Coordination-based polysaccharide curcumin nanoparticles and preparation method and application thereof

Abstract

The present application relates to the field of pharmaceutical technology, specifically to polysaccharide curcumin nanoparticles based on coordination, and preparation methods and applications thereof, wherein the preparation method comprises the following steps: mixing FeCl3 solution and citric acid modified fucoidan solution, under light-proof and protective gas atmosphere conditions, dripping curcumin ethanol solution while stirring, ultrasonic assisted reaction, and obtaining suspension; the suspension is redispersed by centrifugation to obtain; wherein, the amount ratio of curcumin, citric acid modified fucoidan, and FeCl3 is 10mg: 0.1mmol: (0.08-0.12)mmol; the preparation method of citric acid modified fucoidan solution is to take citric acid modified fucoidan and add it to water, and add acetic acid-sodium acetate buffer with a pH value of 5-6.5; the preparation method of curcumin ethanol solution is to take curcumin and add it to anhydrous ethanol. The preparation method of the present application is simple, easy to operate, green and environmentally friendly, and the obtained nanoparticles have stable particle size, uniform dispersion, high drug loading rate, and good biosafety, and can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical technology, and in particular to a coordination-based polysaccharide curcumin nanoparticle and a preparation method and application thereof. Background Art

[0002] Curcumin (Cur for short) is a hydrophobic plant polyphenol derived from the tuberous roots of Curcuma aromatica, the rhizomes of Turmeric, the rhizomes of Curcuma zedoaria and the rhizomes of Acorus calamus of the Araceae family. It is an orange-yellow crystalline powder at room temperature and tastes slightly bitter.

[0003] In recent years, cell experiments, animal experiments and clinical studies have shown that curcumin is a powerful molecule that can play a variety of positive pharmacological effects, including anti-inflammatory, antioxidant, and anti-apoptotic effects. In addition, curcumin has an inhibitory effect on a variety of tumors, such as liver cancer, intestinal cancer, breast cancer, gastric cancer, lymphoma and leukemia. Curcumin has broad application prospects in the prevention and treatment of tumors. However, curcumin still has serious limitations in its anti-tumor applications, such as poor selectivity for tumors and low bioavailability.

[0004] Studies have found that the tumor microenvironment has the characteristics of low pH, high reactive oxygen content, and high glutathione (GSH) levels. Based on the differences between the tumor microenvironment and the normal physiological environment, people have designed stimulus-responsive nanodrug delivery systems (NDDSs), which can be stably transported in vivo. After reaching the tumor target tissue or target cells, the carrier structure responds to the stimulation of the tumor microenvironment, effectively controlling the transport site and release rate of the loaded drug, thereby significantly improving the target drug concentration, increasing its anti-tumor activity and reducing its adverse reactions. However, the introduction of chemical bonds that are sensitive to the tumor microenvironment in NDDSs is highly dependent on skilled and time-consuming organic synthesis, and a large amount of toxic organic reagents are inevitably introduced during the synthesis process, which reduces the biosafety of NDDSs to a certain extent. Summary of the invention

[0005] The present application aims to overcome at least one of the defects of the prior art and provide a polysaccharide-based curcumin nanoparticle based on coordination effect and a preparation method and application thereof, wherein the polysaccharide-based curcumin nanoparticle can respond to the stimulation of the tumor microenvironment and release curcumin, thereby improving the tumor targeting ability and bioavailability of curcumin.

[0006] In the first aspect, the present invention provides a method for preparing polysaccharide curcumin nanoparticles based on coordination, which is achieved by the following technical solutions:

[0007] A method for preparing polysaccharide curcumin nanoparticles based on coordination, comprising the following steps:

[0008] S1. The FeCl3 solution and the citric acid-modified fucoidan (FU) solution were mixed, and the ethanol solution of curcumin was added dropwise while stirring under light-proof and protective gas atmosphere, and the reaction was assisted by ultrasound to obtain a suspension;

[0009] S2. The suspension obtained in step S1 is redispersed by centrifugation to obtain polysaccharide-based curcumin nanoparticles based on coordination;

[0010] Wherein, the usage ratio of the curcumin, the citric acid-modified fucoidan, and the FeCl3 is 10 mg: 0.1 mmol: (0.08-0.12) mmol;

