Curcumin-loaded core-shell nanoparticles and construction method thereof

Abstract

The invention relates to the technical field of nano-carriers, and discloses curcumin-loaded core-shell nano-particles and a construction method thereof.The construction method comprises the following steps that zein is dissolved in an ethanol solution, reducing sugar is added for a reaction, then differential pressure oscillation depolymerization treatment is conducted, and a zein solution is obtained; adding curcumin into the zein solution to obtain a zein solution loaded with curcumin; then adding a CaCl2 solution and a pectin solution, mixing, dropwise adding a phosphate solution, and carrying out a reaction to form a precipitate; and washing with water, centrifuging and freeze-drying to obtain the curcumin-loaded composite nanoparticles. According to the curcumin-loaded core-shell nanoparticle and the construction method thereof, the zein is taken as a core, the calcium phosphate is taken as a shell, and the pectin is wrapped outside through coordination complexation, so that the stability of the nanoparticle is improved, and meanwhile, the nanoparticle has a pH response characteristic.

Description

Technical Field

[0001] The invention relates to the technical field of nanocarriers, in particular to core-shell nanoparticles loaded with curcumin and a construction method thereof. Background Art

[0002] Natural polyphenolic compounds (such as curcumin, anthocyanins and resveratrol) are increasingly being used in the development of nutritionally fortified foods. However, the functional properties of simple polyphenolic additions are limited by many factors, including poor water dispersibility, physicochemical instability, and the influence of the external environment (such as light, oxygen and temperature) and gastrointestinal changes. Green bio-based nanocarriers are very suitable for encapsulating, protecting and delivering polyphenols, thereby improving their bioavailability. Among them, the use of protein / polysaccharide-based nanoparticles to encapsulate polyphenolic compounds has become a hot topic in international research. This is because proteins and polysaccharides are both bio-based materials with high bioaffinity; in addition, polysaccharide coatings can significantly improve the stability of nanoparticles in the gastrointestinal digestive environment, thereby improving their bioavailability.

[0003] Zein is the main storage protein in corn. It is abundant in resources, inexpensive, and has good biocompatibility, biodegradability, and non-immunogenicity. Zein has obvious hydrophobic regions in its structure, is easy to self-assemble, and has unique solubility characteristics. It is an ideal material for preparing nano-delivery systems for encapsulating polyphenolic compounds. However, nanoparticles composed of single zein are unstable near pH=6.2, lose stability in low salt ion and high temperature environments, and the electrolyte components contained in gastrointestinal fluids will destroy its nanostructure. It cannot be redissolved after drying. These drawbacks greatly limit its application. Therefore, research on improving the stability of zein nanoparticles to give full play to its advantages as a delivery system is currently a hot topic. The use of hydrophilic anionic polysaccharides to modify, coat, and stabilize zein nanoparticles is a hot topic.

[0004] Calcium phosphate (CaP) is the main inorganic component of human bones and is widely used as a drug delivery carrier. CaP-based nanocarriers have the following advantages in drug loading: (1) biocompatibility. CaP naturally exists in bones or teeth and has no obvious toxicity to the human body and will not induce autoimmune system attacks; (2) pH responsiveness and good biodegradability. CaP nanomaterials will decompose into biocompatible ionic components Ca at slightly acidic pH. 2+ and PO4 3-, therefore, CaP hardly accumulates in the body; (3) it is easily available and inexpensive; (4) it has a porous structure, which enables the incorporation of active substances into its porous scaffold; and (5) it is biostable, with little effect on the activity of the transported small molecules. These advantages provide great potential for the application of CaP as a nanocarrier.

[0005] Pectin is an anionic polysaccharide derived from plant cell walls. It has a mature production process and is easy to obtain in large quantities. At the same time, pectin has a certain resistance to proteases and amylases present in the upper digestive tract, and is a rational wall material for oral drug delivery systems. In previous studies, self-assembly technology was used to construct core-shell zein / pectin composite nanoparticles for the loading of polyphenolic compounds. However, this nanosystem still has certain limitations, including low encapsulation efficiency, unstable particle structure and poor bioavailability, which are closely related to its system formation mechanism.

