the hydrophobicity of starch, etc. These modification methods also have certain defects, for example, cross-linking may cause excessive hardening of starch particles, affecting their biodegradability; incomplete esterification may result in non-uniform properties of starch micelles; acetylation may reduce the hydrophilicity of starch particles, affecting their application in water environment, and the introduction of acetyl groups may also affect the biocompatibility of starch. Therefore, it is necessary to find a new modification method for starch-based micelles. SUMMARY

[0006] The main purpose of the present application is to provide a disulfide cross-linked starch-based micelle as well as a preparation method and application thereof, so as to overcome the deficiencies of the prior art.

[0007] To achieve the aforementioned purposes, the technical solutions adopted by the present application include:

[0008] The embodiment of the present application provides a preparation method of disulfide cross-linked starch-based micelles, which comprises the following steps:

[0009] The waxy corn starch is subjected to branch-removing treatment by adopting a pullulanase to obtain dextrin;

[0010] The dextrin is subjected to oxidation by adopting a periodate to obtain dialdehyde dextrin;

[0011] The cysteamine is subjected to grafting reaction with the dialdehyde dextrin to obtain cysteamine-dextrin;

[0012] And the cysteamine-dextrin is dissolved in water and subjected to self-assembly to form micelles, and then subjected to ultrasonic thiol oxidation to obtain the disulfide cross-linked starch-based micelles.

[0013] The embodiment of the present application further provides the disulfide cross-linked starch-based micelles prepared by the preparation method.

[0014] The embodiment of the present application further provides a preparation method of disulfide cross-linked starch-based micelles loaded with a hydrophobic functional factor, which comprises the following steps:

[0015] The disulfide cross-linked starch-based micelles are prepared by the preparation method;

[0016] And the hydrophobic functional factor is mixed with the disulfide cross-linked starch-based micelles to obtain the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor.

[0017] The embodiment of the present application further provides the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor prepared by the preparation method.

[0018] The embodiment of the present application further provides the use of the disulfide cross-linked starch-based micelles or the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor in the field of colon targeting or oral delivery.

[0019] Compared with the prior art, the present application has the beneficial effects that:

[0020] (1) In the present application, the natural amino acid cysteamine is selected as a modifier, and the cysteamine is helpful to reduce potential toxicity and side effects on the human body due to its excellent biocompatibility; meanwhile, the free thiol group at the terminal of the structure of the cysteamine can easily form intermolecular and intramolecular disulfide bonds, and this structure significantly enhances the structural stability of the micelles; in addition, the cysteamine also has significant antioxidant properties, which can effectively protect the active ingredients in the micelles from oxidative damage, thereby prolonging the effective period of the active ingredients;

[0021] (2) The ultrasonic treatment in the present application can effectively disperse the agglomerated particles, reduce the size distribution range of the particles, and improve the particle size uniformity, while the ultrasonic waves can cause intense vortex and shear in the liquid, improve the collision frequency of the mercapto groups between different chain segments, and accelerate the process of forming disulfide bonds from mercapto groups;

[0022] (3) The micelles in the present application are mainly stabilized by disulfide bonds and hydrophobic interaction, supplemented by hydrogen bonds, and the content of free mercapto groups can be changed by controlling the addition amount of cysteamine, so as to control the size of the formed micelles. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 is a graph of the change of the particle size of the starch-based micelles in Examples 1-3 and Comparative Example 1 of the present application with pH;

[0025] Figure 2 is a graph of the change of the zeta potential of the starch-based micelles in Examples 1-3 and Comparative Example 1 of the present application with pH;

[0026] Figure 3 is a graph of the change of the particle size of the starch-based micelles in Examples 4-6 and Comparative Example 2 of the present application with ionic strength;

[0027] Figure 4 is a graph of the change of the zeta potential of the starch-based micelles in Examples 4-6 and Comparative Example 2 of the present application with ionic strength;

[0028] Figures 5a-5b is a graph of the change of the cumulative release rate of curcumin with time of the starch-based micelles loaded with curcumin in Example 5 and Comparative Example 2 in the environment with pH of 1.2, 6.0 and 7.4, respectively. DETAILED DESCRIPTION

[0029] In view of the defects of the prior art, the present inventors have long studied and practiced and finally proposed the technical scheme of the present application by developing a new kind of disulfide cross-linked starch-based micelles to load hydrophobic functional factors, which not only improves the water solubility of the hydrophobic functional factors, but also enhances the stability in the gastrointestinal tract. The reaction principle of the present application is to reduce the molecular weight of starch molecules by enzymatic hydrolysis to form small molecular dextrin, so that it is easier to self-assemble, and then introduce cysteamine structure into the starch skeleton to improve the hydrophobicity of dextrin molecules, which are aggregated to form micelles under the action of hydrophobic interaction, and the ultrasonic treatment is used to promote the oxidation of sulfhydryl to form disulfide cross-linked starch-based micelles.

[0030] The technical scheme of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] Specifically, as one aspect of the technical scheme of the present application, a preparation method of a disulfide cross-linked starch-based micelle comprises:

[0032] Purified enzyme is used to carry out branch removal treatment on waxy corn starch to prepare dextrin;

[0033] Potassium periodate is used to oxidize the dextrin to prepare dialdehyde dextrin;

[0034] Cysteamine is grafted with the dialdehyde dextrin to prepare cysteamine-dextrin;

[0035] And the cysteamine-dextrin is dissolved in water and self-assembled to form micelles, and then subjected to ultrasonic sulfhydryl oxidation to prepare disulfide cross-linked starch-based micelles.

