Zein-soluble protein-sodium caseinate-6, 7-dihydroxy coumarin nanoparticle delivery system as well as preparation method and application thereof

Abstract

The invention discloses a zein sodium caseinate 6, 7-dihydroxy coumarin nanoparticle delivery system as well as a preparation method and application thereof, and belongs to the technical field of drug delivery systems. According to the nanoparticle delivery system, zein wraps 6, 7-dihydroxycoumarin as a core, sodium caseinate is adsorbed to an outer layer as a shell, and a core-shell structure is formed. The preparation method comprises the following steps: dissolving zein and 6, 7-dihydroxy coumarin in an ethanol water solution to obtain a mixed solution; dropwise adding the mixed solution into a sodium caseinate aqueous solution, and stirring to form a dispersion liquid; and removing the organic solvent to obtain the product. The nanoparticles are uniform in particle size and good in stability, the water solubility, photo-thermal stability and chemical stability of 6, 7-dihydroxy coumarin can be remarkably improved, sustained and controlled release in the gastrointestinal tract environment is achieved, and the oral bioavailability of 6, 7-dihydroxy coumarin is improved. The method is simple in process, mild in condition and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of drug delivery system technology, specifically to a composite nanoparticle based on zein and sodium caseinate for encapsulating and delivering the hydrophobic active molecule 6,7-dihydroxycoumarin, and its preparation method and application. Background Technology

[0002] With advancements in pharmaceutical technology, novel drug delivery systems, represented by targeted delivery, intelligent controlled release, and biodegradable carriers, are gradually replacing traditional oral and injectable drug delivery methods, becoming the mainstream direction of modern drug development, thanks to their precision, long-lasting effects, and good biocompatibility. Among these, nanoparticle delivery systems systematically address the bottlenecks of traditional drug delivery through unique physicochemical properties and functional design: utilizing their hydrophobic core to solubilize poorly soluble drugs, achieving passive targeted enrichment in tumor tissue through the EPR effect, and relying on biodegradable materials to achieve long-lasting sustained release at the lesion site. This strategy, integrating "solubilization, targeting, and sustained release," provides an ideal solution for the clinical translation of highly toxic or unstable drugs.

[0003] Zein, a major storage protein in corn, is a hydrophobic plant protein with unique solubility properties (insoluble in water but soluble in alcohol). Its excellent biocompatibility has made it highly sought after in the biomedical field. Zein's unique amphiphilic structure allows it to efficiently encapsulate various active substances and facilitates site-specific delivery through surface modification. However, single Zein nanoparticles exhibit poor stability in aqueous solutions. Sodium caseinate (NaCas, Nac), an amphiphilic protein with properties similar to macromolecular surfactants, has been proven to be an ideal component for stabilizing Zein nanoparticle systems.

[0004] 6,7-Dihydroxycoumarin (Esculetin) is a natural coumarin derivative, primarily extracted from the bark of *Fraxinus chinensis*, a plant in the Oleaceae family. The presence of an ortho-dihydroxyl group in its structure endows it with extremely strong biological activity. It possesses significant antioxidant activity, potent anti-inflammatory effects, antitumor activity, hepatoprotective effects, and hypoglycemic and hypolipidemic activities. Despite its potent pharmacological activity, 6,7-dihydroxycoumarin's unique physicochemical properties severely limit its clinical application and oral efficacy. It exhibits poor water solubility and chemical stability. While the ortho-dihydroxyl group in its structure provides antioxidant activity, it also makes the drug itself highly susceptible to oxidation. Under light and air conditions, it easily oxidizes, discolors, and loses its efficacy. The lactone ring in its skeleton may undergo ring-opening hydrolysis in alkaline or strongly acidic environments, leading to structural destruction. Therefore, 6,7-dihydroxycoumarin is a hydrophobic small-molecule polyphenol with significant anti-inflammatory and antioxidant activities, but its extremely low water solubility severely limits its bioavailability. Therefore, developing a novel delivery system that can effectively improve the solubility, stability, and bioavailability of 6,7-dihydroxycoumarin is of great practical significance. Summary of the Invention

[0005] To address the aforementioned issues, this invention aims to propose a zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system, its preparation method, and its applications. It constructs a composite ternary nanoparticle delivery system based on Zein and NaCas, utilizing the stabilizing effect of NaCas in conjunction with the carrier function of Zein. By optimizing the component ratio, it effectively solves the problems of low solubility, poor stability, poor in vivo absorption, and low bioavailability of 6,7-dihydroxycoumarin, while simultaneously achieving targeted drug delivery and long-lasting sustained release.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention proposes a method for preparing a zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system, comprising the following process steps: S1: Dissolve zein in an aqueous ethanol solution, add 6,7-dihydroxycoumarin, stir thoroughly to dissolve, and obtain an ethanol solution of zein-6,7-dihydroxycoumarin. S2: Slowly add the ethanol solution of zein-6,7-dihydroxycoumarin obtained in step S1 to the sodium caseinate aqueous solution and stir continuously to obtain a dispersion; then, remove the organic solvent in the dispersion by vacuum distillation on a rotary evaporator to obtain the nanoparticle delivery system.

