A curcumin delivery system based on trityl nonaethylene glycol / ovalbumin and a preparation method thereof

Abstract

The application belongs to the technical field of preparations for medical, dental or cosmetic use, and specifically discloses a self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin, which is obtained by self-assembly of trityl nonaethylene glycol, ovalbumin and curcumin; and simultaneously discloses a preparation method of the curcumin delivery system. The drug delivery system prepared by the method significantly improves the drug encapsulation dispersion and cell absorption efficiency, and can be widely used in the fields of food, daily chemicals and medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical, dental or cosmetic formulations, and in particular relates to a self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin and a preparation method. BACKGROUND

[0002] Curcumin (Cur) is an active ingredient present in turmeric, which has attracted much attention in recent years due to its antioxidant, anti-inflammatory and anti-aging biological activities. However, curcumin has poor water solubility, is easily photodegradable, has unstable chemical properties, and has low bioavailability, which limits its application in many functional foods, supplements and drugs. Therefore, it is urgent to find ways to improve the solubility, stability and bioavailability of curcumin, such as nano delivery systems, nano emulsion encapsulation, chemical modification and physical dispersion.

[0003] The patent document with publication number CN 117899052 A provides a preparation method for a curcumin nano delivery carrier. The invention uses biological polymers such as zein, sodium caseinate and sodium alginate to prepare nanoparticles that can be used as curcumin delivery carriers using an anti-solvent precipitation technique. The curcumin nano delivery carrier prepared by the above method is mainly aimed at improving the water solubility, antioxidant activity and photochemical stability of curcumin. There is no mention of how to promote cell uptake efficiency. Moreover, the process used in this method requires two layers (casein, sodium alginate) self-assembly, which is complex in itself and has low assembly efficiency.

[0004] Ovalbumin (OVA) is high in content in chicken egg white and has been widely concerned in the development of delivery systems as one of the most abundant protein sources. Due to its excellent properties such as gelation, easy digestion, self-assembly and amphiphilicity, a variety of emulsions and gels based on ovalbumin have been constructed for the delivery of active substances such as curcumin. However, ovalbumin usually needs to be denatured by heat treatment to promote binding with curcumin. The hydrophobic groups of denatured ovalbumin are exposed and prone to flocculation and precipitation, which generally requires further stabilization. Ovalbumin particles are spherical in shape, and general emulsifiers are not easy to bind to the surface of the protein, making it difficult to form small-scale nanoparticles with good dispersion.

[0005] Trityl has the advantages of high selectivity and easy introduction, and is often used as a group protection of amines, alcohols and thiols in traditional organic chemical industry and drug synthesis, but its self-assembly performance has not been utilized. Trityl forms a specific trilobal structure due to its three benzene rings, which can effectively fit the surface of spherical proteins, and is a promising small-molecule protein particle homeostasis structure unit, and has a broad application prospect. Polyethylene glycol molecules have ether bonds and hydroxyl groups, and have good hydrophilicity and biocompatibility, and are widely used as emulsifiers, drugs and hydrophilic modification of materials. In addition, polyethylene glycol is also a commonly used cell fusion inducer, which promotes cell fusion by changing the structure of cell membrane, but the molecular weight usually needs to be 2000 or more. SUMMARY

[0006] To solve the problems in the prior art, the application provides a preparation method of a trityl nonaethylene glycol / ovalbumin-based self-assembled curcumin delivery system. In the method, trityl and nonaethylene glycol are combined through ether bonds to form an amphiphilic molecule, the trilobal structure of trityl effectively combines the hydrophobic surface of spherical ovalbumin through hydrophobic and aromatic ring interaction, significantly improves the dispersion degree of the delivery system, and the polyethylene glycol structure of nonaethylene glycol (molecular weight of nonaethylene glycol ~ 400, much lower than 2000) effectively combines with cells, and significantly promotes the uptake of nanoparticles.

[0007] A trityl nonaethylene glycol / ovalbumin-based self-assembled curcumin delivery system is obtained by self-assembly of trityl nonaethylene glycol, ovalbumin and curcumin.

[0008] .

[0009] Preferably, the delivery system is a self-assembled nanoparticle formed by trityl nonaethylene glycol, ovalbumin and curcumin.

[0010] Preferably, the delivery system is obtained by mixing trityl nonaethylene glycol aqueous solution, ovalbumin aqueous solution and curcumin aqueous solution, heating treatment, and cooling to obtain the self-assembled nanoparticle.

[0011] Preferably, the mass ratio of trityl nonaethylene glycol, ovalbumin and curcumin is (20-40):100:(20-40).

