Curcumin-loaded core-shell nanoparticles and methods of construction thereof

Abstract

The application relates to the technical field of nanocarrier, and discloses curcumin-loaded core-shell type nanoparticles and a construction method thereof, which comprises the following steps: dissolving zein in an ethanol solution, adding a reducing sugar for reaction, then performing pressure differential oscillation depolymerization treatment to obtain a zein solution; adding curcumin into the zein solution to obtain a curcumin-loaded zein solution; then adding a CaCl2 solution and a pectin solution mixture, dropping a phosphate solution, performing reaction, and forming a precipitate; performing water washing, centrifugation and freeze-drying to obtain curcumin-loaded composite nanoparticles. The curcumin-loaded core-shell type nanoparticles and the construction method thereof are used, zein is used as a core, calcium phosphate is used as a shell, and fruit gum is coated on the outside through coordination complexation, so that the nanoparticles have the pH response characteristic while improving the stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanocarrier, in particular to curcumin-loaded core-shell nanoparticles and a construction method thereof. BACKGROUND

[0002] Natural polyphenols (such as curcumin, anthocyanins and resveratrol) are increasingly widely used in the field of nutritionally fortified food development, however, the functional properties of pure polyphenols are limited by many factors, including poor water dispersibility, physical and chemical instability, and the influence of 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 / polymer-based nanoparticles to encapsulate polyphenols has become a hot international research topic. This is because proteins and polysaccharides are both bio-based materials, with high biological affinity; in addition, the polysaccharide coating can significantly improve the stability of the nanoparticles in the gastrointestinal digestion environment, thereby improving their bioavailability.

[0003] Zein is the main storage protein in corn, which is abundant in resources, low in price, and has good biocompatibility, biodegradability and non-immunogenicity. The zein structure has a distinct hydrophobic region, which is easy to self-assemble, and has a unique solubility, making it an ideal material for preparing a nanoparticle delivery system for encapsulating polyphenol compounds. However, nanoparticles composed of single zein are unstable near pH = 6.2, lose stability in low salt ions and high temperature environments, and the electrolyte components in the gastrointestinal fluid can destroy their nanostructure, and they cannot be reconstituted after drying. These drawbacks greatly limit their application. Therefore, research efforts are devoted to improving the stability of zein nanoparticles to fully exploit their advantages as a delivery system, and the use of hydrophilic anionic polysaccharides to modify, coat and stabilize zein nanoparticles is currently a research hotspot.

[0004] Calcium phosphate (CaP) is the main inorganic component of human bone and is widely used as a drug delivery carrier. CaP-based nanocarriers have the following advantages for drug loading: (1) biocompatibility, CaP naturally exists in bone or teeth and has no obvious toxicity to the human body and does not induce the body's immune system to attack; (2) pH response characteristics and good biodegradability, CaP nanomaterials will decompose into biocompatible ionic components Ca 2+ and PO4 3-Therefore, CaP is almost not accumulated in vivo; (3) easy to obtain and low in price; (4) porous structure, which can incorporate active substances in the porous scaffold thereof; (5) biological stability, which has little influence on the activity of transported small molecule substances. These advantages provide great potential for the application of CaP as a nano-carrier.

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

[0006] To solve the above technical problems, the present application constructs a curcumin-loaded core-shell nanoparticle, with zein as the core, calcium phosphate as the shell, and pectin as the outer coating through coordination complexation, so that the nanoparticle has improved stability and pH-responsive properties, solving the problem of poor stability of the existing nanoparticle system. SUMMARY

[0007] The present application aims to provide a curcumin-loaded core-shell nanoparticle and a construction method thereof, solving the problem of poor stability of the existing nanoparticle system. The curcumin-loaded core-shell nanoparticle constructed by the present application has zein as the core, calcium phosphate as the shell, and pectin as the outer coating through coordination complexation, so that the nanoparticle has improved stability and pH-responsive properties.