[0007] Furthermore, in step S1, the mass ratio of 6,7-dihydroxycoumarin to zein is 1:(1-3).

[0008] Furthermore, the volume fraction of ethanol in the ethanol-water solution is 70-90%.

[0009] Furthermore, in step S2, the mass ratio of sodium caseinate to zein is (0.5-2):1, and the solvent is water.

[0010] Furthermore, in step S2, the stirring speed is 500-1000 rpm and the stirring time is 20-40 minutes.

[0011] Secondly, this invention proposes a zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system prepared by the above-mentioned method. The nanoparticle delivery system uses zein to encapsulate 6,7-dihydroxycoumarin as the core and sodium caseinate as the shell, constructing a core-shell structure for delivering 6,7-dihydroxycoumarin composite nanoparticles (ZEN nanoparticles). The composite nanoparticles have a particle size of 150-250 nm, a polydispersity index (PDI) of less than 0.3, and a zeta potential between -40 mV and -50 mV.

[0012] This invention also proposes the application of the above-mentioned zein-casein sodium-6,7-dihydroxycoumarin nanoparticle delivery system in the treatment of anti-inflammatory and antioxidant diseases.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: (1) Significantly improves drug solubility and stability. Addressing the shortcomings of 6,7-dihydroxycoumarin (Esculetin)—its extremely poor water solubility, sensitivity to light and heat, and easy oxidation and discoloration due to its phenolic hydroxyl groups—this invention utilizes the hydrophobic cavities of zein to efficiently encapsulate it, and introduces sodium caseinate (NaCas) to form a dense protective layer. This dual barrier not only significantly improves the saturated solubility of the drug but also effectively isolates it from oxygen and light, greatly enhancing the chemical and storage stability of the drug.

[0014] (2) The composite nanocarrier based on zein and sodium caseinate (NaCas) of the present invention is used for the efficient delivery of the hydrophobic active molecule 6,7-dihydroxycoumarin (Esculetin). This delivery system is constructed through self-assembly technology, which can effectively overcome the defect of poor water solubility of drugs, enhance their photothermal stability, and achieve controlled release and improved bioavailability of drugs in the gastrointestinal tract. Attached Figure Description

[0015] Figure 1 The particle size and PDI of nanoparticles at different Zein-Esc mass ratios in Example 1; Figure 2The zeta potential of nanoparticles with different Zein-Esc mass ratios in Example 1; Figure 3 The particle size and PDI of nanoparticles at different Zein-Esc-Nac mass ratios in Example 1; Figure 4 The zeta potential of nanoparticles with different Zein-Esc-Nac mass ratios in Example 1; Figure 5 The particle size and PDI of ZEN nanoparticles at different pH values ​​in Example 2; Figure 6 The zeta potential of ZEN nanoparticles at different pH values ​​in Example 2; Figure 7 The particle size and PDI of ZEN nanoparticles under different NaCl concentrations in Example 2; Figure 8 The zeta potential of ZEN nanoparticles at different NaCl concentrations in Example 2; Figure 9 The particle size and PDI of ZEN nanoparticles after heating at 80°C in Example 2; Figure 10 The zeta potential of ZEN nanoparticles after heating to 80°C in Example 2; Figure 11 The release rate of ZEN at different in vitro temperatures in Example 3; Figure 12 The release rate of ZEN in the in vitro gastrointestinal digestion simulation in Example 4 is shown. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0017] Example 1: Preparation of a ternary composite nanoparticle delivery system of zein-sodium caseinate-6,7-dihydroxycoumarin (1) Accurately weigh 2 g of Zein using an analytical balance and place it in a 100 mL Erlenmeyer flask containing 80% anhydrous ethanol. Stir to dissolve. Then weigh Esc and add it to the Erlenmeyer flask to make the mass ratio of Zein to Esc 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, and 1:3. Stir at 750 rpm for 30 min to obtain an ethanol-water solution of Zein-Esc. Use a Malvern laser particle size analyzer to measure the particle size, PDI (A), and zeta potential (B) of the nanoparticles to determine the optimal ratio.