[0011] The preparation method of the citric acid-modified fucoidan solution in step S1 is to add citric acid-modified fucoidan into water, and then add acetic acid-sodium acetate buffer with a pH value of 5-6.5;

[0012] The preparation method of the ethanol solution of curcumin in step S1 is to add curcumin into anhydrous ethanol;

[0013] The preparation method of the citric acid-modified fucoidan comprises the following steps:

[0014] Add fucoidan to a citric acid solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC for short), stir, react at a temperature of 70-90°C for 2h-4h, then cool the solution, adjust the pH to 6.5-7.5, dialyze with tap water for 40h-50h, dialyze with distilled water for 20h-30h, and then freeze-dry to obtain citric acid-modified fucoidan;

[0015] The usage ratio of the fucoidan, the citric acid and the EDC is (5-10) mg:(0.04-0.06) mmol:(0.05-0.1) mmol.

[0016] According to a method for preparing polysaccharide curcumin nanoparticles based on coordination according to an embodiment of the present application, there are at least the following beneficial effects:

[0017] The preparation method of the present application adopts a one-pot synthesis method for preparation, and the reactants undergo multi-step reactions in one reactor, avoiding lengthy separation and purification processes in the middle, thereby saving time and resources and improving reaction efficiency. The preparation method is not only simple and easy, but also avoids the use of a large number of toxic organic reagents, and the preparation process is green and environmentally friendly.

[0018] The present application adopts citric acid-modified fucoidan. The citric acid-modified fucoidan has good biocompatibility and will not cause obvious immune response or tissue damage. The solubility of the citric acid-modified fucoidan in water is improved, which is beneficial to its application in pharmaceutical preparations. At the same time, the citric acid-modified fucoidan has better stability and biological activity, such as anti-inflammatory, antioxidant, and anti-tumor effects. It also has the ability to specifically bind to P-selectin on the surface of cancer cells, which can effectively solve the problem of poor solubility of hydrophobic active substances, avoid aggregation in the solution that affects dispersibility, and can accumulate at the tumor site.

[0019] This application utilizes the carbonyl group of curcumin, the sulfate group of citric acid-modified fucoidan and the trace element Fe essential to the human body. 3+ The coordination effect between Fe and Fe was used to drive the self-assembly to prepare polysaccharide curcumin nanoparticles. The good water solubility of citric acid-modified fucoidan was used to adjust the hydrophilic and hydrophobic ratio, which effectively improved the loading amount of curcumin and the stability of polysaccharide curcumin nanoparticles. 3+ The formation of complexes with curcumin and citric acid-modified fucoidan can enhance the structural strength of polysaccharide curcumin nanoparticles. Trivalent iron ions also have antiviral and antitumor biological activities. The prepared polysaccharide curcumin nanoparticles based on coordination are stable under physiological conditions and have GSH responsiveness. The competitive coordination induced by high GSH content in the tumor microenvironment can trigger the effective release of curcumin therein, thereby improving the tumor targeting ability and bioavailability of curcumin. The prepared polysaccharide curcumin nanoparticles based on coordination have stable particle size, uniform dispersion, high drug loading rate, and good biosafety. They can be used to prepare anti-tumor drugs. Coordination-driven self-assembly is easy to combine and integrate and biodegrade due to its rich structure and properties, and has good application prospects in drug delivery.

[0020] In this application, the acetic acid-sodium acetate buffer with a pH value of 5-6.5 is selected to keep the pH value of the reaction solution relatively stable and inhibit Fe 3+ The hydrolysis of curcumin-citric acid modified fucoidan-Fe 3+ The stability of the complex reduces the decomposition of curcumin and promotes the reaction. If the pH value of the reaction system is too high, curcumin is easily decomposed. 3+ It is easy to hydrolyze, which affects the formation of the complex.

[0021] The unsaturation degree of curcumin in the present application is high. By adopting light-proof conditions and using a protective gas atmosphere to reduce contact with air, the stability of curcumin can be improved, the decomposition of curcumin can be reduced, the active ingredients can be retained, and the drug loading rate of curcumin can be improved.