[0012] Preferably, the trityl nonaethylene glycol is obtained by reaction of trityl chloride and nonaethylene glycol as reaction substrates.

[0013] A preparation method of trityl nonaethylene glycol includes the following steps:

[0014] Trityl chloride is used as a modification molecule to react with nonaethylene glycol molecules, and trityl derivative is obtained after reaction and treatment.

[0015] As a preference, in the process of preparing the trityl nonaethylene glycol, a base needs to be added. The base used in the reaction is one of potassium carbonate, DIPEA, triethylamine, imidazole, 2,6-dimethylpyridine, 2,6-pyridine, potassium hydroxide. To improve the conversion rate of the reaction and reduce by-products, triethylamine is further preferred.

[0016] As a preference, in the process of preparing the trityl nonaethylene glycol, the solvent used in the reaction is one of DMF, anhydrous acetonitrile, tetrahydrofuran, t-butanol, dichloromethane, ethyl acetate. To improve the conversion rate of the reaction and reduce by-products, dichloromethane is further preferred.

[0017] A preparation method of a self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin, comprising: mixing a trityl nonaethylene glycol aqueous solution, an ovalbumin aqueous solution, and a curcumin aqueous suspension, and heating treatment, to finally obtain the self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin.

[0018] Further, the ovalbumin solution and the curcumin suspension, and the trityl nonaethylene glycol solution are respectively ultrasonically mixed with water as the solvent, and then heated and treated, and subsequently gradually cooled to room temperature, while denaturation and embedding are completed. Thus, the self-assembled nanoparticles are obtained.

[0019] As a preference, the average particle size of the self-assembled nanoparticles is 30-100 nanometers. As a further preference, the average particle size of the self-assembled nanoparticles is 30-80 nanometers; as a still further preference, the average particle size of the self-assembled nanoparticles is 30-70 nanometers.

[0020] As a preference, the heating treatment temperature is 70-100℃. As a further preference, the heating treatment temperature is 70-90℃. As a still further preference, the heating treatment temperature is 80-100℃. As the best embodiment, the heating treatment temperature is 90℃.

[0021] As a preference, the heating treatment time is 10-30 minutes.

[0022] As a preference, after the heating treatment is completed, the temperature is cooled to 20-30℃ for further reaction for 10-25 minutes, to obtain the self-assembled nanoparticles.

[0023] As a preference, the mass ratio of the trityl nonaethylene glycol, the ovalbumin, and the curcumin is (20-40):100:(20-40). As a preference, the ovalbumin, the trityl nonaethylene glycol, and the curcumin are self-assembled at a mass ratio of 4:1:1, to obtain the drug-loaded nanoparticles.

[0024] As preferred, the concentration of the triphenylmethyl nonaglycerol aqueous solution is 0.15-1 mg / mL; the concentration of the ovalbumin solution is 0.5-2 mg / mL.

[0025] As preferred, the concentration of the curcumin water suspension is 0.15-1 mg / mL. As further preferred, the concentration of the curcumin water suspension is 0.15-0.5 mg / mL; as still further preferred, the concentration of the curcumin water suspension is 0.15-0.35 mg / mL; as yet further preferred, the concentration of the curcumin water suspension is 0.2-0.3 mg / mL; as a specific selection, the concentration of the curcumin water suspension is 0.25 mg / mL.

[0026] As preferred, the concentration of the triphenylmethyl nonaglycerol aqueous solution is 0.15-0.5 mg / mL; as further preferred, the concentration of the triphenylmethyl nonaglycerol aqueous solution is 0.15-0.35 mg / mL; as a specific selection, the concentration of the triphenylmethyl nonaglycerol aqueous solution is 0.25 mg / mL.

[0027] As preferred, the concentration of the ovalbumin solution is 0.5-2 mg / mL; as further preferred, the concentration of the ovalbumin solution is 1-2 mg / mL; as a specific selection, the concentration of the ovalbumin solution is 1 mg / mL.

[0028] The present application uses triphenylmethyl as a modification group and nonaglycerol as a reaction substrate, and the synthesized molecule triphenylmethyl nonaglycerol has good amphiphilicity and biocompatibility. The synthesized molecule is fully self-assembled with raw materials such as ovalbumin and curcumin under heating conditions to form drug-loaded nanoparticles. The self-assembled nanomedicine delivery system based on triphenylmethyl nonaglycerol prepared by the present application has a high-efficiency and easy-to-operate preparation method, significantly improves the drug encapsulation dispersion and absorption efficiency, and can be widely used in the fields of food, daily chemicals and medicine.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] (1) The present application innovatively uses the spherical protein binding performance of triphenylmethyl nonaglycerol for the preparation of self-assembled nanoparticles, and prepares uniform small-molecule drug-loaded self-assembled nanoparticles, which has a simple and easy-to-operate preparation method and greatly improves the drug encapsulation dispersion.