[0008] To achieve the above-mentioned purpose, the present application provides a curcumin-loaded core-shell nanoparticle and a construction method thereof, comprising the following steps:

[0009] Step 1, preparation of a zein solution, dissolving zein in an 80% ethanol solution and adding oligosaccharide syrup for reaction, and then performing pressure differential shock and oscillation depolymerization treatment to obtain a zein solution;

[0010] Step 2, loading of curcumin, adding curcumin to the zein solution and mixing by vortex, and then magnetically stirring the mixed solution at room temperature and centrifuging to obtain a curcumin-loaded zein solution;

[0011] Step 3, mixing CaCl2 solution, curcumin-loaded zein solution and pectin solution in a certain proportion, then dropping into a phosphate solution, stirring and reacting to form a precipitate;

[0012] Step 4, the precipitate is taken out, washed with water, centrifuged, and freeze-dried to obtain the curcumin-loaded composite nanoparticles.

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

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

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

[0016] Further, in step 1, the pressure difference shock oscillation depolymerization treatment step is: after static pressure control treatment for 15 min at room temperature, the pressure is instantaneously released to 0 MPa, which has an oscillation depolymerization effect on protein molecules, wherein the pressure of static pressure control is 125, 150 or 175 MPa.

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

[0018] Further, 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] Further, in step 3, the phosphate solution is an aqueous solution of disodium hydrogen phosphate or diammonium hydrogen phosphate, the pH value is 9.55-9.60, and the concentration is 0.1 mol / L.

[0020] The application also provides the curcumin-loaded core-shell nanoparticles prepared by the construction method.

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

[0022] The curcumin-loaded core-shell nanoparticles and the construction method thereof have the following advantages and positive effects:

[0023] 1. The curcumin-loaded core-shell nanoparticles constructed by the application have zein as the core, calcium phosphate as the shell, and pectin on the outside through coordination complexation, so that the nanoparticles have improved stability and pH response characteristics.

[0024] 2、The reducing sugar is used for modifying the zein in the application, the Maillard reaction between the zein and the reducing sugar is caused, the extension of the zein molecule is promoted, the internal hydrophobic group is exposed, and the curcumin is conveniently loaded; then the zein exposes more hydrophobic groups in the differential pressure oscillation process, so that the zein has a larger loading space, and the loading capacity of the curcumin is remarkably improved.

[0025] 3、The calcium phosphate is deposited and coated on the surface of the zein in the application, the zein and the curcumin are protected, and the calcium ions in the calcium phosphate are coupled with the carboxyl in the pectin through a coordination complex reaction, so that a pectin protective layer is formed on the outer layer of the calcium phosphate, and the thermal stability of the nanoparticles is improved.

[0026] The technical scheme of the application is further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The thermal stability test results of the nanoparticles prepared in the examples of the application;

[0028] Figure 2 The release performance evaluation results of the nanoparticles prepared in the examples of the application in the gastrointestinal tract. DETAILED DESCRIPTION

[0029] The technical scheme of the application is further described in detail below with reference to the drawings and examples.

[0030] Unless otherwise defined, the technical terms or scientific terms used in the application shall have the usual meanings understood by those skilled in the art to which the application pertains.

[0031] Based on the examples in the application, all other examples obtained by those skilled in the art without creative labor shall fall within the scope of protection of the application. The experimental methods not specified in the following examples are usually determined according to national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.

[0032] The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, the general international standard, conventional conditions, or the conditions recommended by the manufacturer are followed.

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

[0034] Example 1

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

[0036] Step 1, preparation of a zein solution, 250 mg of corn zein is dissolved in 25 mL of 80% ethanol solution to obtain a corn zein solution with a mass concentration of 1%, then 10 mg of fructose is added for reaction, the reaction conditions are pH 8-9, temperature 55-60°C, time 15-30 min, the reaction is ended in an ice bath at 4°C, then differential pressure shock depolymerization treatment is performed to obtain the corn zein solution.

[0037] The differential pressure shock depolymerization treatment step is: after static pressure control treatment at room temperature for 15 min, the pressure is instantaneously released to 0 MPa to produce an oscillation depolymerization effect on protein molecules, wherein the static pressure control pressure is 150 MPa.