[0036] In some preferred embodiments, the preparation method specifically comprises: mixing waxy corn starch with phosphate buffer solution with a pH value of 4.0-5.0 to form a waxy corn starch dispersion, then heating and gelatinizing in a boiling water bath, then adding purified enzyme and stirring and reacting in a water bath for 15-24 h, and then carrying out enzyme inactivation, freeze-drying treatment to prepare dextrin.

[0037] Further, the concentration of waxy corn starch in the waxy corn starch dispersion is 3-6 w / v% (g / ml).

[0038] Further, the ratio of the amount of purified enzyme to waxy corn starch is 20-50 U: 1 g.

[0039] In some preferred embodiments, the preparation method specifically comprises: dissolving dextrin in water to form a dextrin solution, then adding a periodate salt, mixing and reacting at room temperature for 1-2 hours in the dark, then adding ethylene glycol and reacting for 1-2 hours, and then performing dialysis and freeze-drying to obtain dialdehyde dextrin.

[0040] Further, the concentration of dextrin in the dextrin solution is 2.5-5.0 w / v%.

[0041] Further, the mass ratio of the periodate salt to dextrin is 10-40:100.

[0042] Further, the periodate salt comprises sodium periodate and / or potassium periodate, and is not limited thereto.

[0043] In some preferred embodiments, the preparation method specifically comprises: dissolving dialdehyde dextrin in water to form a dialdehyde dextrin solution, then adding MES hydrate and cysteamine, adjusting the pH value to 3-4 and stirring and reacting at room temperature for 3 hours, then adding sodium cyanoborohydride and stirring and reacting at room temperature for 72 hours, and then performing dialysis and freeze-drying to obtain cysteamine-dextrin.

[0044] Further, the concentration of dialdehyde dextrin in the dialdehyde dextrin solution is 1.0-2.5 w / v%.

[0045] Further, the mass ratio of cysteamine to dialdehyde dextrin is 50:100.

[0046] Further, the mass ratio of sodium cyanoborohydride to dialdehyde dextrin is 200-400:100.

[0047] In some preferred embodiments, the preparation method specifically comprises: dissolving cysteamine-dextrin in water at 70-85°C for 10-30 minutes to form a cysteamine-dextrin solution, then oscillating at room temperature for 12-24 hours to form a micelle solution, and then performing ultrasonic treatment at a power of 100-400 W for 1-10 minutes to obtain disulfide cross-linked starch-based micelles.

[0048] Further, the concentration of cysteamine-dextrin in the cysteamine-dextrin solution is 0.5-2 w / v%.

[0049] Another aspect of the embodiments of the present application also provides the disulfide cross-linked starch-based micelles prepared by the aforementioned preparation method.

[0050] Another aspect of the embodiments of the present application also provides a preparation method of disulfide cross-linked starch-based micelles loaded with a hydrophobic functional factor, which comprises:

[0051] The disulfide cross-linked starch-based micelles are prepared by the aforementioned preparation method.

[0052] and mixing homogeneously the hydrophobic functional factor with the disulfide cross-linked starch-based micelles to obtain the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor.

[0053] In some preferred embodiments, the preparation method specifically comprises: adding the hydrophobic functional factor into the disulfide cross-linked starch-based micelles and homogenizing for 1N2 min at 8000-12000 rpm, then oscillating at room temperature for 12-24 h, and then removing the un-encapsulated hydrophobic functional factor by centrifugation to obtain the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor.

[0054] Further, the mass-volume ratio of the hydrophobic functional factor to the disulfide cross-linked starch-based micelles is 20-50 mg: 100 ml.

[0055] Further, the hydrophobic functional factor comprises any one or a combination of curcumin, quercetin, catechol, and the like, but is not limited thereto.

[0056] In some more specific embodiments, the preparation method of the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor comprises the following steps:

[0057] (1) debranching waxy corn starch by using pullulanase to obtain dextrin;

[0058] (2) oxidizing the dextrin by using sodium periodate to obtain dialdehyde dextrin;

[0059] (3) grafting cysteamine onto the dialdehyde dextrin according to reductive amination reaction to obtain cysteamine-dextrin;

[0060] (4) dissolving the cysteamine-dextrin in water by heating, then oscillating at room temperature to make hydrophobic segments aggregate with each other under the driving force of interaction, and finally self-assembling to form micelles. Subsequently, ultrasonic treatment is performed to promote oxidation of thiol groups, and disulfide cross-linked starch-based micelles are obtained;

[0061] (5) adding the hydrophobic functional factor into the micelle solution, homogeneously dispersing, and oscillating at room temperature to make the hydrophobic functional factor migrate to the hydrophobic core of the micelles, and finally obtaining the disulfide cross-linked starch-based micelles loaded with the hydrophobic functional factor.

[0062] Further, step (1) specifically comprises: using a phosphate buffer with a pH value of 4.0-5.0 to prepare a starch milk with a concentration of 3-6 w / v%, heating and gelatinizing in a boiling water bath for 30-60 min, adding pullulanase (the ratio of the amount of pullulanase to waxy corn starch is 20-50 U: 1 g) after cooling, stirring in a water bath for 15-24 h, heating at 85-95 ℃ for 10-20 min to inactivate the enzyme after the reaction is completed, and freeze-drying to obtain dextrin powder;

[0063] Further, step (2) specifically comprises: configuring a dextrin solution with a concentration of 2.5-5.0 w / v %, adding sodium periodate (the mass ratio of sodium periodate to dextrin is 10-40:100), mixing and stirring, reacting for 1-2 h at room temperature in the dark, adding 0.5-1 mL / g (dry mass of dextrin) of ethylene glycol to react for 1-2 h to remove unreacted sodium periodate, then dialyzing (dialysis bag with a molecular weight cut-off of 1000-3500 Da) for three days, and freeze-drying to obtain a dialdehyde dextrin powder;