[0031] (2) The present application innovatively uses the cell binding performance of triphenylmethyl nonaglycerol, and the drug-loaded self-assembled nanoparticles significantly improve the cell absorption efficiency, solving the technical problem of low absorption efficiency of traditional methods. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Scanning electron microscope images of curcumin nanodelivery particles based on triphenylmethyl nonaethyleneglycol (EG-OVA-Cur) and ovalbumin (OVA-Cur).

[0033] Figure 2 Graph of cell absorption rate results of EG-OVA-Cur, OVA-Cur and control group (CK) obtained by flow cytometry.

[0034] Figure 3 H-nuclear magnetic resonance spectrum of triphenylmethyl nonaethyleneglycol.

[0035] Figure 4 Mass spectrum of triphenylmethyl nonaethyleneglycol. DETAILED DESCRIPTION

[0036] In order to further illustrate the present application, the present application will be further described in detail below in conjunction with the accompanying drawings and examples, but the embodiments of the present application are not limited thereto.

[0037] Example 1: Preparation of curcumin nanodelivery particles based on triphenylmethyl nonaethyleneglycol

[0038] Nonaethyleneglycol 2.0 eq (2 mmol, 812.73 μL) was dissolved in dichloromethane solvent (3 mL), 1.2 eq of triethylamine (166.34 μL) was added, 1.0 eq of triphenylmethyl chloride (0.2788 μL) was added, and the reaction was carried out at room temperature for 3 h. The product, triphenylmethyl nonaethyleneglycol, was separated by column chromatography. An ovalbumin solution of 1 mg / mL, a triphenylmethyl nonaethyleneglycol solution of 0.25 mg / mL and a curcumin suspension of 0.25 mg / mL were prepared with water as the solvent, respectively. After ultrasonic mixing, they were heated at 90°C for 15 min, and then gradually cooled to 25°C for 15 min, to complete the denaturation and assembly embedding. The obtained self-assembled particles were curcumin nanodelivery particles based on triphenylmethyl nonaethyleneglycol, which were recorded as EG-OVA-Cur.

[0039] The product, triphenylmethyl nonaethyleneglycol, has the following characteristics: 1 H NMR data (400 MHz, DMSO-d6): δ 7.44-7.16 (m, 15 H), 4.57 (t, J = 5.4 Hz, 1 H), 3.62-3.37 (m, 34 H), 3.06 (t, J = 4.8 Hz, 2H), mass spectrum (ESI): [M+Na] 679.52 m / z. + 679.52 m / z.

[0040] Proportion optimization: different concentrations of trityl nonaethylene glycol (0.03 mg / mL, 0.06 mg / mL, 0.12 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL) and 1 mg / mL ovalbumin solution, 0.25 mg / mL curcumin suspension were used. After ultrasonic mixing, heat treatment was carried out at 90℃ for 15 min, followed by gradual cooling to 25℃ for 15 min, and then denaturation and assembly embedding were completed. Nanoparticle size analyzer was used to test the particle size of the nanoparticles, and the particle sizes were 78 nm, 79 nm, 72 nm, 63 nm, 71 nm, and 179 nm, respectively. The particle size of 0.25 mg / mL trityl nonaethylene glycol was the smallest, and it was selected as the optimal proportion.

[0041] Comparative Example 1: Preparation of Ovalbumin-based Curcumin Nanodelivery Particles

[0042] Using water as the solvent, 1 mg / mL ovalbumin solution and 0.25 mg / mL curcumin suspension were prepared, respectively. After ultrasonic mixing, heat treatment was carried out at 90℃ for 15 min, followed by gradual cooling to 25℃ for 15 min, and then denaturation and assembly embedding were completed. The obtained self-assembled particles were ovalbumin-based curcumin nanodelivery particles, denoted as OVA-Cur.

[0043] Performance Test 1: Evaluation of Encapsulation Dispersion of Self-assembled Nanoparticles Based on Trityl Modification

[0044] Nanoparticle size analyzer was used to test the particle size of the two self-assembled nanoparticles prepared in Example 1 and Comparative Example 1. The particle size of EG-OVA-Cur was 63 nm, and the particle size of OVA-Cur was 83 nm. Scanning electron microscope was used to characterize the morphology and structure of the two self-assembled nanoparticles prepared in Example 1 and Comparative Example 1. The test results are shown in Figure 1 Compared with ovalbumin self-assembled curcumin nanoparticles, the surface of self-assembled nanoparticles modified by trityl nonaethylene glycol molecules was smoother and more regular, the edge profile of the particles was clear and sharp, and the overall showed high monodispersity and geometric symmetry, smaller and more uniform particle size, forming nanoparticles with compact structure and good uniformity.