[0022] The curcumin in this application is added by dripping, which can keep the citric acid modified fucoidan and Fe 3+ The dosage is sufficient, the reaction system is more stable, curcumin is not easy to decompose, which is conducive to the curcumin-citric acid modified fucoidan-Fe 3+ The formation of the complex allows the curcumin to react more fully, thereby increasing the drug loading rate of curcumin. Adding the mixture dropwise while stirring can allow the reactants to mix quickly and distribute evenly, thereby accelerating the reaction rate and increasing the utilization rate of the reactants.

[0023] In this application, ultrasound-assisted reaction can promote the reaction, accelerate the combination of reactants, and increase the reaction rate. After the ultrasound is transmitted to the liquid, compression effect and shear effect will be produced. The citric acid-modified fucoidan molecules and curcumin molecules in the liquid will be affected by the pressure change, so that the shape changes, the molecular spacing will also change, the chemical bonds in the molecules will break, and chemical reactions will occur. Ultrasound will also produce tiny bubbles, and the rupture of the bubbles will produce high temperature, high pressure and a large amount of energy, thereby promoting the occurrence of chemical reactions. In addition, ultrasound can also accelerate the impact and interference between reactants, thereby promoting contact and reaction between reactants, accelerating the mixing between reactants, and greatly accelerating the reaction rate.

[0024] According to some embodiments of the present application, the pH value of the acetic acid-sodium acetate buffer is 5.6. The acetic acid-sodium acetate buffer in the present application can maintain the stability of the reaction solution and keep the pH value relatively stable, which is conducive to the preparation of structurally stable polysaccharide curcumin nanoparticles.

[0025] According to some embodiments of the present application, the protective gas described in step S1 is selected from one of nitrogen, helium, neon, and argon. For example, the protective gas described in step S1 is nitrogen. Nitrogen is stable in nature, widely available, and has low production costs. Nitrogen as a protective gas can form an oxygen-free environment to prevent curcumin from being oxidized and destroyed, and reduce the occurrence of side reactions. Rare gases such as helium, neon, and argon are stable in nature and are not easy to participate in chemical reactions, which can reduce the generation of side reactions.

[0026] According to some embodiments of the present application, the dripping method in step S1 is uniform dripping. Maintaining uniform dripping can control the dripping speed, thereby controlling the dosage, avoiding adverse reactions, and ensuring the stability of the reaction from beginning to end. If the dripping speed is unstable, it is easy to cause changes in the reaction conditions, affecting the quality and yield of the prepared polysaccharide curcumin nanoparticles.

[0027] According to some embodiments of the present application, the stirring method in step S1 is magnetic stirring.

[0028] Furthermore, the speed of the magnetic stirring is 500-1000 rpm, for example, the speed of the magnetic stirring is 1000 rpm.

[0029] According to some embodiments of the present application, the stirring time in step S1 is 1-10 min, for example, the stirring time in step S1 is 5 min.

[0030] According to some embodiments of the present application, the ultrasonic power of the ultrasonic-assisted reaction in step S1 is 10-30W, for example, the ultrasonic power of the ultrasonic-assisted reaction in step S1 is 30W.

[0031] According to some embodiments of the present application, the ultrasonic time of the ultrasonic-assisted reaction in step S1 is 1-5 min. For example, the ultrasonic time of the ultrasonic-assisted reaction in step S1 is 5 min.

[0032] According to some embodiments of the present application, the centrifugal rotation speed in step S2 is 5000-14000 rpm, for example, the centrifugal rotation speed in step S2 is 14000 rpm.

[0033] According to some embodiments of the present application, the centrifugation time in step S2 is 5-15 min, for example, the centrifugation time in step S2 is 15 min.

[0034] According to some embodiments of the present application, the centrifugation temperature in step S2 is 0-20°C, for example, the centrifugation temperature in step S2 is 4°C.

[0035] According to some embodiments of the present application, the usage ratio of the curcumin, the citric acid-modified fucoidan, and the FeCl3 is 10 mg:0.1 mmol:0.1 mmol.

[0036] According to some embodiments of the present application, the usage ratio of the citric acid-modified fucoidan to the acetic acid-sodium acetate buffer is (0.04-0.06) mmol:10 mL, for example, the usage ratio of the citric acid-modified fucoidan to the acetic acid-sodium acetate buffer is 0.05 mmol:10 mL.

[0037] According to some embodiments of the present application, the freeze-drying temperature is -70°C.

[0038] According to some embodiments of the present application, the freeze-drying time is 15h-30h.