[0006] In order to solve the above technical problems, the present invention constructs a core-shell nanoparticle loaded with curcumin, with zein as the core and calcium phosphate as the shell, and pectin is wrapped on the outside through coordination complexation, so that the nanoparticles have improved stability and pH response characteristics, solving the problem of poor stability of existing nano systems. Summary of the invention

[0007] The purpose of the present invention is to provide core-shell nanoparticles loaded with curcumin and a construction method thereof, so as to solve the problem of poor stability of existing nano systems. The core-shell nanoparticles loaded with curcumin constructed by the present invention use zein as a core and calcium phosphate as a shell, and are externally wrapped with pectin through coordination complexation, so that the nanoparticles have improved stability and pH response characteristics.

[0008] To achieve the above object, the present invention provides core-shell nanoparticles loaded with curcumin and a method for constructing the same, comprising the following steps:

[0009] Step 1, preparation of a zein solution, dissolving zein in an 80% ethanol solution, and adding oligosaccharide syrup to react, and after the reaction is completed, performing a pressure difference oscillation depolymerization treatment to obtain a zein solution;

[0010] Step 2, loading curcumin, adding curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature, and centrifuging to obtain a zein solution loaded with curcumin;

[0011] Step 3, mixing the CaCl2 solution, the curcumin-loaded zein solution and the pectin solution in proportion, then dropping the phosphate solution into the mixture, stirring to react and forming a precipitate;

[0012] Step 4, taking out the precipitate, washing, centrifuging and freeze-drying to obtain composite nanoparticles loaded with curcumin.

[0013] Furthermore, in step 1, the mass concentration of the zein solution is 1-1.5%, the concentration of the oligosaccharide syrup is 40-50%, and the mass ratio of zein to oligosaccharides is 25:1-4.

[0014] Furthermore, in step 1, the oligosaccharide is selected from one of fructooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, sucrose, maltose and lactose.

[0015] Furthermore, in step 1, the reaction conditions are pH 8-9, temperature 55-60°C, time 15-30 min, and the reaction is terminated in an ice bath at 4°C.

[0016] Furthermore, the pressure difference oscillation depolymerization treatment step in step 1 is: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 125, 150 or 175 MPa.

[0017] Furthermore, the mass ratio of the amount of curcumin added in step 2 to the amount of zein added in step 1 is 15-18:250.

[0018] Furthermore, in step 3, the concentration of the CaCl2 solution is 0.2%, the concentration of the pectin solution is 0.2%, and the volume ratio of the CaCl2 solution, the curcumin-loaded zein solution and the pectin solution is 0.4-0.6:1:1.

[0019] Furthermore, in step 3, the phosphate solution is an aqueous solution of disodium hydrogen phosphate or diammonium hydrogen phosphate, with a pH value of 9.55-9.60 and a concentration of 0.1 mol / L.

[0020] The present invention also provides core-shell nanoparticles loaded with curcumin prepared by the above construction method.

[0021] The present invention also provides application of the core-shell nanoparticles loaded with curcumin in drug delivery.

[0022] The advantages and positive effects of the curcumin-loaded core-shell nanoparticles and the construction method thereof of the present invention are:

[0023] 1. The core-shell nanoparticles loaded with curcumin constructed by the present invention have zein as the core and calcium phosphate as the shell, and are externally wrapped with pectin through coordination complexation, so that the nanoparticles have improved stability and pH response characteristics.

[0024] 2. In the present invention, reducing sugar is used to modify zein, so that zein and reducing sugar undergo Maillard reaction, which promotes the extension of zein molecules and exposes internal hydrophobic groups to facilitate the loading of curcumin; then, during the pressure difference oscillation process, zein further exposes more hydrophobic groups, so that it has a larger loading space, which significantly increases the loading amount of curcumin.

[0025] 3. In the present invention, calcium phosphate is deposited and coated on the surface of zein to protect zein and curcumin, and the calcium ions in the calcium phosphate are coupled with the carboxyl groups in pectin through coordination complex reaction to form a pectin protective layer on the outer layer of the calcium phosphate, thereby improving the thermal stability of the nanoparticles.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The thermal stability test results of the nanoparticles prepared in the embodiments of the present invention;

[0028] Figure 2 These are the release performance evaluation results of the nanoparticles prepared in the examples of the present invention in the gastrointestinal tract. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0030] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0031] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually measured in accordance with national standards. The experimental instruments, equipment and reagents without source indication in the following examples are all commercially available raw materials.

[0032] The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0033] Unless otherwise defined or described, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0034] Example 1

[0035] The method for constructing core-shell nanoparticles loaded with curcumin comprises the following steps:

[0036] Step 1, preparation of alcohol-soluble protein solution, dissolving 250 mg of zein in 25 mL of 80% ethanol solution to obtain a zein solution with a mass concentration of 1%, then adding 10 mg of fructose, reacting under the conditions of pH 8-9, temperature 55-60° C., time 15-30 min, ending the reaction in an ice bath at 4° C., and then performing a pressure difference oscillation depolymerization treatment to obtain a zein solution.

