Lac and zein-curcumine-C8G2 drug-loaded nanoparticles as well as preparation method and application of lac and zein-curcumine-C8G2 drug-loaded nanoparticles

Abstract

The invention relates to a preparation method and an application of a lac and zein-curcumin-C8G2 drug-loaded nano particle. Comprising the following preparation raw materials in parts by mass: 100 to 300 parts of shellac, 100 to 300 parts of zein, 1 to 150 parts of curcumin and 1 to 10 parts of C8G2. And preparing the lac and zein-curcumine-C8G2 drug-loaded nano particles by adopting a high-performance micro-fluidic device. Curcumin is packaged in shellac and zein composite nanoparticles by adopting a high-performance micro-fluidic device, then the nanoparticles and C8G2 are stirred, and the stability of the nanoparticles and the encapsulating / charging efficiency of drugs are improved by optimizing micro-fluidic prescription parameters, controlling the particle size of the nanoparticles and reducing the polydispersity index of the nanoparticles; cell experiments prove that the compound has good cell targeting property, and in-vivo experiments show that the compound provides a promising method for cancer application.

Description

Technical Field

[0001] The invention relates to the technical field of drug-carrying nanoparticles, and more specifically to a preparation method and application of shellac and zein-curcumin-C8G2 drug-carrying nanoparticles. Background Art

[0002] The pursuit of improved living standards has emphasized the need for the advancement of pure and natural foods. Natural compounds extracted from plants have become the focus of the food industry, known for their potential health benefits and reduced adverse reactions. However, many natural plant active extracts have limited solubility in water, which poses a challenge to their oral bioavailability. To address this issue, various delivery systems have been designed, including hydrogels, dendrimers, liposomes, microparticles, and nanoparticles, among which nanoparticles have gained great attention, mainly because of their unique advantages, including high encapsulation efficiency compared with other delivery systems. Nanoparticles made of naturally derived materials such as silk, chitosan, zein, shellac, etc. are widely used as delivery systems for drugs and nutrients, with advantages such as controlled release, increased solubility and stability, and improved in vitro and in vivo delivery.

[0003] Zein is a food raw material with good biodegradability, biocompatibility and low cost, and is an excellent choice for encapsulating bioactive substances. Zein can be easily converted into colloidal nanoparticles by antisolvent coprecipitation due to its inherent hydrophobic properties. Since many bioactive ingredients, such as curcumin, β-carotene, astaxanthin and quercetin, are hydrophobic, they must be encapsulated in delivery systems to improve their bioavailability. Composite nanoparticle systems composed of zein and hydrophobic biopolymers have been studied. The composite particles formed by zein and other encapsulation materials such as shellac have the characteristics of smaller particle size, higher turbidity value and higher thermal stability. Zein and chitosan form compact zein-chitosan nanoparticles and reduce the release of curcumin from the nanoparticles. Nanospheres formed by zein and silk fibroin improve the loading capacity and encapsulation efficiency of paclitaxel (PTX) and curcumin, and last for at least one week in a sustained release manner. Adding sodium alginate coating to zein nanoparticles significantly improved the encapsulation efficiency of curcumin (up to 92.6%). Zein and carboxymethyl dextrin (CMD) nanoparticles have a synergistic effect on the encapsulation efficiency of curcumin. The presence of CMD significantly enhances the photochemical stability, thermal stability and antioxidant activity of curcumin, and delays the release of curcumin in simulated gastrointestinal fluid. Coating alginate oligosaccharides (AOS) on zein nanoparticles also improves the encapsulation efficiency of zein nanoparticles, enhances colloidal stability, and slowly releases in the gastrointestinal tract. These nanoparticles are mainly prepared by antisolvent coprecipitation, continuous stirring and microfluidics. However, most of the zein nanoparticles prepared by the above methods have a large particle size (>100nm), a high PDI (>0.1), and low drug encapsulation and loading efficiency, which seriously limits the bioavailability of drug-loaded zein nanoparticles.

[0004] Shellac (E904) has the properties of being non-toxic, physiologically safe, having good film-forming ability, high gloss, and low permeability to gases, acids, and water vapor. It is often used to extend the shelf life of food by shielding against water, gases, lipids, and microorganisms. Researchers have attempted to improve the barrier properties of shellac by mixing it with zein.