[0045] Performance Test 2: Evaluation of Cell Uptake Efficiency of Self-assembled Nanoparticles Based on Trityl Modification

[0046] Take 30 μL of Saccharomyces cerevisiae culture into a centrifuge tube, add 150 μL of self-assembled nanoparticle suspension (prepared in Example 1), and incubate in a shaker at 200 rpm and 30°C. After incubation for 4 h, stop the reaction, centrifuge at 3000 rpm and 4°C for 3 min to remove the culture medium, then add 50 μL of PBS, centrifuge under the same conditions, and resuspend in PBS.

[0047] After each group of samples was incubated using the above procedures, the fluorescence intensity of the samples was collected using a flow cytometer. Each sample (the negative control group was the cell group without "triphenylmethyl-modified self-assembled nanoparticles", denoted as the CK (Control) group) was diluted with PBS to an appropriate factor before collection. The emission channel of the flow cytometer was set to KO525-A, and each sample was collected 3 times.

[0048] The test results showed that the cell uptake rate (i.e., fluorescence intensity) of ovalbumin-assembled curcumin nanoparticles was 22%, while that of triphenylmethylnonadecanol-assembled curcumin nanoparticles was 32%, indicating a significant increase in cell uptake rate. Figure 2 .

Claims

1. A triphenylmethyl nonaethylene glycol / ovalbumin based self-assembly curcumin delivery system characterized in that, Self-assembly curcumin delivery system based on trityl nonaethyleneglycol / ovalbumin is prepared by self-assembly of trityl nonaethyleneglycol and ovalbumin and curcumin, wherein the trityl nonaethyleneglycol has the following structure: ； The preparation method of the self-assembly curcumin delivery system based on trityl nonaethyleneglycol / ovalbumin comprises the following steps: mixing uniformly mixed trityl nonaethyleneglycol aqueous solution, ovalbumin aqueous solution and curcumin aqueous suspension, heating treatment, cooling, and obtaining the self-assembly curcumin delivery system based on trityl nonaethyleneglycol / ovalbumin. The heating treatment temperature is 70-100 DEG C, and the heating treatment time is 10-30 minutes. After the heating treatment is completed, the temperature is cooled to 20-30 DEG C to continue the reaction for 10-25 minutes to obtain self-assembly nanoparticles.

2. The trityl nonaethylene glycol / ovalbumin based self-assembled curcumin delivery system according to claim 1, characterized in that, The delivery system is self-assembly nanoparticles formed by trityl nonaethyleneglycol, ovalbumin and curcumin.

3. The trityl nonaethylene glycol / ovalbumin based self-assembled curcumin delivery system according to claim 2, characterized in that, The mass ratio of the trityl nonaethyleneglycol, ovalbumin and curcumin is (20-40) : 100 : (20-40).

4. The trityl nonaethylene glycol / ovalbumin based self-assembled curcumin delivery system according to claim 2, wherein, The average particle size of the self-assembly nanoparticles is 30-100 nanometers.

5. A method for preparing the self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin according to any one of claims 1 to 4, comprising: The preparation method of the self-assembly curcumin delivery system based on trityl nonaethyleneglycol / ovalbumin comprises the following steps: mixing uniformly mixed trityl nonaethyleneglycol aqueous solution, ovalbumin aqueous solution and curcumin aqueous suspension, heating treatment, cooling, and obtaining the self-assembly curcumin delivery system based on trityl nonaethyleneglycol / ovalbumin. The heating treatment temperature is 70-100 DEG C, and the heating treatment time is 10-30 minutes. After the heating treatment is completed, the temperature is cooled to 20-30 DEG C to continue the reaction for 10-25 minutes to obtain self-assembly nanoparticles.

6. The method of preparing the self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin according to claim 5, characterized in that, The concentration of the trityl nonaethyleneglycol aqueous solution is 0.15-1 mg / mL; the concentration of the ovalbumin aqueous solution is 0.5-2 mg / mL; and the concentration of the curcumin aqueous suspension is 0.15-1 mg / mL.

7. The method of preparing the self-assembled curcumin delivery system based on trityl nonaethylene glycol / ovalbumin according to claim 5, characterized in that, The trityl nonaethyleneglycol is obtained by reaction of trityl chloride and nonaethyleneglycol as reaction substrates.

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