Magnolol targeting nanoparticle with high drug loading capacity

Abstract

The invention discloses a magnolol targeting nanoparticle with high drug loading capacity, which is composed of a magnolol nanocrystal and a polydopamine layer wrapping the magnolol nanocrystal, and folic acid is modified on the surface of the polydopamine layer. The preparation can slowly release magnolol and target breast cancer cells, and has the advantages of high drug loading capacity and high accumulative release rate.

Description

Technical Field

[0001] The invention relates to the technical field of drug delivery, and in particular to magnolol targeting nanoparticles with high drug loading. Background Art

[0002] The active ingredients of natural plants have attracted the attention of many researchers due to their clinical effects in natural products and herbal formulas. Among them, magnolol is a representative natural polyphenol active ingredient in magnolia bark. In recent years, it has attracted much attention due to its anti-cancer effects on a variety of cancers, including breast cancer, lung cancer, liver cancer and colon cancer. Preclinical studies have shown that magnolol has great therapeutic potential for cancer through multiple pharmacological mechanisms. However, magnolol has poor solubility and rapid metabolism, which affects its efficacy.

[0003] Traditional drug delivery methods face challenges such as poor systemic distribution, degradation, and solubility, which may lead to reduced drug efficacy and increased side effects. In contrast, nanoparticles have advantages such as targeted delivery, controlled release, and enhanced solubility, which can better cope with these challenges. Therefore, nanoparticles are a potential drug delivery method with broad application prospects.

[0004] Related studies have reported that the water solubility and bioavailability of magnolol were improved by using various nano dosage forms, such as solid dispersions, liposomes, nanoparticles, emulsions, mixed micelles and β-cyclodextrin.

[0005] Existing nano-drug delivery systems have low drug loading and cumulative release rates. Drug loading refers to the mass ratio of drug to nano-drug, while cumulative release rate refers to the percentage of loaded drug that can be released by nanoparticles. Low drug loading capacity means that more carrier material must be used to administer the required dose of drug at the target site, which may impose an additional burden on patients to degrade, metabolize and excrete these carriers.

[0006] According to literature reports, in the study of the use of nanoparticles for magnolia phenol delivery, different nanoparticle drug loadings were 2%-20%, and the cumulative release rate was 60%-80%. For example, the drug loading of chondroitin sulfate-coated zeolite-based nanoparticles was 2.04%, and the cumulative release rate was 61.65%; the drug loading of hydrogel nanoparticles was between 5%-20%, and the cumulative release rate was 60%; the drug loading of polylactic acid-glycolic acid copolymer nanoparticles was 6.38±0.13%, and the cumulative release rate was 80%; the drug loading of β-cyclodextrin and polypropylene glycol nanoparticles was 15.29%, and the cumulative release rate was about 70%. The magnolia phenol nanoparticles (polylactic acid-glycolic acid copolymer nanoparticles) in the existing patents have a drug loading of less than 10%, and a cumulative release rate of less than 80%. Summary of the invention

[0007] The purpose of the present invention is to provide a magnolol targeting nanoparticle with a high drug loading, which can slowly release magnolol and target breast cancer cells, and has the advantages of high drug loading and high cumulative release rate.

[0008] The technical solution adopted by the present invention to solve its technical problem is: A magnolol targeted nanoparticle with a high drug loading is composed of magnolol nanocrystals and a polydopamine layer wrapping the magnolol nanocrystals, wherein the surface of the polydopamine layer is modified with folic acid.

[0009] The invention utilizes the physical properties of magnolol and uses an anti-solvent crystallization method to prepare nanoparticles loaded with magnolol, and simultaneously utilizes the self-polymerization property of dopamine to form a polydopamine coating layer on the particle surface, and then connects the folic acid ligand with the polydopamine coating layer through a Michael addition reaction to obtain polydopamine magnolol nanoparticles modified with folic acid. Since folic acid receptors are overexpressed on the surface of cancer cells and can be used as tumor markers, the nanoparticles prepared by the invention can deliver magnolol to cancer cells in a targeted manner for release. In addition, due to the stable structure of polydopamine, the diffusion release rate of magnolol is slowed down, and the nanoparticles have a sustained release performance over a long period of time.