[0039] In a second aspect, the embodiments of the present application provide polysaccharide curcumin nanoparticles based on coordination prepared by the preparation method of the above-mentioned polysaccharide curcumin nanoparticles based on coordination.

[0040] The polysaccharide-based curcumin nanoparticles prepared in the present application based on coordination have stable particle size, uniform dispersion, high drug loading rate and good biosafety.

[0041] According to some embodiments of the present application, the particle size of the coordination-based polysaccharide curcumin nanoparticles is 120-150 nm.

[0042] According to some embodiments of the present application, the PDI of the particle size of the polysaccharide curcumin nanoparticles based on coordination is no more than 0.2. When preparing drugs, if the drug particle size difference is too large, it will affect its bioavailability and efficacy. The PDI of the particle size of the polysaccharide curcumin nanoparticles based on coordination is no more than 0.2, which can improve its bioavailability and efficacy.

[0043] In a third aspect, the present application provides an application of the above-mentioned coordination-based polysaccharide curcumin nanoparticles in the preparation of anti-tumor drugs. The application of the coordination-based polysaccharide curcumin nanoparticles in the preparation of anti-tumor drugs can increase the target drug concentration and increase its anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a transmission electron microscope (TEM) image of the coordination-based polysaccharide curcumin nanoparticles prepared in Example 1 of the present application.

[0045] Figure 2 It is a dynamic light scattering (DLS) graph of the coordination-based polysaccharide curcumin nanoparticles prepared in Example 1 and Comparative Examples 1-3 of the present application.

[0046] Figure 3 It is a dynamic light scattering (DLS) graph of the coordination-based polysaccharide curcumin nanoparticles prepared in Examples 1-3 of the present application.

[0047] Figure 4 This is an in vitro release curve of the coordination-based polysaccharide curcumin nanoparticles prepared in Example 1 of the present application.

[0048] Figure 5 This is a graph showing the toxicity of the coordination-based polysaccharide curcumin nanoparticles prepared in Example 1 of the present application and the agents of Comparative Examples 4-5 to mouse breast cancer cells 4T1. DETAILED DESCRIPTION

[0049] To make the purpose, technical solution and advantages of the present application clearer, the following will be further described in detail in conjunction with specific embodiments. The embodiments described here are only part of the embodiments of the present application and cannot be understood as limiting the scope of protection of the present application.

[0050] Preparation Example 1 of Citric Acid Modified Fucoidan

[0051] The preparation method of citric acid modified fucoidan includes the following steps: adding 8 mg of fucoidan to 5 mL of 10 mM citric acid solution, adding 0.08 mmol of EDC, stirring, reacting at 80°C for 3 hours, then cooling the solution, adjusting the pH to 7.0, dialyzing with tap water for 45 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, dialyzing with distilled water for 25 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, and freeze-drying for 20 hours at -70°C using a vacuum freeze dryer to obtain citric acid modified fucoidan.

[0052] Preparation Example 2 of Citric Acid Modified Fucoidan

[0053] The preparation method of citric acid modified fucoidan includes the following steps: adding 5 mg of fucoidan to 5 mL of 12 mM citric acid solution, adding 0.05 mmol of EDC, stirring, reacting at 90°C for 2 hours, then cooling the solution, adjusting the pH to 7.5, dialyzing with tap water for 40 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, dialyzing with distilled water for 30 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, and freeze-drying for 15 hours at -70°C using a vacuum freeze dryer to obtain citric acid modified fucoidan.

[0054] Preparation Example 3 of Citric Acid Modified Fucoidan

[0055] The preparation method of citric acid modified fucoidan includes the following steps: adding 10 mg of fucoidan to 5 mL of 8 mM citric acid solution, adding 0.1 mmol of EDC, stirring, reacting at a temperature of 70°C for 4 hours, then cooling the solution, adjusting the pH to 6.5, dialyzing with tap water for 50 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, dialyzing with distilled water for 20 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, and then freeze-drying for 30 hours at a temperature of -70°C using a vacuum freeze dryer to obtain citric acid modified fucoidan.