[0005] Anticancer peptides (ACPs) are a class of polypeptides with anticancer activity. They are widely found in mammals, plants, birds, amphibians, fish, insects, microorganisms and other organisms. Anticancer peptides can destroy the membrane structure of tumor cells or inhibit the proliferation and migration of cancer cells and the formation of tumor blood vessels. Compared with traditional chemotherapy drugs, proteins, monoclonal antibodies and other drugs, anticancer peptides are easy to synthesize and modify, have strong ability to penetrate tumors, can be chemically modified to prevent protease hydrolysis, and have a variety of administration methods. At present, some anticancer peptide drugs have entered the clinical trial stage or have been approved for marketing.

[0006] The C8G2 anti-tumor peptide solution takes advantage of the characteristics of peptides, such as good biocompatibility, diverse functions, high in vivo responsiveness, and simple and easy synthetic modification methods. These characteristics enable the C8G2 anti-tumor peptide to construct a targeted drug delivery system, deliver the drug to the tumor area in a targeted manner, and achieve precise release of the drug under the special microenvironment of the tumor tissue or exogenous stimulation. This specific tumor-targeted and stimulus-responsive peptide carrier can maximize the anti-tumor effect of the drug and reduce the toxic side effects of the drug.

[0007] Curcumin (Curcuma cuminor, E100) is a natural lipophilic polyphenol extracted from turmeric, which has antioxidant and antimicrobial properties. Therefore, it is widely used in foods such as candy, cakes, and beverages as a natural food colorant, flavoring agent, and dietary supplement in traditional Asian medicine. In addition, chromium is sensitive to ultraviolet radiation and heating, and changes color under alkaline conditions. When the pH is greater than 8, the color of the curcumin solution changes from orange to red, which can be used as a pH indicator to monitor changes in food quality and freshness. Despite its potential health benefits, it faces challenges such as low water solubility and poor oral bioavailability.

[0008] Curcumin has been shown to be an adjunctive drug for the treatment of ulcerative colitis (UC), a common inflammatory disease of the colon characterized by diarrhea, mucosal ulcers, and rectal bleeding. In recent years, millions of patients worldwide have suffered from this disease, and the incidence of UC has increased rapidly worldwide, especially in many developing countries. However, due to the potential risk of failure of curcumin in the gastrointestinal tract, curcumin usually requires carrier encapsulation for specific colon-targeted delivery, and nanoscale drug delivery systems have very broad prospects.

[0009] Microfluidics refers to the science and technology involved in systems that use microchannels (tens to hundreds of microns in size) to process or manipulate tiny fluids (volumes from nanoliters to attoliters). It is an emerging interdisciplinary subject involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. Because of its miniaturization and integration, microfluidic devices are often called microfluidic chips, also known as labs on a chip (LabonaChip) and micro-total analytical systems (micro-Total Analytical System). This experiment used a microfluidic vortex mixing device to quickly prepare the corresponding nanoparticles by quickly mixing two connected chambers at a relatively low pressure. Summary of the invention

[0010] The invention provides a preparation method and application of lac and zein-curcumin-C8G2 drug-loaded nanoparticles. A high-performance microfluidic device is used to encapsulate curcumin in lac and zein composite nanoparticles, and the prepared nanoparticles are combined with C8G2 anti-tumor peptides through electrostatic force to prepare the lac and zein-curcumin-C8G2 drug-loaded nanoparticles. Cell experiments prove that the lac and zein-curcumin nanoparticles have good biocompatibility and antioxidant effects, and in vivo experiments show that the lac and zein-curcumin nanoparticles loaded with curcumin can effectively relieve enteritis, thereby providing a very promising method for application in the food industry.