[0018] (2) Accurately weigh sodium caseinate (NaC) to achieve a Zein:NaC mass ratio of 2:1, 1.5:1, 1:1, 1.5:1, or 1:2. Place it in the binary composite system with the optimal ratio determined in (1) and stir at 750 rpm for 30 min. Remove ethanol by rotary evaporation in a 40℃ water bath, and centrifuge at 3000 rpm for 10 min to remove larger particles. Similarly, use a Malvern laser particle size analyzer to measure the particle size, PDI (A), and zeta potential (B) of the nanoparticles to determine their optimal ratio.

[0019] Take 1 mL of the composite nanoparticle solution and add it to 80% anhydrous ethanol. Place the solution in an ultrasonic cleaner and sonicate for 30 minutes to disrupt the protein particle structure and release Esc. Measure the absorbance at 345 nm using a UV spectrophotometer and calculate the Esc content based on the standard curve. The encapsulation efficiency (EE) is calculated using the following formula: EE(%) = (Actual drug loading) / (Drug dosage) × 100%.

[0020] Figure 1 and Figure 2 The figures for particle size, PDI (A), and Zeta potential (B) of nanoparticles with different Zein-Esc mass ratios in Example 1 mainly represent the optimization of the Zein-Esc binary system ratio. This demonstrates the effect of different Zein to Esc mass ratios (x-axis set to 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3) on the physicochemical properties of nanoparticles when preparing binary composite nanoparticles of zein and 6,7-dihydroxycoumarin (Esc). Figure 1 This shows the changes in particle size and polydispersity index (PDI) with the mixing ratio. Figure 2 This shows the change in zeta potential with varying sizing ratios.

[0021] Particle size and PDI trend: As the proportion of drug (Esc) increases (i.e., the Zein:Esc ratio decreases), the particle size of the nanoparticles shows a trend of first stabilizing and then increasing. This may be because when the drug is in excess, the hydrophobic cavities of Zein cannot completely encapsulate the drug, leading to the aggregation of unencapsulated drug on the particle surface or the formation of large precipitates, thus causing a sharp increase in particle size and a significant increase in PDI, indicating that the system becomes inhomogeneous. Zeta potential trend: The potential value reflects the stability of the system; as the proportion of drug (Esc) increases, the potential value shows a trend of first increasing and then decreasing. Determination of the optimal ratio: Through... Figures 1-2 The screening criteria (smaller particle size, PDI < 0.3, and larger absolute value of zeta potential indicate more uniform dispersion of the composite system) showed that when Zein:Esc = 1.5:1, the particle size, PDI, and zeta potential of the system reached relative optimality.

[0022] Figure 3 and Figure 4 The optimization of the Zein-Esc-NaCas ternary system demonstrates the effect of different Zein to NaC mass ratios (x-axis set as 2:1, 1.5:1, 1:1, 1:1.5, 1:2) on the properties of nanoparticles when sodium caseinate (NaCas, NaC) is introduced as a stabilizer based on the determined optimal Zein-Esc ratio (i.e., 1.5:1). Figure 3 The figure shows the changes in particle size and PDI with the amount of NaCas added. Figure 4 This shows the change in Zeta potential with the amount of NaCas added.

[0023] Particle size and PDI trends: As the NaCas ratio increases, the particle size first decreases and then tends to plateau. PDI first decreases and then increases. Zeta potential trend: The absolute value of the Zeta potential increases and then decreases. This may be because when NaCas is insufficient (Zein:NaC ratio is too high), NaCas is insufficient to completely cover the Zein particle surface, and hydrophobic interactions between particles dominate, easily leading to aggregation, resulting in larger particle size and poor stability. When NaCas is excessive (Zein:NaC ratio is too low): Excessive NaCas may form empty micelles in the solution or cause bridging flocculation. Although the potential may be higher, it will result in material waste and may affect the calculation of encapsulation efficiency. Determination of the optimal ratio: Through... Figures 3-4 Through screening, it can be observed that when Zein : NaC = 1 : 1, the particle size, PDI, and Zeta potential of the system reach a relative optimum.

[0024] The optimal mass ratio of zein-sodium caseinate-6,7-dihydroxycoumarin ternary composite nanoparticles (ZEN nanoparticles) was finally determined to be m. Zein : mEsc : m NaC With a ratio of 3:2:3, the packaging efficiency is 75.34%.

[0025] Example 2: Stability Study of ZEN Nanoparticles (1) pH stability test Using a precision pH meter, 1.0 mol / L HCl or NaOH solution was slowly added dropwise to the composite nanoparticle solution, adjusting the pH to 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, and 9.0, with three parallel samples at each pH point. After standing for 30 minutes, the particle size, PDI, and Zeta potential of the nanoparticles were measured.