[0038] Step 2, loading of curcumin, 15 mg of curcumin is added to the corn zein solution and mixed by vortex, then the mixed solution is magnetically stirred at room temperature for 4 h, and centrifugation is performed to obtain a corn zein solution loaded with curcumin.

[0039] Step 3, 0.2% CaCl2 solution, corn zein solution loaded with curcumin and 0.2% pectin solution are mixed in a volume ratio of 0.4:1:1, then 0.1 mol / L sodium phosphate dibasic solution is added dropwise, and stirring is performed for 6-8 h to form a precipitate.

[0040] Step 4, the precipitate is taken out, washed with water, centrifuged and freeze-dried to obtain composite nanoparticles loaded with curcumin (Cur@zein)@CaP1.

[0041] Example 2

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

[0043] Step 1, preparation of a zein solution, 250 mg of corn zein is dissolved in 25 mL of 80% ethanol solution to obtain a corn zein solution with a mass concentration of 1%, then 10 mg of fructose is added for reaction, the reaction conditions are pH 8-9, temperature 55-60°C, time 15-30 min, the reaction is ended in an ice bath at 4°C, then differential pressure shock depolymerization treatment is performed to obtain the corn zein solution.

[0044] The differential pressure shock depolymerization treatment step is: after static pressure control treatment at room temperature for 15 min, the pressure is instantaneously released to 0 MPa to produce an oscillation depolymerization effect on protein molecules, wherein the static pressure control pressure is 150 MPa.

[0045] Step 2, loading curcumin, 15 mg of curcumin was added to the zein solution and mixed by vortex, and then the mixture was magnetically stirred at room temperature for 4 h, and the curcumin-loaded zein solution was obtained by centrifugation.

[0046] Step 3, 0.2% CaCl2solution, curcumin-loaded zein solution and 0.2% pectin solution were mixed in a volume ratio of 0.4:1:1, then 0.1 mol / L sodium phosphate dibasic solution was added dropwise, and the reaction was carried out by stirring for 6-8 h to form a precipitate.

[0047] Step 4, the precipitate was taken out, washed with water, centrifuged, and freeze-dried to obtain curcumin-loaded composite nanoparticles (Cur@zein)@CaP2.

[0048] Example 3

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

[0050] Step 1, preparation of zein solution, 250 mg of zein was dissolved in 25 mL of 80% ethanol solution to obtain a zein solution with a mass concentration of 1%, then 16 mg of fructose was added and the reaction was carried out under the conditions of pH 8-9, temperature 55-60°C, time 15-30 min, and the reaction was terminated in an ice bath at 4°C, followed by pressure differential shock depolymerization treatment to obtain a zein solution.

[0051] The pressure differential shock depolymerization treatment step is: after static pressure control at room temperature for 15 min, the pressure is instantaneously released to 0 MPa, which has an oscillating depolymerization effect on protein molecules, and the static pressure control pressure is 150 MPa.

[0052] Step 2, loading curcumin, 15 mg of curcumin was added to the zein solution and mixed by vortex, and then the mixture was magnetically stirred at room temperature for 4 h, and the curcumin-loaded zein solution was obtained by centrifugation.

[0053] Step 3, 0.2% CaCl2solution, curcumin-loaded zein solution and 0.2% pectin solution were mixed in a volume ratio of 0.4:1:1, then 0.1 mol / L sodium phosphate dibasic solution was added dropwise, and the reaction was carried out by stirring for 6-8 h to form a precipitate.

[0054] Step 4, the precipitate was taken out, washed with water, centrifuged, and freeze-dried to obtain curcumin-loaded composite nanoparticles (Cur@zein)@CaP3.

[0055] Comparative Example 1

[0056] A method for constructing curcumin-loaded core-shell nanoparticles, comprising the following steps:

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

[0058] The pressure differential oscillation depolymerization treatment step is: after static pressure control treatment at room temperature for 15 min, instantaneously releasing pressure to 0 MPa to produce oscillation depolymerization effect on protein molecules, wherein the pressure of static pressure control is 150 MPa.