[0064] Further, step (3) specifically comprises: configuring a dialdehyde dextrin solution with a concentration of 1.0-2.5 w / v %, adding MES hydrate and cysteamine (the mass ratio of cysteamine to dialdehyde dextrin is 50:100) to a final concentration of 0.1 M, adjusting the pH value to 3-4 with hydrochloric acid, and stirring for 3 h at room temperature. Then, adding sodium cyanoborohydride (the mass ratio of sodium cyanoborohydride to dialdehyde dextrin is 200-400:100), stirring for 72 h at room temperature, dialyzing (dialysis bag with a molecular weight cut-off of 1000-3500 Da) for three days with distilled water at pH=3 after the reaction is completed, and freeze-drying to obtain a cysteamine-dextrin powder;

[0065] Further, step (4) specifically comprises: configuring a cysteamine-dextrin solution with a concentration of 0.5-2 w / v %, dissolving by heating at 70-85 °C for 10-30 min, and preparing a micelle solution by oscillation at room temperature for 12-24 h. Then, ultrasonicating for 1-10 min under a power of 100-400 W to obtain a two-sulfur cross-linked starch-based micelle solution;

[0066] Further, step (5) specifically comprises: adding curcumin (the mass-volume ratio of curcumin to two-sulfur cross-linked starch-based micelles is 20-50 mg:100 mL) to the two-sulfur cross-linked starch-based micelle solution, homogenizing for 1-2 min at a speed of 8000-12000 rpm, then oscillating at room temperature for 12-24 h to make the curcumin migrate to the hydrophobic core of the micelles, and then centrifuging to remove the un-encapsulated curcumin to obtain a curcumin-loaded micelle solution.

[0067] Another aspect of the embodiments of the present application also provides the two-sulfur cross-linked starch-based micelles prepared by the preparation method described above.

[0068] Another aspect of the embodiments of the present application also provides the use of the two-sulfur cross-linked starch-based micelles or the curcumin-loaded two-sulfur cross-linked starch-based micelles described above in the field of colon-targeted or oral delivery.

[0069] The technical solutions of the present application will be further described in detail below in combination with several preferred embodiments and the accompanying drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0070] The experimental materials used in the following examples are commercially available from conventional biochemical reagent companies, unless otherwise specified.

[0071] Example 1

[0072] 1. Preparation of dextrin

[0073] A 6 w / v% starch solution was prepared using a phosphate buffer with a pH of 4.5, and was gelatinized by heating in a boiling water bath for 30 min. After cooling, pullulanase was added (the ratio of pullulanase to waxy corn starch was 20 U: 1 g), and the solution was stirred in a water bath for 24 h. After the reaction was completed, the enzyme was inactivated by heating at 95°C for 20 min, and the dextrin powder was obtained by freeze-drying.

[0074] 2. Preparation of dialdehyde dextrin

[0075] A 3.0 w / v% dextrin solution was prepared, and sodium periodate was added (the mass ratio of sodium periodate to dextrin was 20: 100). The mixture was stirred and reacted at room temperature for 2 h in the dark. Then, 1 mL / g (dry mass of dextrin) of ethylene glycol was added and reacted for 1 h to remove unreacted sodium periodate. Subsequently, the solution was dialyzed (dialysis bag with a molecular weight cut-off of 1000 Da) for three days, and the dialdehyde dextrin powder was obtained by freeze-drying.

[0076] 3. Preparation of cysteamine-dextrin

[0077] A 1.0 w / v% dialdehyde dextrin solution was prepared, and MES hydrate and cysteamine were added to a final concentration of 0.1 M (the mass ratio of cysteamine to dialdehyde dextrin was 25: 100). The pH was adjusted to 3 using hydrochloric acid, and the solution was stirred at room temperature for 3 h. Then, sodium cyanoborohydride was added (the mass ratio of sodium cyanoborohydride to dialdehyde dextrin was 400: 100), and the solution was stirred at room temperature for 72 h. After the reaction was completed, the solution was dialyzed (dialysis bag with a molecular weight cut-off of 1000 Da) for three days using distilled water with a pH of 3, and the cysteamine-dextrin powder was obtained by freeze-drying.

[0078] 4. Preparation of disulfide cross-linked starch-based micelles

[0079] A 1.0 w / v% cysteamine-dextrin solution was prepared, and was dissolved by heating at 85°C for 10 min. The solution was prepared into a micelle solution by oscillation at room temperature for 24 h. Then, the solution was ultrasonicated for 3 min at a power of 300 W to obtain a disulfide cross-linked starch-based micelle solution.

[0080] Example 2

[0081] 1. Preparation of dextrin

[0082] Dextrin powder was obtained by using 6 w / v% starch solution prepared with pH 4.5 phosphate buffer, heating in boiling water bath for 30 min, adding pullulanase (the ratio of pullulanase to waxy corn starch was 20 U: 1 g) after cooling, stirring in water bath for 24 h, heating at 95 ℃ for 20 min to inactivate the enzyme after the reaction was completed, and freeze-drying;

[0083] 2. Preparation of dialdehyde dextrin

[0084] Dialdehyde dextrin powder was obtained by using 3.0 w / v% dextrin solution, adding sodium periodate (the mass ratio of sodium periodate to dextrin was 20:100), mixing and stirring, reacting at room temperature for 2 h in the dark, adding 1 mL / g (dry basis mass of dextrin) ethylene glycol for 1 h to remove unreacted sodium periodate, then dialyzing (dialysis bag molecular weight cutoff 1000 Da) with distilled water for three days, and freeze-drying;

