Curcumin-loaded shellac liposome double-layer mixed nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a curcumin-loaded shellac liposome double-layer mixed nano-particle, a preparation method and application thereof, and belongs to the technical field of liposome nano-particle preparation. The invention provides a curcumin-loaded shellac lipidosome double-layer mixed nano-particle which comprises a shellac nano-particle loaded with curcumin, and the surface of the shellac nano-particle is coated with a lipidosome double-layer structure. The invention also provides a preparation method of the curcumin-loaded shellac liposome double-layer mixed nanoparticles, which comprises the following steps: dissolving curcumin and shellac in ethanol to prepare an organic phase co-solution, and preparing the curcumin-shellac nanoparticles. The preparation method comprises the following steps: dissolving phospholipid and cholesterol in ethanol to prepare an organic phase co-melting solution; and preparing dispersion liquid with different concentrations of curcumin and lac. And preparing the double-layer mixed nanoparticles of the shellac liposome loaded with the curcumin. The nano-particles are uniform in size, have good biocompatibility and biodegradability, are non-toxic and non-irritant, do not need to be added with a surfactant, and further reduce the toxicity.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome nanoparticle preparation, and specifically relates to a shellac liposome double-layer mixed nanoparticle loaded with curcumin, a preparation method and application thereof. Background Art

[0002] Curcumin (Cur) is a natural diketone compound, belonging to plant polyphenols, mostly extracted from the root of the plant turmeric. Turmeric has a variety of pharmacological effects: antioxidant, anti-inflammatory, anti-tumor, antibacterial and anti-proliferative. Compared with other natural medicines, curcumin has many unique advantages, such as maintaining low biological toxicity even at high doses. In clinical trials, patients still showed good efficacy when taking up to 12g of curcumin per day, without major adverse reactions. Curcumin is a traditional Chinese medicine chemotherapy resistance reversal agent with a wide range of targets, and can enhance the sensitivity of resistant cells to drugs through various pathways. However, the poor stability of curcumin, easy decomposition in light, and extremely poor solubility in water, which is almost insoluble, greatly narrows the scope of use of curcumin. Therefore, it needs to be properly treated to deliver curcumin to the target tissue and improve the bioavailability of curcumin.

[0003] Shellac, as a natural resin, has unique physical and chemical properties, which makes it have certain application prospects in the field of drug delivery. Shellac can be made into nanofibers through electrospinning technology, which can efficiently deliver drugs to the colon. The pH responsiveness of shellac nanofibers enables them to release drugs under specific physiological conditions, thereby improving the bioavailability and therapeutic effect of drugs. In addition, shellac nanofibers can also exert higher anticancer activity than free drugs by blocking cell cycle distribution and inducing apoptosis, showing its application potential in the field of functional foods.

[0004] The structure of liposomes is extremely similar to that of cell membranes, making them highly biocompatible and biodegradable. Therefore, liposomes can protect drugs from being degraded by enzymes before they reach the lesion site. At the same time, drugs are "hidden" inside liposomes in the form of physical encapsulation, which can improve drug stability, reduce drug toxicity, increase drug dosage, and achieve better therapeutic effects. Summary of the invention

[0005] In view of the defects existing in the above-mentioned prior art, in order to solve the problems that curcumin has poor stability, is easily decomposed by light and has poor solubility in water, making it difficult to use as a medicine, the present invention provides a curcumin-loaded shellac liposome bilayer nanoparticle, a preparation method and an application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides a curcumin-loaded shellac liposome bilayer hybrid nanoparticle, wherein the nanoparticle comprises a curcumin-loaded shellac nanoparticle, and a liposome bilayer structure is coated on the surface of the shellac nanoparticle.