[0011] In a first aspect, the present invention provides a lac and zein-curcumin-C8G2 drug-loaded nanoparticles, comprising the following raw materials in parts by weight: 100-300 parts of lac, 100-300 parts of zein, 1-150 parts of curcumin, and 1-10 parts of C8G2 anti-tumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2; a microfluidic device is used to control the total flow rate and the flow rate ratio of the aqueous phase to the organic phase to obtain the lac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0012] Preferably, the preparation comprises the following raw materials in parts by mass: 150-300 parts of shellac, 150-300 parts of zein, 100-150 parts of curcumin, and 6-10 parts of C8G2 anti-tumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2; a microfluidic device is used to control the total flow rate and the flow rate ratio of the aqueous phase to the organic phase to obtain shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0013] Preferably, the preparation comprises the following raw materials in parts by mass: 200-300 parts of lac, 200-300 parts of zein, 120-150 parts of curcumin, and 8-10 parts of C8G2 anti-tumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2; a microfluidic device is used to control the total flow rate and the flow rate ratio of the aqueous phase to the organic phase to obtain lac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0014] Preferably, the preparation comprises the following raw materials in parts by mass: 200 parts of lac, 200 parts of zein, 60 parts of curcumin, and 10 parts of C8G2 anti-tumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2; a microfluidic device is used to control the total flow rate and the flow rate ratio of the aqueous phase to the organic phase to obtain lac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0015] Lac and zein-curcumin-C8G2 drug-loaded nanoparticles overcome the shortcomings of curcumin such as poor water solubility and low bioavailability, provide a method for encapsulating curcumin, can be consumed as a natural beverage pigment, slowly release under certain conditions, reduce beverage oxidation, improve the utilization of zein and curcumin, and have the advantages of prevention and auxiliary treatment of ulcerative colitis.

[0016] In a second aspect, the present invention provides a method for preparing shellac and zein-curcumin-C8G2 drug-loaded nanoparticles, comprising the following steps:

[0017] (1) placing shellac in ethanol and dispersing the shellac evenly until the shellac is completely dissolved to obtain a shellac solution;

[0018] (2) dissolving zein solid particles in ethanol to obtain a zein solution;

[0019] (3) mixing the shellac solution and the zein solution according to a certain ratio to obtain a mixture of shellac solution and zein;

[0020] (4) dissolving curcumin in the shellac solution and the zein mixed solution to obtain a shellac and zein-curcumin mixed solution;

[0021] (5) using a microfluidic device, by adding an aqueous phase and an organic phase into the microfluidic device, the lac and zein-curcumin mixed solution prepared in step (4) as the organic phase, pure water as the aqueous phase, setting the total flow rate TFR of the microfluidic device to 30-60 mL / min, and the flow rate ratio of the aqueous phase to the organic phase to be 1-5:1 to prepare lac and zein-curcumin drug-loaded nanoparticles;

[0022] (6) The prepared nanoparticles were mixed with C8G2 antitumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2 solution and water and stirred overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0023] Preferably, in step (1), shellac is dissolved in 95% ethanol, the concentration of the shellac solution is 20-25 g / L, and the shellac is dispersed uniformly by ultrasonic treatment.

[0024] Preferably, in step (2), the concentration of the zein solution is 10-30 g / L.

[0025] Preferably, in step (2), the concentration of the zein solution is 15-30 g / L.

[0026] Preferably, in step (2), the concentration of the zein solution is 20 g / L.

[0027] Preferably, in step (3), the mixing ratio of the shellac solution to the zein solution is 1-100:100-1.

[0028] Preferably, in step (3), the mixing ratio of the shellac solution to the zein solution is 1-50:100-1.

[0029] Preferably, in step (3), the mixing ratio of the shellac solution to the zein solution is 1-20:100-1.

[0030] Preferably, in step (3), the mixing ratio of the shellac solution to the zein solution is 1:100.

[0031] Preferably, in step (4), the concentration of curcumin is 0-10 g / L.

[0032] Preferably, in step (4), the concentration of curcumin is 6-10 g / L.

[0033] Preferably, in step (4), the concentration of curcumin is 8 g / L.

[0034] Preferably, in step (5), the total flow rate TFR of the microfluidic device is 48-60 mL / min.

[0035] Preferably, in step (5), the total flow rate TFR of the microfluidic device is 48 mL / min.

[0036] Preferably, in step (5), the flow rate ratio of the microfluidic device is aqueous phase: organic phase = 2-5:1.

[0037] Preferably, in step (5), the flow rate ratio of the microfluidic device is aqueous phase: organic phase = 2:1.

[0038] Preferably, in step (6), the particle size of the shellac and zein-curcumin-C8G2 drug-loaded nanoparticles is 70-340 nm.