[0085] 3. Preparation of cysteamine-dextrin

[0086] Cysteamine-dextrin powder was obtained by using 1.0 w / v% dialdehyde dextrin solution, adding 0.1 M MES hydrate and cysteamine (the mass ratio of cysteamine to dialdehyde dextrin was 50:100) to a final concentration, adjusting the pH to 3 with hydrochloric acid, stirring at room temperature for 3 h. Then, sodium cyanoborohydride (the mass ratio of sodium cyanoborohydride to dialdehyde dextrin was 400:100) was added, stirring at room temperature for 72 h, dialyzing (dialysis bag molecular weight cutoff 1000 Da) with pH 3 distilled water for three days after the reaction was completed, and freeze-drying;

[0087] 4. Preparation of disulfide cross-linked starch-based micelles

[0088] Micelle solution was prepared by using 1.0 w / v% cysteamine-dextrin solution, heating at 85 ℃ for 10 min to dissolve, and oscillating at room temperature for 24 h. Then, the solution was ultrasonicated at 300 W for 3 min to obtain disulfide cross-linked starch-based micelle solution.

[0089] Example 3

[0090] 1. Preparation of dextrin

[0091] Dextrin powder was obtained by using 6 w / v% starch solution prepared with pH 4.5 phosphate buffer, heating in boiling water bath for 30 min, adding pullulanase (the ratio of pullulanase to waxy corn starch was 20 U: 1 g) after cooling, stirring in water bath for 24 h, heating at 95 ℃ for 20 min to inactivate the enzyme after the reaction was completed, and freeze-drying;

[0092] 2. Preparation of dialdehyde dextrin

[0093] Prepare a 3.0 w / v dextrin solution, add sodium periodate (sodium periodate to dextrin mass ratio of 20:100), mix and stir, react at room temperature in the dark for 2 h, add 1 mL / g (dextrin dry basis mass) ethylene glycol and react for 1 h to remove unreacted sodium periodate, then dialyze with distilled water (dialysis bag molecular weight cutoff 1000 Da) for three days, and freeze-dry to obtain dialdehyde dextrin powder;

[0094] 3. Preparation of cysteamine-dextrin

[0095] A 1.0 w / v% dialdehyde dextrin solution was prepared, and MES hydrate and cysteamine (cysteamine to dialdehyde dextrin mass ratio of 100:100) were added to a final concentration of 0.1 M. The pH was adjusted to 3 with hydrochloric acid, and the mixture was stirred at room temperature for 3 h. Subsequently, sodium cyanoborohydride (sodium cyanoborohydride to dialdehyde dextrin mass ratio of 400:100) was added, and the mixture was stirred at room temperature for 72 h. After the reaction was completed, the mixture was dialyzed with distilled water at pH=3 (dialysis bag molecular weight cutoff 1000 Da) for three days, and then freeze-dried to obtain cysteamine-dextrin powder.

[0096] 4. Preparation of disulfide-crosslinked starch-based micelles

[0097] A 1.0 w / v cysteamine-dextrin solution was prepared, heated at 85 °C for 10 min to dissolve, and then shaken at room temperature for 24 h to obtain a micelle solution. The solution was then sonicated at 300 W for 3 min to obtain a disulfide-crosslinked starch-based micelle solution.

[0098] Example 4

[0099] 1. Preparation of dextrin

[0100] Prepare a 6 w / v starch milk using a phosphate buffer solution with a pH of 4.5, heat it in a boiling water bath for 30 min to gelatinize it, cool it down and add pullulanase (the ratio of pullulanase to glutinous corn starch is 20 U: 1 g), stir it in a water bath for 24 h, after the reaction is complete, heat it at 95 °C for 20 min to inactivate the enzyme, and freeze dry it to obtain dextrin powder.

[0101] 2. Preparation of dialdehyde dextrin

[0102] A 3.0 w / v dextrin solution was prepared, and sodium periodate (sodium periodate to dextrin mass ratio of 20:100) was added and stirred. The mixture was reacted at room temperature in the dark for 2 h. Then, 1 mL / g (dry basis mass of dextrin) of ethylene glycol was added and reacted for 1 h to remove unreacted sodium periodate. Subsequently, the mixture was dialyzed with distilled water (dialysis bag molecular weight cutoff 1000 Da) for three days and then freeze-dried to obtain dialdehyde dextrin powder.

[0103] 3. Preparation of cysteamine-dextrin

[0104] A solution of dialdehyde-dextrin was prepared at a concentration of 1.0 w / v%, and MES hydrate and cysteamine were added to a final concentration of 0.1 M (mass ratio of cysteamine to dialdehyde-dextrin was 25:100), and the pH was adjusted to 3 with hydrochloric acid. The solution was stirred at room temperature for 3 h. Sodium cyanoborohydride was then added (mass ratio of sodium cyanoborohydride to dialdehyde-dextrin was 400:100), and the solution was stirred at room temperature for 72 h. The reaction was completed by dialysis (dialysis bag with a molecular weight cut-off of 1000 Da) in distilled water at pH 3 for 3 days, and the cysteamine-dextrin powder was obtained by freeze-drying.

[0105] 4. Preparation of disulfide cross-linked starch-based micelles

[0106] A solution of cysteamine-dextrin was prepared at a concentration of 1.0 w / v%, and the solution was dissolved by heating at 85°C for 10 min and then shaken at room temperature for 24 h to obtain a micelle solution. The micelle solution was then ultrasonicated at a power of 300 W for 3 min to obtain a solution of disulfide cross-linked starch-based micelles.