[0010] The innovation of the present invention lies in the structure without drug carriers. It uses drug nanocrystals as the core and directly wraps the functional shell, which not only retains the advantages of targeted sustained release in the prior art, but also achieves high drug loading and cumulative release rate, thereby increasing drug delivery efficiency.

[0011] Magnolia officinalis granules cannot be used directly for encapsulation. Magnolia officinalis granules are purchased as crystalline powders with relatively large particles. Direct encapsulation results in micron-sized particles, which lose the advantages of nanoparticles, such as EPR passive targeting, high solubility and high bioavailability. In addition, particles with too large a particle size are more difficult to disperse and are prone to precipitation and agglomeration. The particle size uniformity obtained by direct encapsulation is poor.

[0012] The preparation method comprises the following steps: (1) adding the magnolol solution to the poloxamer F127 solution, stirring and mixing, then adding the tris(hydroxymethyl)aminomethane solution, stirring and mixing, and finally adding dopamine hydrochloride, stirring continuously at 20-25° C. for 8-10 hours, centrifuging, and collecting the precipitate. The obtained precipitate is magnolol crystal nanoparticles coated with polydopamine; (2) adding the magnolol crystalline nanoparticles coated with polydopamine to the folic acid aqueous solution, stirring for 25-40 minutes, centrifuging, and collecting the precipitate to obtain magnolol targeted nanoparticles with surface modified folic acid; (3) The surface-modified magnolia bark phenol targeted nanoparticles with folic acid are pre-frozen and then vacuum freeze-dried to obtain the finished magnolia bark phenol targeted nanoparticles with a high drug loading.

[0013] The magnolol solution is an ethanol solution of magnolol with a concentration of 8-12 mg / mL.

[0014] The concentration of Poloxamer F127 solution is 1.8-2.2 mg / mL.

[0015] The concentration of the tris(hydroxymethyl)aminomethane solution is 35-45 mg / mL.

[0016] The concentration of folic acid aqueous solution is 1.8-2.2 mg / mL.

[0017] Tris(hydroxymethyl)aminomethane was added to the folic acid aqueous solution to adjust the pH to 8-9.

[0018] The prefreezing parameters are -20℃ for 12h.

[0019] In step (1), the mass ratio of magnolol: poloxamer F127: tris(hydroxymethyl)aminomethane: dopamine hydrochloride is 1:1:3:1.

[0020] In step (2), the mass ratio of magnolol crystalline nanoparticles coated with polydopamine: folic acid is 1:1.

[0021] The beneficial effects of the present invention are: no drug carrier is needed, polydopamine is directly wrapped outside the drug nanocrystals, and the drug loading amount and cumulative release rate of the nanoparticles are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the particle size distribution diagram of particles prepared by different methods; Figure 2 is a transmission electron microscope characterization image of magnolol targeting nanoparticles prepared in Example 3 of the present invention; Figure 3 is the electron diffraction pattern of the crystalline core of magnolol targeting nanoparticles prepared in Example 3 of the present invention; Figure 4 This is a graph showing the in vitro simulated drug release curve of magnolol targeted nanoparticles prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below through specific embodiments.