[0026] like Figure 5 and Figure 6 As shown, under acidic conditions of pH 3-4, the Zeta potential is positive (+25 mV to +10 mV).

[0027] Near pH 5, the Zeta potential is close to 0 mV (around -5 mV), indicating that the isoelectric point (pI) of this complex is located near pH 5. Since both zein and sodium caseinate (NaC) are proteins, their surface charge is pH-regulated. Near pH 5, the net surface charge of the complex is close to zero, electrostatic repulsion disappears, leading to severe isoelectric precipitation between particles, macroscopically manifested as a dramatic increase in particle size.

[0028] When pH > 6 (above pI), the carboxyl groups on the protein surface deprotonate, resulting in a high density of negative charges on the particle surface (Zeta potential < -35 mV). The strong electrostatic repulsion overcomes van der Waals forces, causing the particles to redisperse into uniform nanoparticles. Experiments confirm that pH 6.0 is the optimal environment for maintaining the colloidal stability of this system, where electrostatic stabilization is strongest and avoids excessive protein stretching or hydrolysis that might occur in strongly alkaline environments.

[0029] (2) Ion concentration stability test Prepare NaCl solutions with concentrations of 0, 25, 50, 75, and 100 mmol / L. Mix 5 mL of the composite nanoparticle solution with an equal volume of NaCl solution, and set up three parallel samples for each concentration. Vortex for 30 seconds to ensure uniform dispersion, and after standing for 30 minutes, measure the particle size, PDI, and Zeta potential.

[0030] like Figure 7 and Figure 8 As shown, NaCl, as a strong electrolyte, introduces a large number of ions (Na+, Na ...+ These ions compress the electrical double layer on the surface of nanoparticles, significantly reducing the Zeta potential due to the electrostatic shielding effect. When the absolute value of the Zeta potential decreases to a critical value due to salt shielding, the electrostatic repulsion between particles is insufficient to resist van der Waals forces, leading to aggregation or flocculation of the system. This complex maintains excellent dispersion stability and a high Zeta potential in neutral and weakly alkaline environments (pH 6.0–9.0), especially at pH 6.0 where the particle size is smallest and the distribution is narrowest. This characteristic makes it particularly suitable for intestinal targeted release (intestinal pH approximately 6–7) or for dispensing neutral food systems. The system exhibits a certain tolerance to ionic strength, maintaining nanoscale sizes (<250 nm) without aggregation in environments with NaCl concentrations below 40 mmol / L.

[0031] (3) Thermal stability test 10 mL of the composite nanoparticle solution was heated in an 80°C constant temperature water bath, and three parallel samples were prepared. Samples were taken at 30, 60, 90, 120, and 150 minutes, and particle size, PDI, and Zeta potential were measured using a Malvern laser particle size analyzer.

[0032] like Figure 9 and Figure 10 As shown, the composite nanoparticles exhibit excellent thermal stability during the first 60 minutes of heating. The particle size only slightly increased from approximately 165 nm initially to 185 nm, and the PDI slowly increased from 0.21 to around 0.25. This indicates that the particles can still maintain a good dispersion state under short-term high-temperature treatment. In the later stage (80-160 min): as the heating time was extended to 80 minutes, both the particle size and PDI showed a clear linear growth trend. By 160 minutes, the average particle size increased to approximately 290 nm, and the PDI increased to 0.35. Throughout the entire 160-minute heating process, the Zeta potential remained at a high level.

[0033] Example 3: In vitro drug release study The drug release behavior of ZEN complex under different physiological conditions was studied using dialysis.

[0034] The specific steps are as follows: 5 mL of a 1 mg / mL ZEN solution was placed into a dialysis bag with a molecular weight cutoff of 14 kDa, and then placed in 30 mL of PBS buffer. To simulate different physiological environments, the pH was set to 7.4, and the temperatures were 15 ℃ and 37 ℃, respectively. The release system was placed in a constant-temperature shaker at 120 rpm for continuous shaking. Every hour, 3 mL of the release medium was taken, and an equal volume of fresh buffer under the same conditions was added to maintain a constant system volume. The absorbance was measured at 345 nm using a UV-Vis spectrophotometer, and the cumulative release of Esc was calculated according to the standard curve. Three parallel samples were set up for each experiment to ensure the reliability and repeatability of the data.