[0107] 5. Preparation of curcumin-loaded disulfide cross-linked starch-based micelles

[0108] Curcumin was added to the solution of disulfide cross-linked starch-based micelles (mass-volume ratio of curcumin to disulfide cross-linked starch-based micelles was 50 mg:100 mL), and the solution was homogenized at a speed of 12000 rpm for 1-2 min. The curcumin was then migrated to the hydrophobic core of the micelles by shaking at room temperature for 24 h. The curcumin that was not encapsulated was removed by centrifugation to obtain a solution of curcumin-loaded micelles.

[0109] Example 5

[0110] 1. Preparation of dextrin

[0111] A starch solution was prepared at a concentration of 6 w / v% using a phosphate buffer at a pH of 4.5. The starch was gelatinized by heating in a boiling water bath for 30 min. After cooling, pullulanase was added (dose ratio of pullulanase to waxy corn starch was 20 U:1 g), and the solution was stirred in a water bath for 24 h. The enzyme was inactivated by heating at 95°C for 20 min after the reaction was completed. The dextrin powder was obtained by freeze-drying.

[0112] 2. Preparation of dialdehyde-dextrin

[0113] A dextrin solution was prepared at a concentration of 3.0 w / v%, and sodium periodate was added (mass ratio of sodium periodate to dextrin was 20:100). The solution was stirred and reacted at room temperature in the dark for 2 h. Ethylene glycol was added at a concentration of 1 mL / g (dry mass of dextrin) to remove unreacted sodium periodate, and the solution was reacted for 1 h. The dialdehyde-dextrin powder was obtained by dialysis (dialysis bag with a molecular weight cut-off of 1000 Da) in distilled water for 3 days and freeze-drying.

[0114] 3. Preparation of cysteamine-dextrin

[0115] A solution of dialdehyde-dextrin was prepared at a concentration of 1.0 w / v%, and MES hydrate and cysteamine were added to a final concentration of 0.1 M (mass ratio of cysteamine to dialdehyde-dextrin was 50:100), and the pH was adjusted to 3 with hydrochloric acid. The solution was stirred at room temperature for 3 h. Sodium cyanoborohydride was then added (mass ratio of sodium cyanoborohydride to dialdehyde-dextrin was 400:100), and the solution was stirred at room temperature for 72 h. The reaction was completed by dialysis (dialysis bag with a molecular weight cut-off of 1000 Da) in distilled water at pH 3 for 3 days, and the cysteamine-dextrin powder was obtained by freeze-drying.

[0116] 4. Preparation of disulfide cross-linked starch-based micelles

[0117] A solution of cysteamine-dextrin was prepared at a concentration of 1.0 w / v%, and the solution was dissolved by heating at 85°C for 10 min and then shaken at room temperature for 24 h to obtain a micelle solution. The micelle solution was then ultrasonicated at a power of 300 W for 3 min to obtain a solution of disulfide cross-linked starch-based micelles.

[0118] 5. Preparation of curcumin-loaded disulfide cross-linked starch-based micelles

[0119] Curcumin was added to the solution of disulfide cross-linked starch-based micelles (mass-volume ratio of curcumin to disulfide cross-linked starch-based micelles was 50 mg:100 mL), and the solution was homogenized at a speed of 12000 rpm for 1-2 min. The curcumin was then migrated to the hydrophobic core of the micelles by shaking at room temperature for 24 h. The un-encapsulated curcumin was removed by centrifugation to obtain a solution of curcumin-loaded micelles.

[0120] Example 6

[0121] 1. Preparation of dextrin

[0122] A starch solution was prepared at a concentration of 6 w / v% using a phosphate buffer at a pH of 4.5. The starch was gelatinized by heating in a boiling water bath for 30 min. After cooling, pullulanase was added (dose ratio of pullulanase to waxy corn starch was 20 U:1 g), and the solution was stirred in a water bath for 24 h. The enzyme was inactivated by heating at 95°C for 20 min after the reaction was completed. The dextrin powder was obtained by freeze-drying.

[0123] 2. Preparation of dialdehyde-dextrin

[0124] A dextrin solution was prepared at a concentration of 3.0 w / v%, and sodium periodate was added (mass ratio of sodium periodate to dextrin was 20:100). The solution was stirred and reacted at room temperature in the dark for 2 h. 1 mL / g (dry mass of dextrin) of ethylene glycol was added to the solution and reacted for 1 h to remove unreacted sodium periodate. The solution was then dialyzed (dialysis bag with a molecular weight cut-off of 1000 Da) in distilled water for 3 days, and the dialdehyde-dextrin powder was obtained by freeze-drying.

[0125] 3. Preparation of cysteamine-dextrin

[0126] A 1.0 w / v% dialdehyde dextrin solution was prepared, and MES hydrate and cysteamine (cysteamine to dialdehyde dextrin mass ratio of 100:100) were added to a final concentration of 0.1 M. The pH was adjusted to 3 with hydrochloric acid, and the mixture was stirred at room temperature for 3 h. Subsequently, sodium cyanoborohydride (sodium cyanoborohydride to dialdehyde dextrin mass ratio of 400:100) was added, and the mixture was stirred at room temperature for 72 h. After the reaction was completed, the mixture was dialyzed with distilled water at pH=3 (dialysis bag molecular weight cutoff 1000 Da) for three days, and then freeze-dried to obtain cysteamine-dextrin powder.

[0127] 4. Preparation of disulfide-crosslinked starch-based micelles

[0128] A 1.0 w / v cysteamine-dextrin solution was prepared, heated at 85 °C for 10 min to dissolve, and then shaken at room temperature for 24 h to obtain a micelle solution. The solution was then sonicated at 300 W for 3 min to obtain a disulfide-crosslinked starch-based micelle solution.