[0024] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0025] Embodiment 1: A high-drug-loading magnolol targeted nanoparticle, which is composed of magnolol nanocrystals and a polydopamine layer encapsulating the magnolol nanocrystals, wherein the surface of the polydopamine layer is modified with folic acid; the preparation method is as follows: (1) Preparation of magnolol solution: Dissolve magnolol in ethanol, stir to dissolve, prepare a solution with a magnolol concentration of 8 mg / mL, and then filter with a 0.22 μm microporous filter membrane to obtain a magnolol solution; (2) Preparation of poloxamer F127 solution: Dissolve poloxamer F127 in ultrapure water, stir to dissolve, and prepare a poloxamer F127 concentration of 1.8 mg / mL, then filter with a 0.22 μm microporous filter membrane to obtain a poloxamer F127 solution; (3) Preparation of tris(hydroxymethyl)aminomethane solution: dissolve tris(hydroxymethyl)aminomethane in ultrapure water, stir to dissolve, and prepare a tris(hydroxymethyl)aminomethane concentration of 35 mg / mL, and then filter with a 0.22 μm microporous filter membrane to obtain a tris(hydroxymethyl)aminomethane solution; (4) Preparation of folic acid solution: Dissolve folic acid in ultrapure water, stir to dissolve, add tris(hydroxymethyl)aminomethane to adjust the pH to 8, and prepare a folic acid concentration of 1.8 mg / mL, and then filter with a 0.22 μm microporous filter membrane to obtain a folic acid solution; (5) Preparation of nanoparticles: a: adding the magnolol solution obtained in step (1) to the poloxamer F127 solution obtained in step (2), stirring rapidly at 800 rpm for 2 minutes, adding the tris(hydroxymethyl)aminomethane solution obtained in step (3), stirring rapidly at 800 rpm for 2 minutes, and then adding dopamine hydrochloride; stirring continuously at 20 degrees Celsius for 10 hours; after the reaction is completed, centrifuging the obtained emulsion at 15,000 rpm for 20 minutes, and the obtained precipitate is magnolol crystal nanoparticles coated with polydopamine; wherein the mass ratio of magnolol: poloxamer F127: tris(hydroxymethyl)aminomethane: dopamine hydrochloride is 1:1:3:1; b: adding the nanoparticles obtained in step (a) to the folic acid aqueous solution obtained in step (4), stirring for 25 minutes, centrifuging at 15,000 rpm for 20 minutes, and collecting the precipitate to obtain nanoparticles with surface modified folic acid; the mass ratio of magnolol crystal nanoparticles coated with polydopamine: folic acid is 1:1; c: The nanoparticles obtained in step (b) were placed in a culture dish, pre-frozen at -20°C for 12 hours, and then vacuum freeze-dried at -50°C for 48 hours to obtain the finished nanoparticles. The drug loading was 54.26±9.1%, the cumulative release rate was 92%, the particle size was 183.5±87.9nm, and the surface potential was -16.0±18.9 mV.

[0026] Embodiment 2: A high-drug-loading magnolol targeted nanoparticle, which is composed of magnolol nanocrystals and a polydopamine layer encapsulating the magnolol nanocrystals, wherein the surface of the polydopamine layer is modified with folic acid; the preparation method is as follows: (1) Preparation of magnolol solution: Dissolve magnolol in ethanol, stir to dissolve, prepare a solution with a magnolol concentration of 12 mg / mL, and then filter with a 0.45 μm microporous filter membrane to obtain a magnolol solution; (2) Preparation of poloxamer F127 solution: Dissolve poloxamer F127 in ultrapure water, stir to dissolve, and prepare a poloxamer F127 concentration of 2.2 mg / mL, then filter with a 0.45 μm microporous filter membrane to obtain a poloxamer F127 solution; (3) Preparation of tris(hydroxymethyl)aminomethane solution: dissolve tris(hydroxymethyl)aminomethane in ultrapure water, stir to dissolve, and prepare a tris(hydroxymethyl)aminomethane concentration of 45 mg / mL, and then filter with a 0.45 μm microporous filter membrane to obtain a tris(hydroxymethyl)aminomethane solution; (4) Preparation of folic acid solution: Dissolve folic acid in ultrapure water, stir to dissolve, add tris(hydroxymethyl)aminomethane to adjust the pH to 9, and prepare a folic acid concentration of 2.2 mg / mL, then filter with a 0.45 μm microporous filter membrane to obtain a folic acid solution; (5) Preparation of nanoparticles: a: adding the magnolol solution obtained in step (1) to the poloxamer F127 solution obtained in step (2), stirring rapidly at 800 rpm for 2 minutes, adding the tris(hydroxymethyl)aminomethane solution obtained in step (3), stirring rapidly at 800 rpm for 2 minutes, and then adding dopamine hydrochloride; stirring continuously at 25 degrees Celsius for 8 hours; after the reaction is completed, centrifuging the obtained emulsion at 15,000 rpm for 20 minutes, and the obtained precipitate is magnolol crystal nanoparticles coated with polydopamine; wherein the mass ratio of magnolol: poloxamer F127: tris(hydroxymethyl)aminomethane: dopamine hydrochloride is 1:1:3:1; b: adding the nanoparticles obtained in step (a) to the folic acid aqueous solution obtained in step (4), stirring for 40 minutes, centrifuging at 15,000 rpm for 20 minutes, and collecting the precipitate to obtain nanoparticles with surface modified folic acid; the mass ratio of magnolol crystal nanoparticles coated with polydopamine: folic acid is 1:1; c: The nanoparticles obtained in step (b) were placed in a culture dish, pre-frozen at -20°C for 12 hours, and then vacuum freeze-dried at -50°C for 48 hours to obtain the finished nanoparticles. The drug loading was 56.71±12.57%, the cumulative release rate was 86%, the particle size was 187±93.7nm, and the surface potential was -15.2±20.2 mV.