[0059] Step 2, loading curcumin, 15 mg of curcumin was added to the corn zein solution and mixed by vortex, and then the mixed solution was magnetically stirred at room temperature for 4 h, and centrifugation was performed to obtain a curcumin-loaded corn zein solution.

[0060] Step 3, 0.2% CaCl2 solution, curcumin-loaded corn zein solution and 0.2% pectin solution were mixed in a volume ratio of 0.4:1:1, then 0.1 mol / L sodium phosphate dibasic solution was added dropwise, and stirring was performed for 6-8 h to form a precipitate.

[0061] Step 4, the precipitate was taken out, washed with water, centrifuged, and freeze-dried to obtain curcumin-loaded composite nanoparticles (Cur@zein)@CaP A .

[0062] Comparative Example 2

[0063] A method for constructing curcumin-loaded core-shell nanoparticles, comprising the following steps:

[0064] Step 1, preparation of a zein solution, 250 mg of corn zein was dissolved in 25 mL of 80% ethanol solution to obtain a corn zein solution with a mass concentration of 1%, and then subjected to pressure differential oscillation depolymerization treatment to obtain a corn zein solution.

[0065] The pressure differential oscillation depolymerization treatment step is: after static pressure control treatment at room temperature for 15 min, instantaneously releasing pressure to 0 MPa to produce oscillation depolymerization effect on protein molecules, wherein the pressure of static pressure control is 150 MPa.

[0066] Step 2, loading curcumin, 15 mg of curcumin was added to the corn zein solution and mixed by vortex, and then the mixed solution was magnetically stirred at room temperature for 4 h, and centrifugation was performed to obtain a curcumin-loaded corn zein solution.

[0067] Step 3, 0.2% CaCl2 solution and curcumin loaded zein solution were mixed at a volume ratio of 0.4:1, then dropped into 0.1 mol / L sodium phosphate dibasic solution, and stirred to react for 6-8 h to form a precipitate.

[0068] Step 4, the precipitate was taken out, washed with water, centrifuged, and freeze-dried to obtain curcumin loaded composite nanoparticles (Cur@zein)@CaP B .

[0069] Performance test

[0070] Embedding rate and encapsulation rate determination:

[0071] 1 g of the composite nanoparticles prepared in Examples 1-3 and Comparative Example 1 was mixed with 4 mL of anhydrous ethanol, ultrasonic assisted extraction, then centrifuged at 10000 rpm for 10 min, the supernatant was taken, and the concentration of curcumin was measured at 426 nm by ultraviolet spectrophotometer, and the encapsulation rate and loading capacity were calculated.

[0072] The calculation formula of encapsulation rate and loading capacity is as follows:

[0073]

[0074] The results are shown in Table 1:

[0075] Table 1 encapsulation rate and loading capacity results

[0076]

[0077]

[0078] As shown in Table 1, the encapsulation rate of the composite nanoparticles prepared in Examples 1-3 is more than 7%, and the loading capacity is more than 89%, compared with Comparative Example 1, the encapsulation rate and loading capacity are significantly increased, which indicates that the Maillard reaction occurs between zein and fructose, the zein molecules are extended, the internal hydrophobic groups are exposed, and the pressure differential shock treatment further exposes more hydrophobic groups of zein, so that it has a larger loading space, and significantly improves 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, heated to 75℃ and 85℃ in a water bath for 20 min respectively. Then, the heated sample was quickly cooled to 25℃ with ice water. Then, the concentration of curcumin was measured at 426 nm by ultraviolet visible spectrophotometer, and the calculation formula of curcumin retention rate is as follows:

[0081]