[0129] 5. Preparation of starch-based micelles loaded with curcumin and cross-linked with disulfide

[0130] Curcumin (with a mass-to-volume ratio of 50 mg curcumin to 100 mL disulfide-crosslinked starch micelles) was added to a disulfide-crosslinked starch micelle solution. The mixture was homogenized at 12,000 rpm for 1–2 min, followed by shaking at room temperature for 24 h to allow curcumin to migrate into the hydrophobic core of the micelles. The unencapsulated curcumin was then removed by centrifugation to obtain a curcumin-loaded micelle solution.

[0131] Comparative Example 1

[0132] 1. Preparation of dextrin

[0133] Prepare a 6 w / v starch milk using a phosphate buffer solution with a pH of 4.5, heat it in a boiling water bath for 30 min to gelatinize it, cool it down and add pullulanase (the ratio of pullulanase to glutinous corn starch is 20 U: 1 g), stir it in a water bath for 24 h, after the reaction is complete, heat it at 95 °C for 20 min to inactivate the enzyme, and freeze dry it to obtain dextrin powder.

[0134] 2. Preparation of dialdehyde dextrin

[0135] Prepare a 3.0 w / v dextrin solution, add sodium periodate (sodium periodate to dextrin mass ratio of 20:100), mix and stir, react at room temperature in the dark for 2 h, add 1 mL / g (dextrin dry basis mass) ethylene glycol and react for 1 h to remove unreacted sodium periodate, then dialyze with distilled water (dialysis bag molecular weight cutoff 1000 Da) for three days, and freeze-dry to obtain dialdehyde dextrin powder;

[0136] 3. Preparation of dialdehyde dextrin micelles

[0137] A concentration of 1.0 w / v% dialdehyde dextrin solution was prepared, dissolved at 85°C for 10 min, and prepared into a micelle solution by oscillation at room temperature for 24 h. Subsequently, the dialdehyde dextrin micelle solution was obtained by ultrasonic treatment at 300 W for 3 min.

[0138] Comparative Example 2

[0139] 1. Preparation of dextrin

[0140] A concentration of 6 w / v% starch milk was prepared using a phosphate buffer having a pH of 4.5, and gelatinized by heating in a boiling water bath for 30 min. After cooling, pullulanase was added (the amount ratio of pullulanase to waxy corn starch was 20 U: 1 g), and stirred in a water bath for 24 h. After the reaction was completed, the enzyme was inactivated by heating at 95°C for 20 min, and freeze-dried to obtain a dextrin powder.

[0141] 2. Preparation of dialdehyde dextrin

[0142] A concentration of 3.0 w / v% dextrin solution was prepared, and mixed and stirred with the addition of sodium periodate (the mass ratio of sodium periodate to dextrin was 20:100). The reaction was carried out at room temperature in the dark for 2 h, 1 mL / g (dry basis mass of dextrin) of ethylene glycol was added to remove unreacted sodium periodate, and then dialyzed (dialysis bag molecular weight cutoff 1000 Da) for three days. The dialdehyde dextrin powder was obtained by freeze-drying.

[0143] 3. Preparation of dialdehyde dextrin micelles

[0144] A concentration of 1.0 w / v% dialdehyde dextrin solution was prepared, dissolved at 85°C for 10 min, and prepared into a micelle solution by oscillation at room temperature for 24 h. Subsequently, the dialdehyde dextrin micelle solution was obtained by ultrasonic treatment at 300 W for 3 min.

[0145] 4. Preparation of curcumin-loaded dialdehyde dextrin micelles

[0146] Curcumin was added to the dialdehyde dextrin micelle solution (the mass-volume ratio of curcumin to dialdehyde dextrin micelles was 50 mg: 100 mL), and homogenized at a speed of 12,000 rpm for 1-2 min. Subsequently, curcumin was migrated to the hydrophobic core of the micelles by oscillation at room temperature for 24 h, and then the un-encapsulated curcumin was removed by centrifugation to obtain a curcumin-loaded micelle solution.

[0147] Comparative Example 3

[0148] The method was the same as in Example 2, except that the ultrasonic treatment process in step 4 was omitted.

[0149] Comparative Example 4

[0150] The method was the same as in Example 5, except that the ultrasonic treatment time in step 4 was 30 min.

[0151] To further illustrate the technical effects of the present application, the relevant products obtained from Examples 1-6 and Comparative Examples 1-4 were measured.

[0152] I. Determination of free thiol content and disulfide bond content of prepared disulfide crosslinked starch-based micelles

[0153] Ellman reagent method and sodium borohydride reduction method were used to determine the free thiol and disulfide bond content.

[0154] (1) Cysteamine-dextrin micelles (5.00 mg / mL, w / v, 0.2 mL) were mixed with phosphate buffer (0.5 M, pH 8.0, 1.8 mL) and DTNB (2 mL, 0.03%, w / v, dissolved in phosphate buffer (0.5 M, pH 8.0)). The mixture was reacted and stored in the dark for 2 h, and the absorbance of the sample at 412 nm was measured. The free thiol concentration was calculated according to the Lambert-Beer law. The formula is as follows:

[0155] Free thiol content (μmol / g) = 73.53 x A 512 x D / C

[0156] where 73.53 is derived from 106 / (1.36 x 10 4 ), 1.36 x 10 4 is the molar absorbance, 10 6 is the conversion factor, A 412 is the absorbance of the supernatant at 412 nm, D is the dilution factor, and C is the sample concentration (mg / mL).

[0157] (2) The disulfide bond content was determined by reducing the polymer dispersion with sodium borohydride (NaBH4) to convert the disulfide bond to free thiol. Cysteamine-dextrin micelles (5.00 mg / mL, w / v, 0.2 mL) were mixed with phosphate buffer (0.05 M, pH 6.8, 1.3 mL), and freshly prepared NaBH4 (4%, w / v, 2 mL) was added for reduction at 37°C for 1 h. The mixture was neutralized with phosphate buffer solution (1 M, pH 8.5, 2 mL). Finally, the disulfide bond content was calculated according to the above determination method.