[0039] Preferably, in step (6), the particle size of the shellac and zein-curcumin-C8G2 drug-loaded nanoparticles is 340 nm.

[0040] In a third aspect, the present invention provides an application of lac and zein-curcumin-C8G2 drug-loaded nanoparticles, and use of lac and zein-curcumin-C8G2 nanoparticles in the preparation of drugs for treating cancer.

[0041] The present invention adopts microfluidic technology and anti-solvent co-precipitation principle, and shellac and zein form a composite material as an encapsulation material, so that curcumin is embedded in shellac and zein, with the purpose of slowly releasing curcumin in beverages and other foods to achieve a preservation effect. After entering the body, the curcumin is released in the small intestine and colorectum to achieve the effect of auxiliary treatment of ulcerative colitis.

[0042] In summary, the present invention has the following beneficial effects:

[0043] 1. The lac and zein-curcumin-C8G2 drug-loaded nanoparticles prepared in the present invention are spherical, with controllable particle size and uniform particle size. Its main components, lac and zein, have good biocompatibility and biodegradability, and its main components, proteins and polysaccharides, have good biological inertness and are removed from the body through common metabolic pathways. It has good stability, is conducive to its stable release in vivo, and its components are non-toxic and non-irritating, and can be used as a good delivery material.

[0044] 2. The present invention overcomes the problem of poor solubility of curcumin, improves the bioavailability of curcumin, increases the therapeutic effect, and has a good sustained release effect. Compared with naked curcumin, nanoparticles are more easily taken up by cells and have a stronger effect of inhibiting the growth of tumor cells. Anti-tumor peptides have unique anti-tumor activity, which is not easy for cancer cells to develop drug resistance and is an ideal substitute for chemotherapy drugs. The addition of surface functionalized anti-tumor peptides makes the nanoparticles more uniform and enhances the killing effect on cells.

[0045] 3. The preparation method of the present invention has simple process and is easy to operate.

[0046] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] 1. Figure 1This is an application effect diagram of the shellac and zein-curcumin drug-loaded nanoparticles prepared by the present invention using microfluidic technology and anti-solvent co-precipitation principle;

[0048] 2. Figure 2 The following are (a) state comparison, (b) particle size and polydispersity index comparison, (c) potential comparison, (d) encapsulation efficiency comparison, and (e) drug loading comparison of lac and zein-curcumin nanoparticles prepared under the same total flow rate, the same flow rate ratio, different ratios of lac and zein, the same ethanol concentration, and the same curcumin concentration in Examples 1-18 of the present invention and Comparative Examples 1-3;

[0049] 3. Figure 3 The following are (a) state comparison, (b) particle size and polydispersity index comparison, (c) potential comparison, (d) encapsulation efficiency comparison, and (e) drug loading comparison of lac and zein-curcumin nanoparticles prepared under the same total flow rate, the same flow rate ratio, the same ratio of lac and zein, the same ethanol concentration, and different curcumin concentrations in Example 5 and Examples 10-13 of the present invention;

[0050] 4. Figure 4 Examples 17-21 of the present invention, with different particle sizes and PDI, Figure 2 (b) In contrast, the particle sizes increased by about 20 nm, proving that C8G2 was successfully bound to the surface of lac and zein-curcumin nanoparticles, and lac and zein-curcumin-C8G2 drug-loaded nanoparticles were successfully prepared;

[0051] 5. Figure 5 Embodiment 18 of the present invention is a transmission electron microscope (TEM) image and a scanning electron microscope (SEM) image of shellac and zein-curcumin-C8G2 drug-loaded nanoparticles;

[0052] 6. Figure 6 It is a comparative diagram of the release of a shellac and zein-curcumin drug-loaded nanoparticles under different pH conditions in Comparative Examples 1-2 of the present invention and Examples 2, 5, and 8 of the present invention;

[0053] 7. Figure 7 A stability comparison diagram of shellac and zein-curcumin drug-loaded nanoparticles in Examples 2, 5, 8, 10-13 of the present invention;

[0054] 8. Figure 8 This is a comparison chart of the cytotoxicity of shellac and zein-curcumin-C8G2 drug-loaded nanoparticles in Examples 5 and 15 of the present invention. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0056] Example

[0057] Example 1

[0058] A method for preparing shellac and zein-curcumin drug-loaded nanoparticles comprises the following steps:

[0059] (1) Preparation of shellac solution: Weigh 200 mg of shellac and dissolve it in 10 mL of 95% ethanol to a concentration of 20 g / L. Ultrasonicate for 2-3 hours until the shellac solid is completely dissolved to obtain a shellac solution with a concentration of 20 g / L in the form of a yellow transparent liquid.