[0037] The steps of pressure difference oscillation depolymerization treatment are: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 150 MPa.

[0038] Step 2, loading curcumin, adding 15 mg of curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature for 4 hours, and centrifuging to obtain a zein solution loaded with curcumin.

[0039] Step 3, 0.2% CaCl2 solution, curcumin-loaded zein solution and 0.2% pectin solution are mixed in a volume ratio of 0.4:1:1, and then 0.1 mol / L disodium hydrogen phosphate solution is added dropwise, and the mixture is stirred for reaction for 6-8 hours to form a precipitate.

[0040] Step 4, taking out the precipitate, washing, centrifuging, and freeze-drying to obtain composite nanoparticles (Cur@zein)@CaP1 loaded with curcumin.

[0041] Example 2

[0042] The method for constructing core-shell nanoparticles loaded with curcumin comprises the following steps:

[0043] Step 1, preparation of alcohol-soluble protein solution, dissolving 250 mg of zein in 25 mL of 80% ethanol solution to obtain a zein solution with a mass concentration of 1%, then adding 13 mg of fructose, reacting under the conditions of pH 8-9, temperature 55-60° C., time 15-30 min, ending the reaction in an ice bath at 4° C., and then performing pressure difference oscillation depolymerization treatment to obtain a zein solution.

[0044] The steps of pressure difference oscillation depolymerization treatment are: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 150 MPa.

[0045] Step 2, loading curcumin, adding 15 mg of curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature for 4 hours, and centrifuging to obtain a zein solution loaded with curcumin.

[0046] Step 3, 0.2% CaCl2 solution, curcumin-loaded zein solution and 0.2% pectin solution are mixed in a volume ratio of 0.4:1:1, and then 0.1 mol / L disodium hydrogen phosphate solution is added dropwise, and the mixture is stirred for reaction for 6-8 hours to form a precipitate.

[0047] Step 4, taking out the precipitate, washing, centrifuging, and freeze-drying to obtain composite nanoparticles (Cur@zein)@CaP2 loaded with curcumin.

[0048] Example 3

[0049] The method for constructing core-shell nanoparticles loaded with curcumin comprises the following steps:

[0050] Step 1, preparation of alcohol-soluble protein solution, dissolving 250 mg of zein in 25 mL of 80% ethanol solution to obtain a zein solution with a mass concentration of 1%, then adding 16 mg of fructose, reacting under the conditions of pH 8-9, temperature 55-60° C., time 15-30 min, ending the reaction in an ice bath at 4° C., and then performing a pressure difference oscillation depolymerization treatment to obtain a zein solution.

[0051] The steps of pressure difference oscillation depolymerization treatment are: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 150 MPa.

[0052] Step 2, loading curcumin, adding 15 mg of curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature for 4 hours, and centrifuging to obtain a zein solution loaded with curcumin.

[0053] Step 3, 0.2% CaCl2 solution, curcumin-loaded zein solution and 0.2% pectin solution are mixed in a volume ratio of 0.4:1:1, and then 0.1 mol / L disodium hydrogen phosphate solution is added dropwise, and the mixture is stirred for reaction for 6-8 hours to form a precipitate.

[0054] Step 4, taking out the precipitate, washing, centrifuging, and freeze-drying to obtain composite nanoparticles (Cur@zein)@CaP3 loaded with curcumin.

[0055] Comparative Example 1

[0056] The method for constructing core-shell nanoparticles loaded with curcumin comprises the following steps:

[0057] Step 1, preparation of a prolamin solution, dissolving 250 mg of zein in 25 mL of 80% ethanol solution to obtain a zein solution with a mass concentration of 1%, and then performing a pressure differential oscillation depolymerization treatment to obtain a zein solution.

[0058] The steps of pressure difference oscillation depolymerization treatment are: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 150 MPa.

[0059] Step 2, loading curcumin, adding 15 mg of curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature for 4 hours, and centrifuging to obtain a zein solution loaded with curcumin.

[0060] Step 3, 0.2% CaCl2 solution, curcumin-loaded zein solution and 0.2% pectin solution are mixed in a volume ratio of 0.4:1:1, and then 0.1 mol / L disodium hydrogen phosphate solution is added dropwise, and the mixture is stirred for reaction for 6-8 hours to form a precipitate.