[0008] In a second aspect, the present invention provides a method for preparing the curcumin-loaded shellac liposome bilayer mixed nanoparticles, comprising the following steps:

[0009] (1) weighing shellac and curcumin, dissolving them in a polar organic solvent, ultrasonically dissolving to obtain a mixed solution, filtering to remove impurities, and loading the mixed solution into syringe a as an organic phase; passing pure water through a membrane and loading it into syringe b as an aqueous phase, performing a microfluidic reaction, and obtaining shellac nanoparticles loaded with curcumin;

[0010] (2) Weigh DSPC (Chinese name: distearoylphosphatidylcholine) and cholesterol, dissolve them in a polar organic solvent, dissolve them by ultrasonication, filter out impurities, and load them into syringe 1 as an organic phase;

[0011] (3) dialyzing the curcumin-loaded shellac nanoparticles obtained in step (1) to remove the polar organic solvent, centrifuging to remove free curcumin, taking the supernatant, passing it through a membrane, and diluting it with pure water to obtain a diluted curcumin-loaded shellac nanoparticle solution, which is loaded into syringe 2 as an aqueous phase;

[0012] (4) The organic phase and the aqueous phase obtained in the above steps (2) and (3) are subjected to a microfluidic reaction to obtain curcumin-loaded shellac liposome bilayer mixed nanoparticles.

[0013] In the preparation method, the conditions of the microfluidic reaction in step (1) are: the flow rate ratio of the organic phase to the aqueous phase is 1:(1-5), and the total flow rate is 2-48 mL / min.

[0014] In the preparation method, the concentration of shellac in the mixed solution in step (1) is 0-10 mg / mL, and the concentration of curcumin is 1-5 mg / mL.

[0015] In the preparation method, the polar organic solvent in steps (1) and (2) is ethanol.

[0016] In the preparation method, the mass ratio of DSPC to cholesterol in step (2) is 2:1;

[0017] The volume ratio of the total mass of the DSPC and cholesterol to the polar organic solvent is 4:1 mg / mL.

[0018] In the preparation method, the concentration of the diluted curcumin-loaded shellac nanoparticle solution in step (3) is 0.1-0.5 mg / mL.

[0019] In the preparation method, the conditions of the microfluidic reaction in step (4) are: the flow rate ratio of the organic phase to the aqueous phase is 2:1-1:10, and the total flow rate is 4-48 mg / mL.

[0020] In a third aspect, the present invention provides the use of the curcumin-loaded shellac liposome bilayer mixed nanoparticles, or the curcumin-loaded shellac liposome bilayer mixed nanoparticles obtained by any of the preparation methods described in the preparation of anti-tumor drugs;

[0021] Preferably, the tumor is breast cancer.

[0022] In a fourth aspect, the present invention provides an anticancer drug comprising the curcumin-loaded shellac liposome bilayer mixed nanoparticles.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The nanoparticles prepared by the present invention are spherical, have controllable particle size, uniform particle size, and good stability, which is conducive to their stable release in vivo, are non-toxic and non-irritating, and no surfactant needs to be added during the preparation process of the nanoparticles, further reducing toxicity.

[0025] 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 tumor cell growth. The nanoparticles prepared by the present invention have good stability, which is conducive to its stable release in vivo.

[0026] 3. The preparation method of the present application is simple in process and easy to operate. With the help of the micro-quantification and precision of the microfluidic chip, the preparation time is greatly shortened by adjusting different experimental parameters. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Microfluidic preparation of curcumin-loaded shellac liposome bilayer nanoparticles and their particle size distribution diagram for Example 1-5;

[0028] Figure 2 This is a graph showing the potential comparison results of different nanoparticles prepared in Example 5 and Comparative Examples 1-4;

[0029] Figure 3 This is a diagram showing the parameter optimization results of preparing blank shellac particles by microfluidics in Comparative Example 1;

[0030] Figure 4 This is a diagram showing the parameter optimization results of preparing blank liposome particles by microfluidics in Comparative Example 2;

[0031] Figure 5 The result diagram of the effect of curcumin concentration on the particle size of shellac loaded with curcumin in Comparative Example 3;

[0032] Figure 6 TEM schematic diagram of different nanoparticles prepared in Example 5 and Comparative Example 3.

[0033] Figure 7 Schematic diagram of MTT toxicity of different nanoparticles prepared in Example 5 and Comparative Examples 3 and 4. DETAILED DESCRIPTION

[0034] The present invention will be further described below by means of drawings and embodiments.