[0060] (2) Preparation of zein solution: Weigh 200 mg of zein and dissolve it in 10 ml of ethanol to obtain a 20 g / L zein solution in the form of a white transparent liquid.

[0061] (3) Preparation of a mixture of shellac and zein: The shellac solution obtained in (1) and the zein solution obtained in (2) were mixed in a mixing ratio of 95:5 (zein solution: shellac solution).

[0062] (4) Preparation of a mixture of lac and zein-curcumin: 10 mL of the mixture of lac and zein obtained in (3) was taken and 60 mg of curcumin was added, with a curcumin concentration of 6 g / L.

[0063] (5) Solution membrane: The shellac and zein-curcumin mixed solution obtained in step (4) is passed through a 0.22 μm organic membrane, and the shellac and zein-curcumin mixed solution after membrane is loaded into a 20 mL disposable syringe for standby use; pure water is passed through a 0.22 μm water membrane, and the pure water after membrane is loaded into a 20 mL syringe for standby use.

[0064] (5) Fixing the solution on the microfluidic pump: fixing the shellac and zein-curcumin mixed solution on pump 1 of the microfluidic pump, and fixing pure water on pump 2 of the microfluidic pump.

[0065] (6) Installing the chip lab: Place the microfluidic chip made of polymer material in the middle of a specific polymer material clamp and fix it with screws at the four corners.

[0066] (7) Connecting the solution and the chip lab: The two solutions fixed in the above step (5) are connected to the chip lab using a PVC hose according to the principle of high flow rate and straight flow.

[0067] (8) Exhaust air: Select Pump 1, adjust the flow rate to 0.5 g / L, click Start, and wait until the solution flows out through the chip laboratory, then click Stop; Select Pump 2, adjust the flow rate to 0.5 g / L, click Start, and wait until the solution flows out through the chip laboratory, then click Stop. At this time, exhaust air is completed.

[0068] (9) Preparation of drug-loaded nanoparticles: The total flow rate was adjusted to TFR = 48 mL / min, the flow rate ratio was set to FRR = 2:1 (aqueous phase: organic phase), and pump 1 and pump 2 of the microfluidic pump were turned on at the same time to prepare shellac and zein-curcumin drug-loaded nanoparticles. The particle size of shellac and zein-curcumin drug-loaded nanoparticles was 340 nm, and the polydispersity index was 0.17.

[0069] Example 2

[0070] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 90:10.

[0071] Example 3

[0072] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 80:20.

[0073] Example 4

[0074] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 70:30.

[0075] Example 5

[0076] Compared with Example 1, the difference is that the mixing ratio of the shellac solution and the zein solution is 50:50.

[0077] Example 6

[0078] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 30:70.

[0079] Example 7

[0080] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 20:80.

[0081] Example 8

[0082] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 10:90.

[0083] like Figure 8 As shown, it was demonstrated that lac and zein-curcumin-C8G2 had stronger anti-tumor effects, and nanoparticles containing C8G2 had stronger anti-tumor effects.

[0084] Example 9

[0085] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 5:95.

[0086] Example 10

[0087] Compared with Example 1, the difference is that the mixing ratio of the shellac solution and the zein solution is 50:50, and the concentration of curcumin is 2 mg / mL.

[0088] Embodiment 11

[0089] Compared with Example 1, the difference is that the mixing ratio of the shellac solution and the zein solution is 50:50, and the concentration of curcumin is 4 mg / mL.

[0090] Example 12

[0091] Compared with Example 1, the difference is that the mixing ratio of the shellac solution and the zein solution is 50:50, and the concentration of curcumin is 8 mg / mL.