[0035] like Figure 11 As shown, experimental results indicate that the ternary composite nanoparticles prepared in this invention exhibit significant temperature-dependent release behavior. This characteristic endows the formulation with dual advantages: at 15°C, drug release is slow, maintaining activity during short-distance transport. At physiological body temperature (37°C), drug release is significantly accelerated, exhibiting a long-lasting sustained-release characteristic of initial rapid release followed by slower release. The rapid release in the early stage (approximately 50% within 12 hours) facilitates the rapid achievement of effective blood drug concentrations, resulting in rapid onset of action; the sustained release in the later stage helps maintain long-lasting efficacy and reduces the frequency of dosing.

[0036] Example 4: In vitro gastrointestinal digestion simulation By simulating the three stages of digestion in the mouth, stomach, and intestines, the in vitro digestion behavior of nanoparticles was systematically studied.

[0037] Oral digestion: Prepare a simulated oral solution containing 0.896 mg / mL KCl, 0.298 mg / mL NaCl, and 0.600 mg / mL α-amylase. Mix the initial sample solution (1 mg / mL) with the oral solution at a 1:1 ratio, adjust the pH to 6.8, and stir magnetically at 37°C for 2 minutes.

[0038] Gastric digestion: Prepare simulated gastric juice containing 2.0 mg / mL NaCl and 3.2 mg / mL pepsin, and preheat to 37 °C. Mix the oral digested sample with the gastric juice at a 1:1 ratio, adjust the pH to 2.5, and stir at 37 °C for 1 hour.

[0039] Intestinal digestion: Prepare a simulated intestinal fluid containing 36.7 mg / mL CaCl2, 218.7 mg / mL NaCl and 24 mg / mL trypsin. Neutralize the gastric digested sample to pH 7.0 and mix it with the simulated intestinal fluid at a 1:1 ratio.

[0040] like Figure 12As shown, the results indicate that ZEN ternary composite nanoparticles possess significant gastrointestinal sustained-release characteristics: this nanocarrier exhibits excellent structural stability in simulated gastric fluid, with a drug release rate of less than 10%, effectively avoiding premature drug degradation or potential irritation to the gastric mucosa under acidic conditions, thus achieving protective drug delivery into the stomach. In simulated intestinal fluid, this composite system exhibits rapid release behavior responsive to pH and enzymes. More importantly, thanks to the solubilizing effect of the nanocarrier, this system significantly improves the solubility and release rate of the poorly soluble drug aescin in the intestinal environment, with its intestinal release rate increasing by approximately 70% compared to the free drug group.

Claims

1. A method of preparing a zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system, characterized in that, The preparation method comprises the following steps: S1: dissolving zein in an aqueous ethanol solution, adding 6,7-dihydroxycoumarin, stirring to dissolve, and obtaining an ethanol solution of zein-6,7-dihydroxycoumarin; S2: slowly adding the ethanol solution of zein-6,7-dihydroxycoumarin obtained in step S1 into an aqueous sodium caseinate solution, and continuously stirring to obtain a dispersion; then, removing the organic solvent in the dispersion by distillation under reduced pressure on a rotary evaporator, and obtaining the nanoparticle delivery system.

2. A process for the preparation of a zein-sodium caseinate-6,7- dihydroxycoumarin nanoparticle delivery system according to claim 1, characterized in that, The mass ratio of 6,7-dihydroxycoumarin to zein in step S1 is 1:(1-3).

3. A process for the preparation of a zein-sodium caseinate-6,7- dihydroxycoumarin nanoparticle delivery system according to claim 1, characterized in that, The volume fraction of ethanol in the aqueous ethanol solution is 70-90%.

4. A process for the preparation of a zein-sodium caseinate-6,7- dihydroxycoumarin nanoparticle delivery system according to claim 1, characterized in that, The mass ratio of sodium caseinate to zein in step S2 is (0.5-2):1, and the solvent is water.

5. The method for preparing a zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system according to claim 1, characterized in that, The stirring speed in step S2 is 500-1000 rpm, and the stirring time is 20-40 minutes.

6. A zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system characterized in that, The preparation method is prepared by using any one of claims 1-5.

7. The zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system according to claim 6, wherein, The complex nanoparticles with core-shell structure for delivering 6,7-dihydroxycoumarin are constructed by using zein-wrapped 6,7-dihydroxycoumarin as the core and sodium caseinate adsorption as the shell.

8. The zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system according to claim 7, wherein, The particle size of the complex nanoparticles is 150-250 nm, the polydispersity index PDI is less than 0.3, and the Zeta potential is between-40 mV and-50 mV.

9. The zein-sodium caseinate-6,7-dihydroxycoumarin nanoparticle delivery system according to any one of claims 6-8 is used for treating anti-inflammatory and antioxidant diseases.

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