[0035] Embodiment 1:

[0036] A method for preparing curcumin-loaded shellac liposome bilayer nanoparticles comprises the following steps:

[0037] (1) Weigh 100 mg of shellac and 30 mg of curcumin and dissolve them in 10 mL of ethanol. Use ultrasound to completely dissolve the shellac. Use a 0.22 μm organic filter membrane to remove impurities in the solution and load it into a 20 mL syringe a. Pass pure water through a water membrane (0.22 μm) and load it into syringe b (diameter 19.13 mm). Install syringe a and syringe b into the microfluidic device, set the flow rate ratio to 1:3, and set the total flow rate to 20 mL / min. After the outlet solution flows out steadily, collect the shellac nanoparticles loaded with curcumin at the outlet and store at 4°C.

[0038] (2) Weigh 40 mg of phospholipid and cholesterol (phospholipid: cholesterol mass ratio 2:1) and dissolve them in 10 mL of anhydrous ethanol. Sonicate to completely dissolve them. Use a 0.22 μm organic filter to remove impurities in the solution. Load the solution into a 20 mL syringe 1 (as the organic phase) and set aside.

[0039] (3) The shellac nanoparticles loaded with curcumin prepared in step (1) were dialyzed for 24 h to remove the ethanol solution, and then centrifuged to remove free curcumin. The supernatant was taken, passed through a membrane, and diluted with pure water to prepare a solution of the shellac nanoparticles loaded with curcumin with a concentration of 0.1 mg / mL, which was loaded into a 20 mL syringe 2 (as an aqueous phase) for standby use.

[0040] (4) The syringe 1 and the syringe 2 obtained in steps (2) and (3) were installed in a microfluidic device, and the flow rate ratio of the organic phase to the aqueous phase was set to 1:3, and the total flow rate was 20 mg / mL. After the product solution at the outlet was stabilized, the curcumin-loaded shellac liposome bilayer nanoparticles were collected at the outlet and stored at 4°C.

[0041] Embodiment 2:

[0042] The preparation method of curcumin-loaded shellac liposome bilayer nanoparticles-2 is the same as that of Example 1, except that the concentration of the curcumin-loaded shellac nanoparticle solution in step (3) is 0.2 mg / mL.

[0043] Embodiment 3:

[0044] The preparation method of curcumin-loaded shellac liposome bilayer nanoparticles-3 is the same as that of Example 1, except that the concentration of the curcumin-loaded shellac nanoparticle solution in step (3) is 0.3 mg / mL.

[0045] Embodiment 4:

[0046] The preparation method of curcumin-loaded shellac liposome bilayer nanoparticles-4 is the same as that of Example 1, except that the concentration of the curcumin-loaded shellac nanoparticle solution in step (3) is 0.4 mg / mL.

[0047] Embodiment 5:

[0048] The preparation method of curcumin-loaded shellac liposome bilayer nanoparticles-5 is the same as that of Example 1, except that the concentration of the curcumin-loaded shellac nanoparticle solution in step (3) is 0.5 mg / mL.

[0049] Example 1-5 Microfluidic preparation of curcumin-loaded shellac liposome bilayer nanoparticles and their particle size distribution Figure 1 As shown in FIG. 1 , the particle size increases with the concentration of the shellac particles loaded with curcumin. To ensure the curcumin loading, 0.5 mg / mL was finally selected. The potential comparison results of different nanoparticles prepared in Example 5 and Comparative Examples 1-4 are shown in FIG. Figure 2 As shown, it can be seen that according to the different potentials, it is proved that the shellac liposome bilayer nanoparticles loaded with curcumin prepared in Example 5 are successfully prepared. Figure 2 In the figure, cur represents the curcumin particles prepared in Comparative Example 4, shellac represents the shellac nanoparticles prepared in Comparative Example 1, DSPC represents the liposome nanoparticles prepared in Comparative Example 2, cur@shellac represents the curcumin-loaded shellac nanoparticles prepared in Comparative Example 3, and Nps represents the curcumin-loaded shellac liposome bilayer nanoparticles-5 prepared in Example 5.

[0050] Comparative Example 1:

[0051] A preparation method of shellac nanoparticles comprises the following steps:

[0052] (1) Weigh 100 mg of shellac and dissolve it in 10 mL of anhydrous ethanol. Sonicate to completely dissolve the shellac. Use a 0.22 μm organic filter to remove impurities in the solution. Place the solution in a 20 mL syringe 1.

[0053] (2) Pure water is passed through a water membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device.