[0092] Embodiment 13

[0093] Compared with Example 1, the difference is that the mixing ratio of the shellac solution and the zein solution is 50:50, and the concentration of curcumin is 10 mg / mL.

[0094] Embodiment 14

[0095] The nanoparticle solution prepared in Example 2 was stirred with a 100 μg / mL C8G2 aqueous solution overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0096] Embodiment 15

[0097] The nanoparticle solution prepared in Example 5 was stirred with a 100 μg / mL C8G2 aqueous solution overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0098] Example 16

[0099] The nanoparticle solution prepared in Example 8 was stirred with a 100 μg / mL C8G2 aqueous solution overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0100] Comparative Example

[0101] Comparative Example 1

[0102] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 100:0.

[0103] Comparative Example 2

[0104] Compared with Example 1, the difference is that the mixing ratio of the shellac solution to the zein solution is 0:100.

[0105] Comparative Example 3

[0106] Compared with Example 1, the difference is that the mixing ratio of the shellac solution and the zein solution is 50:50, and curcumin is not added.

[0107] Comparative Example 4

[0108] Compared with Comparative Example 1, the prepared nanoparticle solution was stirred with a 100 μg / mL C8G2 aqueous solution overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0109] Comparative Example 5

[0110] Compared with Comparative Example 2, the prepared nanoparticle solution was stirred with a 100 μg / mL C8G2 aqueous solution overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0111] 1. The following tests were performed on Examples 1-13, 15 and Comparative Examples 1-3:

[0112] (1) Particle size, polydispersity index and potential detection:

[0113] The particle size, polydispersity index (PDI) and zeta potential of the nanoparticles were determined by dynamic light scattering (Zetasizer Nano ZS, Malvern UK). 0.2 mL of nanoparticles were diluted in 0.8 mL of ultrapure water and measured at 25 °C and reported as the average of three independent tests.

[0114] (2) Encapsulation efficiency detection:

[0115] The encapsulation efficiency is defined as the portion of the initial antioxidants encapsulated by the shellac and zein nanoparticles. When the shellac and zein loaded nanoparticles aggregate and sink at pH = 2, the unencapsulated antioxidants are left in the supernatant, and then a refrigerated centrifuge (Germany, Eppendorf, Centrifuge5418R) is used to centrifuge for 30 min at 12000 rpm to completely sink the nanoparticles, separate the supernatant, and calculate its concentration by measuring the ultraviolet-visible absorption of the supernatant, which is linearly proportional to the antioxidant concentration. The calculation formula is as follows:

[0116]

[0117] Cf: amount of free drug

[0118] Ct: Total amount of drug in nanosuspension

[0119] The encapsulation efficiency of the shellac and zein-curcumin drug-loaded nanoparticles prepared in Examples 1-9 was 54%-98%.

[0120] (3) Detection of drug loading:

[0121] 0.7 mg of freeze-dried shellac and zein-curcumin drug-loaded nanoparticles were weighed using an electronic balance (Satorius, USA), added with hexanol and placed in a numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), and dissolved under ultrasonic power of 100 W for 30 min. The concentration was calculated by measuring the UV-visible absorption of the solution, which was linearly proportional to the antioxidant concentration. The calculation formula is as follows:

[0122]

[0123] We: the amount of drug encapsulated by the drug-carrying nanoparticles;

[0124] Wm: total weight of drug-loaded nanoparticles;

[0125] It can be calculated that the drug loading amounts of the shellac and zein-curcumin drug-loaded nanoparticles prepared in Examples 1-9 are 18%-24%.

[0126] The particle size, dispersibility index, potential, encapsulation efficiency and drug loading of lac and zein-curcumin drug-loaded nanoparticles prepared at different mixing ratios of lac solution and zein solution were tested.

[0127] Figure 2The present invention is a comparison of (a) state, (b) particle size and polydispersity index, (c) potential, (d) encapsulation efficiency, and (e) drug loading of lac and zein-curcumin nanoparticles prepared under the same total flow rate, the same flow rate ratio, different ratios of lac and zein, the same ethanol concentration, and the same curcumin concentration in Examples 1-18 of the present invention and Comparative Examples 1-3.