[0061] Step 4: Take out the precipitate, wash it with water, centrifuge it, and freeze-dry it to obtain the composite nanoparticles (Cur@zein)@CaP loaded with curcumin. A .

[0062] Comparative Example 2

[0063] The method for constructing core-shell nanoparticles loaded with curcumin comprises the following steps:

[0064] Step 1, preparation of alcohol-soluble protein solution, dissolving 250 mg of zein in 25 mL of 80% ethanol solution to obtain a zein solution with a mass concentration of 1%, then adding 13 mg of fructose, reacting under the conditions of pH 8-9, temperature 55-60° C., time 15-30 min, ending the reaction in an ice bath at 4° C., and then performing pressure difference oscillation depolymerization treatment to obtain a zein solution.

[0065] The steps of pressure difference oscillation depolymerization treatment are: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 150 MPa.

[0066] Step 2, loading curcumin, adding 15 mg of curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature for 4 hours, and centrifuging to obtain a zein solution loaded with curcumin.

[0067] Step 3, 0.2% CaCl2 solution and curcumin-loaded zein solution are mixed in a volume ratio of 0.4:1, and then 0.1 mol / L disodium hydrogen phosphate solution is added dropwise, and the mixture is stirred for reaction for 6-8 hours to form a precipitate.

[0068] Step 4: Take out the precipitate, wash it with water, centrifuge it, and freeze-dry it to obtain the composite nanoparticles (Cur@zein)@CaP loaded with curcumin. B .

[0069] Performance Testing

[0070] Determination of embedding rate and encapsulation rate:

[0071] 1 g of the composite nanoparticles prepared in Examples 1-3 and Comparative Example 1 was mixed with 4 mL of anhydrous ethanol, subjected to ultrasonic-assisted extraction, and then centrifuged at 10,000 rpm for 10 min. The supernatant was taken and the concentration of curcumin was measured using an ultraviolet spectrophotometer at 426 nm, and the encapsulation rate and loading amount were calculated.

[0072] The calculation formulas for encapsulation rate and loading are as follows:

[0073]

[0074] The results are shown in Table 1:

[0075] Table 1 Encapsulation rate and loading results

[0076]

[0077]

[0078] As can be seen from Table 1, the composite nanoparticles prepared in Examples 1-3 of the present invention have an encapsulation rate of more than 7% and a loading capacity of more than 89%. Compared with Comparative Example 1, the encapsulation rate and loading capacity are significantly increased, indicating that the Maillard reaction between zein and fructose causes the zein molecules to be extended, exposing the internal hydrophobic groups. After the pressure difference oscillation technology treatment, more hydrophobic groups of zein are further exposed, giving it a larger loading space, thereby significantly improving the encapsulation rate and loading capacity of curcumin.

[0079] Thermal stability test:

[0080] The composite nanoparticle samples prepared in Examples 1-3 and Comparative Example 2 were placed in a transparent glass vial and heated to 75°C and 85°C in a water bath for 20 min each. Then, the heated samples were quickly cooled to 25°C with ice water. Then, the concentration of curcumin was measured using a UV-visible spectrophotometer at 426 nm. The curcumin retention rate was calculated as follows:

[0081]

[0082] The results are as follows Figure 1 As shown, free curcumin lacks protection and has a low retention rate at high temperature (less than 30%). Curcumin is wrapped in nanoparticles of zein, calcium phosphate and pectin, which significantly improves the retention rate of curcumin and significantly improves the thermal stability of the nanoparticles. Compared with Comparative Example 2, the thermal stability of the nanoparticles prepared in Examples 1-3 is better, because the calcium ions in the calcium phosphate as the shell are coupled with the carboxyl groups in the pectin through a coordination complex reaction to form a pectin calcium gel protective layer, which protects zein and curcumin and improves the thermal stability of the nanoparticles.