[0027] Embodiment 3: A high-drug-loading magnolol targeted nanoparticle, which is composed of magnolol nanocrystals and a polydopamine layer encapsulating the magnolol nanocrystals, wherein the surface of the polydopamine layer is modified with folic acid; the preparation method is as follows: (1) Preparation of magnolol solution: Dissolve magnolol in ethanol, stir to dissolve, prepare a solution with a magnolol concentration of 10 mg / mL, and then filter with a 0.22 μm microporous filter membrane to obtain a magnolol solution; (2) Preparation of poloxamer F127 solution: Dissolve poloxamer F127 in ultrapure water, stir to dissolve, and prepare a poloxamer F127 concentration of 2 mg / mL, then filter with a 0.22 μm microporous filter membrane to obtain a poloxamer F127 solution; (3) Preparation of tris(hydroxymethyl)aminomethane solution: dissolve tris(hydroxymethyl)aminomethane in ultrapure water, stir to dissolve, and prepare a tris(hydroxymethyl)aminomethane concentration of 40 mg / mL, and then filter with a 0.22 μm microporous filter membrane to obtain a tris(hydroxymethyl)aminomethane solution; (4) Preparation of folic acid solution: Dissolve folic acid in ultrapure water, stir to dissolve, add tris(hydroxymethyl)aminomethane to adjust the pH to 8.5, and prepare a folic acid concentration of 2 mg / mL, then filter with a 0.22 μm microporous filter membrane to obtain a folic acid solution; (5) Preparation of nanoparticles: a. Add the magnolol solution obtained in step (1) to the poloxamer F127 solution obtained in step (2), and stir rapidly at 800 rpm for 2 minutes; then add the tris(hydroxymethyl)aminomethane solution obtained in step (3); stir rapidly at 800 rpm for 2 minutes, and then add dopamine hydrochloride; stir continuously at 20 degrees Celsius for 9 hours; after the reaction is completed, centrifuge the obtained emulsion at 15,000 rpm for 20 minutes, and the obtained precipitate is magnolol crystal nanoparticles coated with polydopamine; wherein the mass ratio of magnolol: poloxamer F127: tris(hydroxymethyl)aminomethane: dopamine hydrochloride is 1:1:3:1; b: adding the nanoparticles obtained in step (a) to the folic acid aqueous solution obtained in step (4), stirring for 35 minutes, centrifuging at 15,000 rpm for 20 minutes, and collecting the precipitate to obtain nanoparticles with surface modified folic acid; the mass ratio of magnolol crystal nanoparticles coated with polydopamine: folic acid is 1:1; c: The nanoparticles obtained in step (b) were placed in a culture dish, pre-frozen at -20°C for 12 hours, and then vacuum freeze-dried at -50°C for 48 hours to obtain the finished nanoparticles. The drug loading was 58.59±1.97%, the cumulative release rate was 90%, the particle size was 154.3±9.5nm, and the surface potential was -30.0±4.0 mV.

[0028] Comparative Example 1 This example is different from Example 3 in that: a: 5 mL of the magnolol solution obtained in step (1) was added to 25 mL of the poloxamer F127 solution obtained in step (2), and the mixture was rapidly stirred at 800 rpm for 2 minutes. 3 mL of ultrapure water (ultrapure water was used instead of the tris(hydroxymethyl)aminomethane solution) was added, and 60 mg of dopamine hydrochloride was added after rapid stirring at 800 rpm for 2 minutes. The mixture was stirred continuously at 20 degrees Celsius for 9 hours. After the reaction was completed, the obtained emulsion was centrifuged at 15,000 rpm for 20 minutes, and the obtained precipitate was magnolol crystal nanoparticles coated with polydopamine.

[0029] The rest is the same as in Example 3.

[0030] Comparative Example 2 This example is different from Example 3 in that: (1) Preparation of magnolia phenol suspension: Add magnolia phenol into ultrapure water, stir and disperse, and prepare a suspension with a magnolia phenol concentration of 8-12 mg / mL to obtain a magnolia phenol suspension.