[0128] like Figure 2 As shown, it can be seen that with the addition of a small amount of lac, the particle size can be reduced, and after a large amount of addition, it increases slightly. The potential is below -20mV, and the encapsulation efficiency and drug loading increase with the increase in the proportion of lac incorporation. According to the different comprehensive judgments of the mixing ratios of lac solution and zein solution in Examples 2-8, the drug-loaded nanoparticles prepared under the condition that the mixing ratio of lac solution to zein solution is 50:50 (zein solution: lac solution) are the best.

[0129] like Figure 3 As shown, the lac and zein-curcumin nanoparticles prepared under the same total flow rate, the same flow rate ratio, the same ratio of lac and zein, the same ethanol concentration, and different curcumin concentrations in Example 5 of the present invention and Examples 10-13 show (a) state comparison, (b) particle size and polydispersity index comparison, (c) potential comparison, (d) encapsulation efficiency comparison, and (e) drug loading comparison.

[0130] according to Figure 3 Example 15 can be measured simultaneously, and the difference in potential proves that the lac and zein-curcumin nanoparticles are successfully connected with the C8G2 anti-tumor peptide to form lac and zein-curcumin-C8G2 drug-loaded nanoparticles.

[0131] 2. Example 15 performs the following tests

[0132] (1) Transmission electron microscopy (TEM) detection:

[0133] Before TEM detection, the sample needs to be dialyzed in ultrapure water for 24 hours through a dialysis bag (molecular weight cutoff 3.5 kDa, Shanghai Titan Technology Co., Ltd.) until there is no organic solvent such as ethanol in the dialysis bag, and then freeze-dried (FreeZone2.5L, Beijing Zhaoshengxing Instrument Equipment Co., Ltd., USA). The dialyzed drug-loaded nanoparticles are taken and the micromorphology of the sample is evaluated by microscopy at an accelerating voltage of 100 kV. The sample solution was diluted 10 times and deposited on a carbon-coated copper grid for transmission electron microscopy analysis after dilution. The estimation of particle size was studied using software, such as Figure 5 As shown in (a), it can be seen from the figure that the particle size is round and there are small white particles in the drug-loaded nanoparticles, which are curcumin, proving that the encapsulation of curcumin is successful and evenly dispersed.

[0134] (2) Scanning electron microscope (SEM) detection:

[0135] The dialyzed and freeze-dried nanoparticles were observed using an ultra-high field emission scanning electron microscope and the particle size was measured using a program. Figure 5 As shown in (b), it can be seen that the lac and zein-curcumin-C8G2 drug-loaded nanoparticles prepared under the optimal conditions are uniformly dispersed and have uniform particle sizes.

[0136] 5. Examples 2, 5, 8, Comparative Example 1 and Comparative Example 2 were subjected to the following tests:

[0137] (1) Release detection

[0138] The nanoparticles were placed in phosphate buffers of different pH values ​​and placed in a shaker. Samples were taken out at different time points and centrifuged. The supernatant was taken for absorbance detection and brought into the standard curve to calculate the release content.

[0139] (2) Stability test

[0140] The nanoparticles were dialyzed to remove residual organic solvent and excess curcumin and then stored under light-protected conditions at 4 °C. Their stability was monitored by regular assessment of particle size, polydispersity index, and zeta potential, providing insights into potential changes in particle size distribution and aggregation behavior.

[0141] Figure 6 The effect of zein:lac composition on the release of curcumin from lac and zein-curcumin loaded nanoparticles was revealed. Both zein nanoparticles and lac nanoparticles showed slow release of curcumin, which exceeded the equilibrium of 50:50 with increasing lac addition ratio. This phenomenon may stem from two factors: larger size and polydispersity index of high zein ratio and lack of compact structure with excess lac. Therefore, the stability of the nanoparticles was affected by the skew ratio, resulting in faster curcumin release. In addition, Figure 6 The release rate at pH 7.8 was higher than that at pH 7.4, which may be attributed to the enhanced degradation of shellac and zein under alkaline conditions, which promoted the diffusion of curcumin.

[0142] Figure 7 Minimal changes in nanoparticle size and polydispersity index over time were shown, further solidifying their strong stability. Figure 7 The efficacy of shellac and zein composites to encapsulate and stabilize curcumin was demonstrated, with the size characteristics showing impressive temporal stability.