[0082] Results as shown in Figure 1 The free curcumin lacks protection and has a low retention rate at high temperature (less than 30%), and the retention rate of curcumin encapsulated in the nanoparticles of zein, calcium phosphate and pectin is significantly improved, and the thermal stability of the nanoparticles is obviously improved. Compared with Comparative Example 2, the nanoparticles prepared in Examples 1-3 have better thermal stability, because the calcium ions in the calcium phosphate as the shell are coupled with the carboxyl groups in the pectin through coordination complexation to form a pectin calcium gel protective layer, which protects the 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 gastrointestinal tract in the nanoparticle sample, the nanoparticle dispersion was diluted 10 times with simulated gastric fluid (SGF, pH = 4, 1 mg / mL pepsin) and digested at 37°C for 2h. Then the mixture was diluted 10 times with simulated intestinal fluid (SIF, pH = 7.4, 4 mg / mL trypsin) and continued to be digested at 37°C for 4h, and the calculation formula of the release rate of curcumin is as follows:

[0085]

[0086] Results as shown in Figure 2 The release rate of encapsulated curcumin is lower than that of free curcumin. During the process of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), the nanoparticles prepared in Examples 1-3 have better sustained-release performance, and the release rate of the nanoparticles prepared in Examples 1-3 is about 60% at the end of the whole digestion process, which indicates that the nanoparticles prepared in Examples 1-3 can provide a large amount of curcumin at the distal position of the gastrointestinal tract, which is helpful to its bioavailability. Compared with Examples 1-3, the nanoparticles prepared in Comparative Example 2 have poor sustained-release performance, because the surface of the calcium phosphate in Comparative Example 2 is not coupled with pectin, and the calcium phosphate as the 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 in Comparative Example 1 are coupled with the carboxyl groups in the pectin through coordination complexation to form a pectin calcium gel protective layer, although the curcumin loading capacity is low, but the sustained-release performance is not much different from that of Examples 1-3.

[0087] Therefore, the core-shell type nanoparticles loaded with curcumin and the construction method thereof are adopted to solve the problem of poor stability of the existing nanosystem, and the core-shell type nanoparticles loaded with curcumin constructed by the present application have corn protein zein as the core, calcium phosphate as the shell, and pectin as the outer layer through coordination complexation, so that the nanoparticles have improved stability and pH response characteristics.

[0088] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for the construction of curcumin loaded core-shell nanoparticles characterized in that, The method comprises the following steps: Step 1, preparation of a prolamin solution, dissolving corn prolamin in an 80% ethanol solution and adding a reducing sugar for reaction, after the reaction is completed, differential pressure shock depolymerization treatment is performed to obtain a corn prolamin solution; Step 2, loading curcumin, adding curcumin to the corn prolamin solution and mixing by vortex, then magnetically stirring the mixed solution at room temperature, and centrifuging to obtain a curcumin-loaded corn prolamin solution; Step 3, mixing CaCl2 solution, curcumin-loaded corn prolamin solution and pectin solution in a certain proportion, then dropping phosphate solution, stirring and reacting to form a precipitate; Step 4, taking out the precipitate, washing with water, centrifuging and freeze-drying to obtain curcumin-loaded composite nanoparticles; The mass concentration of the corn prolamin solution in step 1 is 1-1.5%, and the mass ratio of corn prolamin to reducing sugar is 25:1-4; The reducing sugar in step 1 is selected from one of glucose, fructose, galactose, lactose and maltose; The reaction conditions in step 1 are pH 8-9, temperature 55-60℃, time 15-30 min, and reaction is completed in an ice bath at 4℃; The differential pressure shock depolymerization treatment step in step 1 is: after static pressure control treatment at room temperature for 15 min, the pressure is instantaneously released to 0 MPa to produce an oscillation depolymerization effect on protein molecules, wherein the static pressure control pressure is 125, 150 or 175 MPa.

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

250.

3. The method for constructing curcumin loaded core-shell nanoparticle as claimed in claim 1 wherein: The concentration of the CaCl2 solution in step 3 is 0.2%, the concentration of the pectin solution is 0.2%, and the volume ratio of the CaCl2 solution, curcumin-loaded corn prolamin solution and pectin solution is 0.4-0.6:1:

1.

4. The method for constructing curcumin loaded core-shell nanoparticle as claimed in claim 1 wherein: 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.

5. The curcumin-loaded core-shell nanoparticles prepared by the construction method of any one of claims 1-4.

6. The use of the curcumin-loaded core-shell nanoparticles of claim 5 in drug delivery.

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