[0031] The rest is the same as in Example 3.

[0032] In the present invention, the use of tris(hydroxymethyl)aminomethane is to form the core of nanoparticles. Without the addition of tris(hydroxymethyl)aminomethane, the agglomeration and precipitation of the grains will result in failure of subsequent encapsulation. Figure 1 ): Using tris(hydroxymethylaminomethane) can prepare nanoparticles with an average particle size of 70.2 nm (Example 3), while the average particle size of the particles prepared without using tris(hydroxymethylaminomethane) is 1169.3 nm (Comparative Example 1), and the average particle size of the particles directly obtained by using the aqueous suspension of magnolol raw material is about 10 μm (Comparative Example 2). The latter two particles are defined as micron particles according to their size and cannot form nanoparticles. According to common sense (Chang TL, Zhan H, Liang D, et al. Nanocrystal technology for drug formulation and delivery [J]. Frontiers of Chemical Science and Engineering, 2015, 9: 1-14), it can be known that drug nanoparticles have better solubility, bioavailability and drug targeting than micron particles.

[0033] Figure 2 It can be seen that a clear core and shell structure can be seen under a transmission electron microscope, and the particle size is also at the nanometer level. The results of Example 1 and Example 2 are consistent with Example 3. Figure 3 It can be seen that the core of the nanoparticles is crystalline, which proves the successful preparation of magnolol nanocrystals. The results of Example 1 and Example 2 are consistent with Example 3. Figure 4 It can be seen that the drug begins to release and shows a sustained release characteristic within 72 hours. In the end, about 90% of the drug is released, that is, the cumulative release rate is about 90%.

[0034] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A high-drug-loaded magnolol targeted nanoparticle, characterized in that: The invention consists of magnolol nanocrystals and a polydopamine layer wrapping the magnolol nanocrystals, and the surface of the polydopamine layer is modified with folic acid.

2. The magnolol targeting nanoparticles according to claim 1, characterized in that: The preparation method comprises the following steps: (1) adding the magnolol solution to the poloxamer F127 solution, stirring and mixing, then adding the tris(hydroxymethyl)aminomethane solution, stirring and mixing, and finally adding dopamine hydrochloride, stirring continuously at 20-25° C. for 8-10 hours, centrifuging, and collecting the precipitate. The obtained precipitate is magnolol crystal nanoparticles coated with polydopamine; (2) adding the magnolol crystalline nanoparticles coated with polydopamine to the folic acid aqueous solution, stirring for 25-40 minutes, centrifuging, and collecting the precipitate to obtain magnolol targeted nanoparticles with surface modified folic acid; (3) The surface-modified magnolia bark phenol targeted nanoparticles with folic acid are pre-frozen and then vacuum freeze-dried to obtain the finished magnolia bark phenol targeted nanoparticles with a high drug loading.

3. The magnolol targeting nanoparticles according to claim 2, characterized in that: The magnolol solution is an ethanol solution of magnolol with a concentration of 8-12 mg / mL.

4. The magnolol targeting nanoparticles according to claim 2, characterized in that: The concentration of Poloxamer F127 solution is 1.8-2.2 mg / mL.

5. The magnolol targeting nanoparticles according to claim 2, characterized in that: The concentration of the tris(hydroxymethyl)aminomethane solution is 35-45 mg / mL.

6. The magnolol targeting nanoparticles according to claim 2, characterized in that: The concentration of folic acid aqueous solution is 1.8-2.2 mg / mL.

7. The magnolol targeting nanoparticles according to claim 6, characterized in that: Tris(hydroxymethyl)aminomethane was added to the folic acid aqueous solution to adjust the pH to 8-9.

8. The magnolol targeting nanoparticles according to claim 2, characterized in that: The prefreezing parameters are -20℃ for 12h.

9. The magnolol targeting nanoparticles according to claim 2, characterized in that: In step (1), the mass ratio of magnolol: poloxamer F127: tris(hydroxymethyl)aminomethane: dopamine hydrochloride is 1:1:3:

1.

10. The magnolol targeting nanoparticles according to claim 2, characterized in that: In step (2), the mass ratio of magnolol crystalline nanoparticles coated with polydopamine: folic acid is 1:1.

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