[0056] Comparative Example 1 of Citric Acid Modified Fucoidan

[0057] The preparation method of citric acid modified fucoidan includes the following steps: adding 8 mg of fucoidan to 5 mL of 10 mM citric acid solution, adding 0.01 mmol of EDC, stirring, reacting at 80°C for 3 hours, then cooling the solution, adjusting the pH to 7.0, dialyzing with tap water for 45 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, dialyzing with distilled water for 25 hours using a dialysis bag with a molecular weight cutoff of 10,000 Daltons, and freeze-drying for 20 hours at -70°C using a vacuum freeze dryer to obtain citric acid modified fucoidan.

[0058] The citric acid-modified fucoidan prepared in the citric acid-modified fucoidan preparation examples 1-3 and the citric acid-modified fucoidan comparative example 1 and the unmodified fucoidan were taken, and their solubilities in 100 mL of water were measured respectively. The measured solubility results are shown in Table 1.

[0059] Table 1

[0060]

[0061] It can be seen from Table 1 that when the amount of catalyst EDC added in the preparation of citric acid-modified fucoidan comparative example 1 is too small, the solubility of the prepared citric acid-modified fucoidan is significantly lower than the solubility of the citric acid-modified fucoidan prepared in citric acid-modified fucoidan preparation example 1, which indicates that adding an appropriate amount of EDC during the preparation of citric acid-modified fucoidan can control the reaction process, promote the reaction, introduce carboxyl groups in citric acid, and achieve the modification of the solubility and stability of fucoidan.

[0062] Example 1

[0063] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0064] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 2 mM;

[0065] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0066] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0067] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0068] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 15 min at 4°C to obtain polysaccharide curcumin nanoparticles based on coordination, named FFC-NPs.

[0069] Example 2

[0070] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0071] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 2 mM;

[0072] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 12mM;

[0073] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0074] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0075] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 15 min at 4° C. to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0076] Example 3

[0077] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0078] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 2 mM;

[0079] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 8mM;

[0080] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0081] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0082] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 15 min at 4° C. to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0083] Example 4

[0084] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0085] Preparation of citric acid-modified fucoidan solution: Take 0.04 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 2 mM;

[0086] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0087] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0088] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0089] S2. The suspension obtained in step S1 was centrifuged at 10,000 rpm for 15 min at 4° C. to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0090] Example 5

[0091] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0092] Preparation of citric acid-modified fucoidan solution: Take 0.06 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 2 mM;

[0093] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0094] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0095] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0096] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 5 min at 4°C to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0097] Comparative Example 1

[0098] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0099] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 0.2 mM;

[0100] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0101] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0102] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0103] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 15 min at 4° C. to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0104] Comparative Example 2

[0105] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0106] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 0.5 mM;

[0107] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0108] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0109] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0110] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 15 min at 4° C. to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0111] Comparative Example 3

[0112] The preparation of polysaccharide curcumin nanoparticles based on coordination comprises the following steps:

[0113] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 1 mM;

[0114] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0115] Preparation of curcumin ethanol solution: Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare a curcumin ethanol solution with a concentration of 10 mg / mL;

[0116] S1. Mix 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution, and under the condition of light protection and nitrogen protection, magnetically stir at a speed of 1000 rpm for 5 min, while uniformly adding 1 mL of curcumin ethanol solution, and perform ultrasound-assisted reaction at an ultrasonic power of 30 W for 5 min to obtain a suspension;

[0117] S2. The suspension obtained in step S1 was centrifuged at 14000 rpm for 15 min at 4° C. to obtain polysaccharide-based curcumin nanoparticles based on coordination.

[0118] Comparative Example 4

[0119] FU-Fe 3+ The preparation comprises the following steps:

[0120] Preparation of citric acid-modified fucoidan solution: Take 0.05 mmol of citric acid-modified fucoidan prepared in Preparation Example 1 and add it to 10 mL of acetic acid-sodium acetate buffer with a pH value of 5.6, add appropriate amount of deionized water, stir, and prepare a citric acid-modified fucoidan solution with a concentration of 2 mM;

[0121] Preparation of FeCl3 solution: Take 1mmolFeCl3, add appropriate amount of deionized water, stir, and prepare a FeCl3 solution with a concentration of 10mM;

[0122] 10 mL of FeCl3 solution and 50 mL of citric acid-modified fucoidan solution were mixed and stirred magnetically at 1000 rpm for 5 min under light-proof and nitrogen protection conditions to obtain FU-Fe 3+ .