[0054] (3) The total flow rate was set to 2-48 mL / min, and the flow rate ratio of the organic phase to the aqueous phase was set to 1:(1-10). After the product solution at the outlet was stable, the shellac nanoparticles were collected at the outlet and stored at 4°C.

[0055] The particle size and dispersion coefficient (PDI) of shellac nanoparticles prepared at different flow rate ratios and total flow rates are shown in Figure 2. Figure 3 As shown, the flow rate ratio (ETOH / H2O) of 1:3 and the total flow rate of 20 mL / min were determined as the final preparation conditions.

[0056] Comparative Example 2:

[0057] A preparation method of liposomes comprises the following steps:

[0058] (1) Weigh 40 mg of phospholipid and cholesterol (phospholipid: cholesterol mass ratio 2:1) and dissolve them in 10 mL of anhydrous ethanol. Sonicate to completely dissolve them. Use a 0.22 μm organic filter to remove impurities in the solution and load it into a 20 mL syringe 1.

[0059] (2) Pure water is passed through a water membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device.

[0060] (3) The total flow rate is set to 4-48 mL / min, and the flow rate ratio of the organic phase to the aqueous phase is set to 2:1-1:10. After the product solution at the outlet is stable, the liposome nanoparticles are collected at the outlet and stored at 4°C.

[0061] The particle size and PDI of liposome nanoparticles prepared at different flow rate ratios and different total flow rates are shown in Figure 2. Figure 4 As shown, it can be seen that the flow rate ratio of 1:3 (ETOH / H2O) and the total flow rate of 30 mL / min are the optimal preparation conditions.

[0062] Comparative Example 3:

[0063] A method for preparing curcumin-loaded shellac nanoparticles comprises the following steps:

[0064] (1) Weigh 100 mg of shellac and 10-50 mg of curcumin and dissolve them in 10 mL of ethanol. Ultrasonicate to completely dissolve the shellac. Use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0065] (2) Pure water was passed through a water membrane (0.22 μm) and loaded into syringe 2 (diameter 19.13 mm). Syringes 1 and 2 were installed in the microfluidic device, and the flow rate ratio was set to 1:3, and the total flow rate was set to 20 mL / min. After the outlet solution flowed out steadily, the shellac nanoparticles loaded with curcumin were collected at the outlet and stored at 4°C.

[0066] Shellac@curcumin nanoparticles prepared at different concentrations of curcumin Figure 5 As shown, it can be seen that the curcumin concentration of 3 mg / mL is the optimal choice.

[0067] Comparative Example 4:

[0068] A preparation method of a curcumin solution comprises the following steps:

[0069] (1) Weigh 30 mg of curcumin and dissolve it in 10 mL of anhydrous ethanol. Sonicate to completely dissolve it. Use a 0.22 μm organic filter to remove impurities in the solution. Place the solution in a 20 mL syringe 1.

[0070] (2) Pure water is passed through a water membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the flow rate ratio of the organic phase to the aqueous phase is controlled to remain constant at 1:3.

[0071] (3) The flow rate ratio of the organic phase to the aqueous phase was set to 1:3. After the product solution at the outlet was stable, the curcumin particles were collected at the outlet and stored at 4°C.

[0072] Embodiment 6:

[0073] The products obtained in Example 5 and Comparative Example 3 were subjected to transmission electron microscopy (TEM) detection, as follows:

[0074] 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 6As shown (Comparative Example 3, Example 5), it can be seen from the figure that the particle size is round, and there are small white particles in the drug-loaded nanoparticles, that is, the shellac nanoparticles loaded with curcumin, which proves that the shellac liposome bilayer mixed nanoparticles loaded with curcumin are successfully prepared.

[0075] Embodiment 7:

[0076] The MTT cytotoxicity test was performed on the products prepared in Example 5 and Comparative Examples 3 and 4, as follows:

[0077] 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 for 24 hours in a humidified 5% CO2 incubator, the culture medium was replaced with fresh DMEM solutions (Example 5 and Comparative Examples 3 and 4) containing different nanoparticles of different concentrations of curcumin (5, 10, 20, 40 and 60 μM). Each concentration gradient was repeated 6 times. Wells containing only DMEM but no cells were used as blank controls, while wells containing only DMEM were used as negative controls. Further incubation was performed under the same conditions for 24 hours, followed by washing with phosphate buffered saline (PBS). Subsequently, 150 μL of DMEM with MTT (5 mg / mL) added 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-shielded multifunctional plate reader (Varioskan LUX, Thermo Fisher). Then, the cell viability was determined by measuring the optical density at 490 nm, as shown in the following formula (1):

[0078]

[0079] Among them, ODs represents the sample group. b represents the blank control group, and ODc represents the negative control group.