[0158] II. The loading capacity of curcumin in the micelles was determined by UV-visible spectrophotometry

[0159] The absorbance of curcumin-loaded cysteamine-dextrin micelles was measured at 422 nm using a UV-visible spectrophotometer. The concentration of Cur was calculated according to the standard curve. Then the loading capacity of Cur encapsulated in the micelles was calculated according to the formula.

[0160] Loading capacity (%) = mass of encapsulated curcumin / mass of micelle carrier x 100%

[0161] Four, the particle size and zeta potential of the disulfide cross-linked starch-based micelles with different free thiol content were determined by zeta potential analyzer, and the pH stability and ionic strength stability of the micelles were evaluated.

[0162] Five, the in vitro release behavior of curcumin in Example 5 was determined by dialysis method

[0163] The simulated gastrointestinal release medium was hydrochloric acid solution (pH 1.2, simulating gastric pH), PBS (pH 7.4, simulating small intestinal pH), and PBS (pH 6.0, simulating colonic pH). Due to the low water solubility of curcumin, 1% (v / v) Tween 80 was added to the release medium. The cysteamine-dextrin micelle solution (10 mL) was placed in a dialysis bag (molecular weight cutoff 3500 Da) and immersed in the release medium (50 mL). Every other time, the release medium (2 mL) was removed and replaced with an equal volume of fresh phosphate buffer. According to the curcumin standard curve, the cumulative release amount of curcumin was calculated by ultraviolet spectrophotometry.

[0164] Table 1 Free thiol and disulfide bond content of starch-based micelles in Examples 1-3 and Comparative Examples 1, 3, 4

[0165]

[0166] As can be seen from Table 1, the free thiol and disulfide bond content of the disulfide cross-linked starch-based micelles of Examples 1-3 is gradually increasing, while the dialdehyde dextrin micelles of Comparative Example 1 do not contain free thiol. The change in free thiol and disulfide bond content between Example 2 and Example 3 is small, indicating that when the amount of cysteamine added reaches a certain amount, the reaction reaches saturation. As can be seen from Example 2, Comparative Example 3 and Comparative Example 4, ultrasonic treatment can promote the conversion of free thiol to disulfide bond.

[0167] Table 2 Loading amount of curcumin in starch-based micelles in Examples 4-6 and Comparative Example 2

[0168] Name Example 4 Example 5 Example 6 Comparative Example 2 Loadings (%) 0.286±0.005 0.463±0.001 0.501±0.005 0.039±0.001

[0169] As can be seen from Table 2, the curcumin loading amount of the dialdehyde dextrin micelles of Comparative Example 2 is only 0.039%, while the curcumin loading amount of the disulfide cross-linked starch-based micelles of Examples 4-6 is 0.286%, 0.463%, and 0.501% respectively, which is increased by 7.33-12.85 times. This is because the increase in the number of hydrophobic disulfide bonds promotes the hydrophobic interaction between the micelles and curcumin, thereby increasing the loading amount.

[0170] Table 3 Particle size and zeta potential of micelles in Examples 1-6 and Comparative Examples 1-4

[0171] Name Size (nm) Zeta potential (mV) Example 1 92.52±1.90 23.58±0.89 Example 2 120.12±3.60 29.70±1.29 Example 3 157.12±3.68 29.90±2.16 Example 4 168.72±2.26 21.97±0.72 Example 5 143.60±2.85 26.25±0.77 Example 6 141.00±1.25 26.75±1.06 Comparative Example 1 152.67±3.39 -1.84±0.15 Comparative Example 2 209.87±5.44 -11.90±0.62 Comparative Example 3 189.45±6.25 25.63±0.22 Comparative Example 4 204.22±1.49 22.14±1.09

[0172] From Table 3, it can be seen that compared with Comparative Examples 1 and 2, the particle sizes of Examples 1-6 are smaller and the zeta potential values are higher, indicating that the stability of the disulfide cross-linked starch-based micelles is stronger. With the increase of the number of free thiol groups, the particle sizes of Examples 1-3 show a trend of increase, because with the increase of the content of free thiol groups, the probability of collision between hydrophobic segments to form disulfide bonds increases, leading to the occurrence of connection polymerization to form larger micelles. The particle sizes of Examples 4-6 after loading curcumin increase because the hydrophobic curcumin occupies the hydrophobic core of the micelles, leading to the increase of the volume. The particle sizes of Comparative Examples 3 and 4 are obviously larger than those of Examples 2 and 5, which indicates that the ultrasonic treatment can break larger micelle aggregates into smaller micelles through cavitation effect.

[0173] From Figure 1 and Figure 2 it can be seen that with the increase of pH value, the particle sizes of Examples 1-3 and Comparative Examples show a trend of first increase and then decrease. This is because the decrease of zeta potential value reduces the electrostatic repulsion between micelles, and the particle aggregation increases. After the pH value reaches 7.4, the particle size decreases due to the charge screening effect of counter ions, leading to the decrease of particle size.

[0174] From Figure 3 and Figure 4 it can be seen that except for Example 1, the rest of the samples show good stability under a salt ion concentration of 200 mM or less. The particle size increases with the increase of salt ion concentration, and the absolute value of zeta potential gradually decreases with the increase of salt ion concentration. The increase of particle size is due to the shielding of salt ions to the surface charge of micelles and the decrease of electrostatic repulsion between particles.