[0123] Comparative Example 5

[0124] Take 10 mg of curcumin, add an appropriate amount of anhydrous ethanol, stir, and prepare an ethanol solution of curcumin with a concentration of 10 mg / mL.

[0125] Experimental example

[0126] The polysaccharide curcumin nanoparticles prepared in Example 1 were observed using a transmission electron microscope. Figure 1 As shown, the particle size of polysaccharide-based curcumin nanoparticles based on coordination is stable and the dispersion is uniform.

[0127] The polysaccharide curcumin nanoparticles based on coordination prepared in Example 1 and Comparative Examples 1-3 were tested for their hydrated particle sizes using a Zeta potential instrument. The results are as follows: Figure 2 As shown, the average diameter of FFC-NPs in Example 1 is 140nm, and the PDI is 0.15, the average diameter of the polysaccharide curcumin nanoparticles based on coordination in Comparative Example 1 is 460nm, and the PDI is 0.4, the average diameter of the polysaccharide curcumin nanoparticles based on coordination in Comparative Example 2 is 350nm, and the PDI is 0.37, and the average diameter of the polysaccharide curcumin nanoparticles based on coordination in Comparative Example 3 is 250nm, and the PDI is 0.22. Compared with Example 1, Comparative Examples 1-3 differ only in the concentration of the citric acid-modified fucoidan solution, indicating that the concentration of the citric acid-modified fucoidan solution in the present application is very important for the preparation of polysaccharide curcumin nanoparticles based on coordination. The polysaccharide curcumin nanoparticles based on coordination prepared by using the citric acid-modified fucoidan solution concentration in Example 1 of the present application not only have a smaller particle size, but also have a more uniform distribution, which can improve its bioavailability and efficacy.

[0128] The polysaccharide curcumin nanoparticles prepared in Example 1-3 based on coordination were tested for their hydrated particle size using a Zeta potential instrument. The results are as follows: Figure 3 As shown, the average diameter of the FFC-NPs in Example 1 is 140 nm, and the PDI is 0.15, the average diameter of the polysaccharide curcumin nanoparticles based on coordination in Example 2 is 150 nm, and the PDI is 0.11, and the average diameter of the polysaccharide curcumin nanoparticles based on coordination in Example 3 is 125 nm, and the PDI is 0.19, indicating that the polysaccharide curcumin nanoparticles based on coordination prepared in Examples 1-3 of the present application not only have small particles, but also have PDIs below 0.2, uniform particle size distribution, and better bioavailability and efficacy.

[0129] The drug loading capacity of the polysaccharide-based curcumin nanoparticles prepared in Examples 1-5 and Comparative Examples 1-3 based on coordination was tested, and the test results are shown in Table 2.

[0130] Table 2

[0131]

[0132] As can be seen from Table 2, the drug loading of the polysaccharide curcumin nanoparticles based on coordination prepared in Examples 1-5 of the present application is better than that in Comparative Examples 1-3, indicating that the selection of the amount of raw materials used in the preparation of the present application is very important, and the combination of the appropriate amount of raw materials and a specific preparation method can improve the drug loading of the polysaccharide curcumin nanoparticles based on coordination.

[0133] The in vitro release curve of the polysaccharide curcumin nanoparticles based on coordination prepared in Example 1 is as follows: Figure 4 As shown in the figure, FFC-NPs exhibited different response behaviors in GSH with a concentration of 10 mM and PBS buffer with a pH of 7.4, respectively; at 24 h, the curcumin release of FFC-NPs in PBS buffer with a pH of 7.4 was only 29.7%, while it reached 77.0% in GSH with a concentration of 10 mM, indicating that the competitive coordination effect induced by the high content of GSH in the tumor microenvironment can trigger the effective release of curcumin in FFC-NPs.