[0083] Evaluation of release performance in the gastrointestinal tract:

[0084] In order to evaluate the release of curcumin in the nanoparticle samples in the gastrointestinal tract, the nanoparticle dispersion was diluted 10 times with simulated gastric fluid (SGF, pH = 4, 1 mg / mL pepsin) and digested at 37 ° C for 2 hours. The mixture was then diluted 10 times with simulated intestinal fluid (SIF, pH = 7.4, with 4 mg / mL pancreatin) and continued to digest at 37 ° C for 4 hours. The release rate of curcumin was calculated as follows:

[0085]

[0086] The results are as follows Figure 2 As shown, the release rate of curcumin after encapsulation is lower than that of free curcumin. In the process of simulated gastric juice (SGF) and simulated intestinal juice (SIF), the sustained release performance of the nanoparticles prepared in Examples 1-3 is good. After the entire digestion process, the release rate of the nanoparticles prepared in Examples 1-3 is about 60%, indicating that the nanoparticles prepared in Examples 1-3 can provide a large amount of curcumin at the distal position of the gastrointestinal tract, which helps its bioaccessibility. Compared with Examples 1-3, the sustained release performance of the nanoparticles prepared in Comparative Example 2 is poor. This is because the calcium phosphate surface of Comparative Example 2 is not coupled with pectin. The calcium phosphate as a shell is decomposed in the stomach, and the curcumin inside the nanoparticles is released, resulting in poor sustained release performance. The calcium ions in the calcium phosphate of Comparative Example 1 are coupled with the carboxyl groups in pectin through a coordination complex reaction to form a pectin calcium gel protective layer. Although the curcumin loading is low, the sustained release performance is not much different from that of Examples 1-3.

[0087] Therefore, the present invention adopts the above-mentioned core-shell nanoparticles loaded with curcumin and the construction method thereof to solve the problem of poor stability of the existing nano system. The core-shell nanoparticles loaded with curcumin constructed by the present invention use zein as the core and calcium phosphate as the shell, and are externally wrapped with pectin through coordination complexation, so that the nanoparticles have improved stability and pH response characteristics.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for constructing core-shell nanoparticles loaded with curcumin, characterized in that: The following steps are involved: Step 1, preparation of a zein solution, dissolving zein in an 80% ethanol solution, adding reducing sugar, and reacting, and after the reaction is completed, performing a pressure difference oscillation depolymerization treatment to obtain a zein solution; Step 2, loading curcumin, adding curcumin to the zein solution, and mixing by vortexing, and then magnetically stirring the mixture at room temperature, and centrifuging to obtain a zein solution loaded with curcumin; Step 3, mixing the CaCl2 solution, the curcumin-loaded zein solution and the pectin solution in proportion, then dropping the phosphate solution into the mixture, stirring to react and forming a precipitate; Step 4, taking out the precipitate, washing, centrifuging and freeze-drying to obtain composite nanoparticles loaded with curcumin.

2. The method for constructing the core-shell nanoparticles loaded with curcumin according to claim 1, characterized in that: The mass concentration of the zein solution in step 1 is 1-1.5%, and the mass ratio of zein to reducing sugar is 25:1-4.

3. The method for constructing curcumin-loaded core-shell nanoparticles according to claim 1, characterized in that: In step 1, the reducing sugar is selected from one of glucose, fructose, galactose, lactose and maltose.

4. The method for constructing curcumin-loaded core-shell nanoparticles according to claim 1, characterized in that: The reaction conditions in step 1 are pH 8-9, temperature 55-60°C, time 15-30 min, and the reaction is terminated in an ice bath at 4°C.

5. The method for constructing the core-shell nanoparticles loaded with curcumin according to claim 1, characterized in that: The pressure difference oscillation depolymerization treatment step in step 1 is: after static pressure control treatment at room temperature for 15 minutes, the pressure is instantly released to 0 MPa to produce an oscillating depolymerization effect on the protein molecules, wherein the static pressure control pressure is 125, 150 or 175 MPa.

6. The method for constructing curcumin-loaded core-shell nanoparticles according to claim 1, characterized in that: The mass ratio of the amount of curcumin added in step 2 to the amount of zein added in step 1 is 15-18:

250.

7. The method for constructing curcumin-loaded core-shell nanoparticles according to claim 1, characterized in that: In step 3, the concentration of the CaCl2 solution is 0.2%, the concentration of the pectin solution is 0.2%, and the volume ratio of the CaCl2 solution, the curcumin-loaded zein solution and the pectin solution is 0.4-0.6:1:

1.

8. The method for constructing curcumin-loaded core-shell nanoparticles according to claim 1, characterized in that: The phosphate solution in step 3 is an aqueous solution of disodium hydrogen phosphate or diammonium hydrogen phosphate, with a pH value of 9.5-9.6 and a concentration of 0.1 mol / L.

9. core-shell nanoparticles loaded with curcumin prepared by the construction method as described in any one of claims 1 to 8.

10. Use of the curcumin-loaded core-shell nanoparticles according to claim 9 in drug delivery.

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