[0143] 7. MTT cytotoxicity test was performed on Examples 2 and 15.

[0144] Human breast cancer cells (MCF-7) in the proliferation phase were seeded in 96-well plates at a rate of 5000 cells per well. After incubation at 37°C in a humidified 5% CO2 incubator for 24 hours, the culture medium was replaced with fresh DMEM containing different concentrations of curcumin (15, 20, 25, 30 and 35 μM), which were encapsulated by Z+S nanoparticles at different ratios. Each concentration gradient was repeated 6 times. Wells containing only DMEM without cells served as blank controls, while wells containing only DMEM served as negative controls. They were further incubated for 24 hours under the same conditions and then washed with phosphate-buffered saline (PBS). Subsequently, 150 μL of DMEM supplemented with MTT (5 mg / mL) was added to each well and incubated for 4 hours at 37°C and 5% CO2. The culture medium was then replaced with DMSO and the plate was shaken at 160 rpm for 10 minutes in a light-proof multifunctional plate reader (VarioskanLUX, ThermoFisher). Cell viability was then determined by measuring the optical density at 490 nm using the following formula:

[0145]

[0146] ODs: represents sample group; ODb: blank control group; ODc: negative control group.

[0147] like Figure 8 As shown, the MTT method was used to detect the cytotoxicity of curcumin-loaded nanoparticles with different ratios of lac to zein on MCF-7 cells, which proved that nanoparticles containing C8G2 antitumor peptide had a stronger antitumor effect.

[0148] The above is only an exemplary embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A lac and zein-curcumin-C8G2 drug-loaded nanoparticle, characterized in that: The invention comprises the following raw materials in parts by weight: 100-300 parts of lac, 100-300 parts of zein, 1-150 parts of curcumin, and 1-10 parts of C8G2 anti-tumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2; a microfluidic device is used to control the total flow rate and the flow rate ratio of the aqueous phase to the organic phase to obtain lac and zein-curcumin-C8G2 drug-loaded nanoparticles.

2. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 1, characterized in that: The following steps are involved: (1) placing shellac in ethanol and dispersing the shellac evenly until the shellac is completely dissolved to obtain a shellac solution; (2) dissolving zein solid particles in ethanol to obtain a zein solution; (3) mixing the shellac solution and the zein solution according to a certain ratio to obtain a mixture of shellac solution and zein; (4) dissolving curcumin in the shellac solution and the zein mixed solution to obtain a shellac and zein-curcumin mixed solution; (5) using a microfluidic device, by adding an aqueous phase and an organic phase into the microfluidic device, the lac and zein-curcumin mixed solution prepared in step (4) as the organic phase, pure water as the aqueous phase, setting the total flow rate TFR of the microfluidic device to 30-60 mL / min, and the flow rate ratio of the aqueous phase to the organic phase to be 1-5:1 to prepare lac and zein-curcumin drug-loaded nanoparticles; (6) The prepared nanoparticles were mixed with C8G2 antitumor peptide CH3(CH2)CO-GIIKKIIKKI-NH2 solution and water and stirred overnight to prepare shellac and zein-curcumin-C8G2 drug-loaded nanoparticles.

3. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (1), shellac is dissolved in 95% ethanol, the concentration of the shellac solution is 20-25 g / L, and the shellac is dispersed uniformly by ultrasonic treatment.

4. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (2), the concentration of the zein solution is 10-30 g / L.

5. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (3), the volume ratio of the shellac solution to the zein solution is 1-100:100-1.

6. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (4), the concentration of curcumin is 1-10 g / L.

7. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (5), the total flow rate TFR of the microfluidic device is 48-60 mL / min.

8. The method for preparing the lac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (5), the flow rate ratio of the aqueous phase to the organic phase in the microfluidic device is 2-5:

1.

9. The method for preparing shellac and zein-curcumin-C8G2 drug-loaded nanoparticles according to claim 2, characterized in that: In step (6), the particle size of the shellac and zein-curcumin-C8G2 drug-loaded nanoparticles is 70-340 nm.

10. An application of shellac and zein-curcumin-C8G2 drug-loaded nanoparticles, characterized in that: Use of the shellac and zein-curcumin-C8G2 nanoparticles according to claim 1 in the preparation of a drug for treating cancer.

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