[0134] The polysaccharide curcumin nanoparticles based on coordination prepared in Example 1 and the agents of Comparative Examples 4-5 were used to determine their cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The specific method is as follows: mouse breast cancer cells 4T1 were inoculated in a 96-well plate (1×10 4 / well) and incubated overnight, then added sample solutions of different concentrations of Example 1 and Comparative Examples 4-5 and co-cultured with cells for 24 hours, washed with PBS, added 100 μL of 0.5 mg / mL MTT solution to each well for 4 hours, removed the MTT solution, added 200 μL of dimethyl sulfoxide to each well to dissolve the purple crystals at the bottom of the well plate, and after shaking, read the absorbance of each well at a wavelength of 490 nm with an ELISA reader; cells not treated with drugs were used as the control group, and their theoretical survival rate was 100%; with reference to the control group, the cell survival rates at different time points after treatment with different concentrations of drugs were calculated. The results are shown in Figure 5 As shown, FU-Fe 3+ It has no obvious toxicity to mouse breast cancer cells 4T1 and has good in vitro biocompatibility; FFC-NPs and Cur have dose-dependent antitumor activity, and the half-inhibitory concentration (IC50) for 4T1 cells is 12.06μg / mL and 15.31μg / mL, respectively, indicating that FFC-NPs has higher tumor cell toxicity than Cur.

[0135] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions or variations may be made to the embodiments without departing from the principles and purpose of the present application, and that the technical solutions after these changes, modifications, substitutions or variations will fall within the scope of protection of the present application.

Claims

1. A method for preparing polysaccharide curcumin nanoparticles based on coordination, characterized in that: The following steps are involved: S1. The FeCl3 solution and the citric acid-modified fucoidan solution were mixed, and the ethanol solution of curcumin was added dropwise while stirring under light-proof and protective gas atmosphere, and the reaction was assisted by ultrasound to obtain a suspension; S2. The suspension obtained in step S1 is redispersed by centrifugation to obtain polysaccharide curcumin nanoparticles based on coordination; Wherein, the usage ratio of the curcumin, the citric acid-modified fucoidan, and the FeCl3 is 10 mg: 0.1 mmol: (0.08-0.12) mmol; The preparation method of the citric acid-modified fucoidan solution in step S1 is to add citric acid-modified fucoidan into water, and then add acetic acid-sodium acetate buffer with a pH value of 5-6.5; The preparation method of the ethanol solution of curcumin in step S1 is to add curcumin into anhydrous ethanol; The preparation method of the citric acid-modified fucoidan comprises the following steps: Add fucoidan to a citric acid solution, add EDC, stir, react at a temperature of 70-90°C for 2h-4h, then cool the solution, adjust the pH to 6.5-7.5, dialyze with tap water for 40h-50h, dialyze with distilled water for 20h-30h, and then freeze-dry to obtain citric acid-modified fucoidan; The usage ratio of the fucoidan, the citric acid and the EDC is (5-10) mg:(0.04-0.06) mmol:(0.05-0.1) mmol.

2. The method for preparing polysaccharide curcumin nanoparticles based on coordination according to claim 1, characterized in that: The pH value of the acetic acid-sodium acetate buffer is 5.

6.

3. The method for preparing polysaccharide curcumin nanoparticles based on coordination according to claim 1, characterized in that: The freeze-drying temperature is -70°C, and / or the freeze-drying time is 15h-30h.

4. The method for preparing polysaccharide curcumin nanoparticles based on coordination according to claim 1, characterized in that: The dosage ratio of the citric acid-modified fucoidan to acetic acid-sodium acetate buffer is (0.04-0.06) mmol:10 mL.

5. The method for preparing polysaccharide curcumin nanoparticles based on coordination according to claim 1, characterized in that: The stirring speed in step S1 is 500-1000 rpm, and / or the stirring time in step S1 is 1-10 min.

6. The method for preparing polysaccharide curcumin nanoparticles based on coordination according to claim 1, characterized in that: The ultrasonic power of the ultrasonic-assisted reaction in step S1 is 10-30 W, and / or the ultrasonic time of the ultrasonic-assisted reaction in step S1 is 1-5 min.

7. The method for preparing polysaccharide curcumin nanoparticles based on coordination according to claim 1, characterized in that: The centrifugal speed in step S2 is 5000-14000 rpm, and / or the centrifugal time in step S2 is 5-15 min.

8. Coordination-based polysaccharide curcumin nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. The polysaccharide-based curcumin nanoparticles based on coordination according to claim 8, characterized in that: The particle size of the polysaccharide curcumin nanoparticles based on coordination is 120-150 nm, and / or the PDI of the particle size of the polysaccharide curcumin nanoparticles based on coordination is not more than 0.

2.

10. Use of the coordination-based polysaccharide curcumin nanoparticles according to claim 8 or 9 in the preparation of anti-tumor drugs.

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