[0175] From Figures 5a-5b it can be seen that at pH 1.2, the cumulative release rate of curcumin of Example 5 is low, only 17.21% is released in 2 h, because the micelles have a high surface potential under acidic conditions, and the system is stable under the action of strong electrostatic repulsion. At pH 6.0 and pH 7.4, the total release amount of curcumin of Example 5 is 42.57% and 26.63% respectively, showing a colon-targeted release. In contrast, Comparative Example 2 shows very high release of curcumin under different pH conditions, indicating that its release characteristics are not suitable for gastrointestinal delivery.

[0176] In summary, the dithio cross-linked starch-based micelles obtained by the technical scheme described above use natural amino acid-cysteamine as a modifier, and good self-assembled micelles can be obtained by heating dissolution, room temperature shaking and ultrasonic treatment, and the method is simple, efficient and safe. The prepared product has a nanoscale size, and can be used to encapsulate hydrophobic functional factors such as curcumin, quercetin and catechol, greatly improving the water solubility thereof. The micelles also have good environmental stress stability such as pH and ionic strength stability. In vitro model release shows that the dithio cross-linked starch-based micelles loaded with curcumin can well prevent premature drug leakage in the stomach, can efficiently deliver to the small intestine and colon, and achieve efficient delivery. The prepared product is expected to be widely used in the fields of oral delivery, controlled release and biological activity maintenance of nutrients.

[0177] In addition, the inventors of the present application also refer to the foregoing examples, and other raw materials, process operations and process conditions described in the specification to conduct tests, and all obtain relatively ideal results.

[0178] It should be understood that the technical scheme of the present application is not limited to the specific implementation cases described above, and any technical modification made according to the technical scheme of the present application without departing from the purpose of the present application and the scope protected by the claims falls within the protection scope of the present application.

Claims

1. A method for preparing disulfide-crosslinked starch-based micelles loaded with hydrophobic functional factors, characterized in that, include: Pullulanase was used to debranch glutinous corn starch to obtain dextrin; periodate was used to oxidize the dextrin to obtain dialdehyde dextrin; cysteine ​​was grafted onto the dialdehyde dextrin to obtain cysteine-dextrin; the cysteine-dextrin was dissolved in water and self-assembled to form micelles, followed by ultrasonic thiol oxidation to obtain disulfide-crosslinked starch-based micelles. Furthermore, the hydrophobic functional factor is mixed and homogenized with the disulfide-crosslinked starch-based micelles to obtain disulfide-crosslinked starch-based micelles loaded with the hydrophobic functional factor.

2. The preparation method according to claim 1, characterized in that, Specifically, it includes: Waxy corn starch was mixed with phosphate buffer solution with a pH of 4.0-5.0 to form a waxy corn starch dispersion. Then, it was heated in a boiling water bath to gelatinize. Pullulanase was then added and the mixture was stirred in a water bath for 15-24 hours. After enzyme inactivation and freeze drying, dextrin was obtained. The concentration of glutinous corn starch in the glutinous corn starch dispersion is 3-6 w / v %. The ratio of pullulanase to glutinous corn starch is 20-50 U: 1g.

3. The preparation method according to claim 1, characterized in that, Specifically, it includes: Dextrin was dissolved in water to form a dextrin solution, then periodate was added, mixed and stirred and reacted at room temperature in the dark for 1-2 hours. Then ethylene glycol was added and reacted for 1-2 hours. After dialysis and freeze-drying, dialdehyde dextrin was obtained. The concentration of dextrin in the dextrin solution is 2.5~5.0 w / v % %. The mass ratio of periodate to dextrin is 10~40:100; The periodate includes sodium periodate and / or potassium periodate.

4. The preparation method according to claim 1, characterized in that, Specifically, it includes: Dialdehyde dextrin was dissolved in water to form a dialdehyde dextrin solution. Then, MES hydrate and cysteamine were added, the pH was adjusted to 3-4, and the mixture was stirred at room temperature for 3 hours. Then, sodium cyanoborohydride was added and the mixture was stirred at room temperature for 72 hours. Finally, the mixture was dialyzed and freeze-dried to obtain cysteamine-dextrin. The concentration of dialdehyde dextrin in the dialdehyde dextrin solution is 1.0~2.5 w / v % %. The mass ratio of cysteamine to dialdehyde dextrin is 50:100; The mass ratio of sodium cyanoborohydride to dialdehyde dextrin is 200~400:

100.

5. The preparation method according to claim 1, characterized in that, Specifically, it includes: Cysteine-dextrin was dissolved in water by heating at 70-85℃ for 10-30 min to form a cysteine-dextrin solution. The solution was then shaken at room temperature for 12-24 h to form a micelle solution. Finally, the solution was sonicated at 100-400W for 1-10 min to obtain disulfide-crosslinked starch-based micelles. The concentration of cysteamine-dextrin in the cysteamine-dextrin solution is 0.5~2 w / v.

6. The preparation method according to claim 1, characterized in that, Specifically, it includes: The hydrophobic functional factor was added to the disulfide-crosslinked starch micelles and homogenized at 8000~12000 rpm for 1~2 min. Then, the mixture was shaken at room temperature for 12~24 h. The unencapsulated hydrophobic functional factor was removed by centrifugation to obtain disulfide-crosslinked starch micelles loaded with hydrophobic functional factor. The mass-to-volume ratio of the hydrophobic functional factor to the disulfide-crosslinked starch-based micelles is 20-50 mg: 100 ml. The hydrophobic functional factor includes any one or more combinations of curcumin, quercetin, and catechin.

7. Disulfide-crosslinked starch micelles loaded with hydrophobic functional factors, prepared by any one of claims 1-6.

8. Use of the disulfide-crosslinked starch micelles loaded with hydrophobic functional factors as described in claim 7 in the preparation of colon-targeted drugs or orally delivered drugs.

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