[0080] The results of MTT assay for the cytotoxicity of curcumin, curcumin-loaded shellac nanoparticles, and curcumin-loaded shellac liposome bilayer hybrid nanoparticles on MCF-7 cells are shown in the figure. Figure 7 As shown, it was demonstrated that the shellac liposome bilayer hybrid nanoparticles loaded with curcumin had a stronger antitumor effect on MCF-7.

[0081] from Figure 7 It can be seen that the cell survival rate of curcumin is lower than that of the curcumin-loaded shellac liposome particles, indicating that the curcumin-loaded shellac liposome nanoparticles increase the anticancer efficacy.

[0082] The above is only an exemplary embodiment of the present disclosure, but the protection scope of the present disclosure 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 in the present disclosure should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A curcumin-loaded shellac liposome bilayer mixed nanoparticle, characterized in that: The nanoparticles include shellac nanoparticles loaded with curcumin, and the surface of the shellac nanoparticles is coated with a liposome structure.

2. The method for preparing a curcumin-loaded shellac liposome bilayer mixed nanoparticle according to claim 1, characterized in that: The following steps are involved: (1) weighing shellac and curcumin, dissolving them in a polar organic solvent, ultrasonically dissolving to obtain a mixed solution, filtering to remove impurities, and loading the mixed solution into syringe a as an organic phase; passing pure water through a membrane and loading it into syringe b as an aqueous phase, performing a microfluidic reaction, and obtaining shellac nanoparticles loaded with curcumin; (2) Weigh DSPC and cholesterol, dissolve them in a polar organic solvent, dissolve them by ultrasonication, filter out impurities, and load them into syringe 1 as an organic phase; (3) dialyzing the curcumin-loaded shellac nanoparticles obtained in step (1) to remove the polar organic solvent, centrifuging to remove free curcumin, taking the supernatant, passing it through a membrane, and diluting it with pure water to obtain a diluted curcumin-loaded shellac nanoparticle solution, which is loaded into syringe 2 as an aqueous phase; (4) The organic phase and the aqueous phase obtained in the above steps (2) and (3) are subjected to a microfluidic reaction to obtain curcumin-loaded shellac liposome bilayer mixed nanoparticles.

3. The preparation method according to claim 2, characterized in that: The conditions of the microfluidic reaction in step (1) are: the flow rate ratio of the organic phase to the aqueous phase is 1:(1-5), and the total flow rate is 2-48 mL / min.

4. The preparation method according to claim 2, characterized in that: The concentration of shellac in the mixed solution in step (1) is 10 mg / mL, and the concentration of curcumin is 1-5 mg / mL.

5. The preparation method according to claim 2, characterized in that: The polar organic solvent in steps (1) and (2) is ethanol.

6. The preparation method according to claim 2, characterized in that: The mass ratio of DSPC to cholesterol in step (2) is 2:1; The volume ratio of the total mass of the DSPC and cholesterol to the polar organic solvent is 4:1 mg / mL.

7. The preparation method according to claim 2, characterized in that: The concentration of the diluted curcumin-loaded shellac nanoparticle solution in step (3) is 0.1-0.5 mg / mL.

8. The preparation method according to claim 2, characterized in that: The conditions of the microfluidic reaction in step (4) are: the flow rate ratio of the organic phase to the aqueous phase is 2:1-1:10, and the total flow rate is 4-48 mg / mL.

9. Use of the curcumin-loaded shellac liposome bilayer hybrid nanoparticles according to claim 1, or the curcumin-loaded shellac liposome bilayer hybrid nanoparticles obtained by the preparation method according to any one of claims 2 to 8 in the preparation of antitumor drugs; Preferably, the tumor is breast cancer.

10. An anticancer drug, characterized in that: The invention relates to a shellac liposome bilayer mixed nanoparticle containing curcumin as claimed in claim 1.

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