Preparation method of cannabidiol nanoparticles based on soybean protein refolding

Abstract

The invention discloses nanoparticles for efficiently and stably delivering cannabidiol as well as a preparation method and application of the nanoparticles, and belongs to the technical field of bioactive substance delivery. The method is characterized in that a soybean protein structure is induced to generate controllable folding and re-folding by adopting a pH migration method, cannabidiol is efficiently encapsulated by utilizing the hydrophobic effect of protein in the process, and composite nanoparticles are formed through self-assembly. According to the method, when the mass ratio of the soybean protein to the cannabidiol is 10: 1, the encapsulation efficiency can reach 96.01%, the particle size of the obtained nanoparticles is uniform, and the average particle size is smaller than or equal to 100 nm. The stability of the cannabidiol can be effectively improved, the retention rate of the cannabidiol is still larger than or equal to 90% after the cannabidiol is heated in water at 80 DEG C for 2 hours, the antioxidant activity of the cannabidiol is remarkably enhanced, and the in-vitro bioavailability of the cannabidiol is improved. The whole preparation process is mild in condition, an organic solvent or chaotropic agent does not need to be used, safety is achieved, the green production concept is met, and the obtained nanoparticles have wide application prospects in medicine.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance delivery and nanoparticle technology, specifically relating to a composite nanoparticle constructed based on protein pH-driven refolding technology, and particularly to a core-shell structured soybean protein-cannabidiol nanoparticle, its preparation method, and its applications. The nanoparticles encapsulate cannabidiol through the refolding of soybean protein, exhibiting both sustained-release properties and antioxidant activity, effectively improving the stability and bioavailability of cannabidiol, and are suitable for the pharmaceutical field. Background Technology

[0002] Cannabidiol (CBD), the main non-psychoactive component extracted from hemp plants, possesses anti-inflammatory, antioxidant, and non-addictive physiological activities, demonstrating significant potential in the medical field. Specifically, its anti-cancer and neuroprotective effects have been confirmed by numerous studies, showing potential therapeutic value, particularly for central nervous system diseases such as epilepsy and cognitive impairment. However, CBD's extremely low oral bioavailability (approximately 6%) and its physicochemical instability due to sensitivity to light, heat, and oxygen severely limit its practical application and product development. Encapsulating CBD in suitable carriers using encapsulation technology is an effective strategy to address these issues. Among these, protein carriers have attracted considerable attention due to their excellent nutritional and functional properties and affinity for hydrophobic active ingredients. Soy protein, as a widely available, inexpensive plant protein with a balanced amino acid composition, has significant advantages in this field. Although numerous protein-based encapsulation systems have been reported, existing processes generally suffer from high complexity, unsatisfactory encapsulation efficiency, high energy consumption, or the need for organic solvents, limiting their large-scale application. Therefore, there is an urgent need in this field to develop a novel protein-based encapsulation method that is simpler, greener, safer, and easier to industrialize, in order to deliver cannabidiol efficiently and stably. Summary of the Invention

[0003] I. Purpose of the Invention Based on the shortcomings of existing cannabidiol delivery systems in terms of encapsulation efficiency, stability, and biosafety as pointed out in the background art, the primary objective of this invention is to provide a composite nanoparticle with cannabidiol as the active ingredient and soy protein as the carrier. Another objective of this invention is to provide a method for preparing the aforementioned nanoparticles, which is simple, mild, environmentally friendly, safe, and easily scalable. Through the above-described nanoparticles and preparation methods, this invention aims to solve at least one or more of the following key technical problems: First, to provide a nanoencapsulation system with good biocompatibility and no risk of biotoxicity, avoiding the safety hazards caused by the use of organic solvents or liquid release agents; Second, to construct a multifunctional nanoparticle with excellent antioxidant capacity, utilizing the synergistic effect of soybean protein and cannabidiol to more effectively address oxidative stress and inflammatory responses in trauma microenvironments; Third, to provide a nanodelivery system that can achieve efficient loading, good protection, and controlled sustained release of cannabidiol, fundamentally overcoming the inherent defects of cannabidiol itself, such as poor water solubility, low chemical stability, and poor oral bioavailability, thereby fully releasing and maintaining its pharmacological potential; Fourth, to provide a simple, mild, and highly reproducible method for preparing protein-based polyphenol nanoparticles, overcoming the problems of complex processes, strict parameter control, and difficulty in large-scale scaling in existing technologies; Fifth, to provide a protein-based nanoparticle formulation with excellent physical and storage stability, solving the technical problem of easy aggregation, precipitation, or degradation of active ingredients in nano-dispersion systems during storage.

[0004] II. Technical Solution To achieve the above-mentioned objectives, the key components and process parameters of this invention have been optimized as follows: The concentration of the soybean protein solution is 5-20 mg / mL; the concentration of the cannabidiol ethanol solution is 2-10 mg / mL; the mass ratio of soybean protein to cannabidiol is 5:1 to 20:1; the volume ratio of cannabidiol ethanol solution to unfolded soybean protein solution is 1:4; the pH value of the solution during the unfolding stage is in the strongly alkaline range of 10.0-12.5; the pH value of the solution during the refolding stage is 6.5-7.5.

[0005] The present invention also provides a method for preparing the nanoparticles, characterized by comprising the following steps: 1. Soy protein unfolding: Disperse soybean protein in ultrapure water, stir to dissolve, and adjust the pH to 10.0-12.5 with 1 M NaOH solution to obtain a denatured protein stock solution with a concentration of 5-20 mg / ml.

[0006] 2. Protein-polyphenol self-assembly: Cannabidiol was dissolved in anhydrous ethanol to prepare a 2-10 mg / mL cannabidiol ethanol stock solution. This stock solution was then slowly added to the protein stock solution obtained in step 1 with stirring. The volume ratio of the cannabidiol ethanol solution to the unfolded soy protein solution was 1:4. The mixture was stirred for 30 min under alkaline conditions.

[0007] 3. Soy protein folding and nanoparticle formation: Under stirring, 1 M HCl solution was slowly added dropwise to adjust the pH of the system back to 6.5-7.5, driving the protein to refold and encapsulate cannabidiol to form composite nanoparticles.

[0008] 4. Purification and concentration of nanoparticles: Ethanol was removed by rotary evaporation, and after standing at room temperature for 4-12 h, unencapsulated cannabidiol was removed by low-speed centrifugation at 3000-6000 rpm for 10-20 min, and the pure nanoparticle dispersion was collected.

[0009] 5. Product solidification and storage: The purified dispersion is pre-frozen and then freeze-dried to obtain solid nanoparticle powder.

[0010] III. Beneficial Effects Compared with the prior art, the technical solution provided by the present invention has the following significant advantages and beneficial effects: 1. Excellent Encapsulation Efficiency: This invention achieves highly efficient encapsulation of cannabidiol (CBD) through precisely controlled pH shift and refolding technology. Experimental data show that when the mass ratio of soy protein to CBD is 20:1-5:4, the encapsulation efficiency is significantly higher than that of traditional soy protein encapsulation methods without refolding treatment. When the soy protein to CBD ratio is 10:1, the encapsulation efficiency can reach 96.01%.

[0011] 2. Excellent Stability: The nanoparticles prepared in this invention exhibit outstanding thermal and physical stability. After heating in an 80°C water bath for 120 min, the retention rate of free cannabidiol (CBD) dropped sharply to 27.41%, while the retention rate of soybean protein-CBD nanoparticles remained as high as 90.09%, demonstrating a significant slowdown in thermal degradation rate and proving that they provide strong protection for CBD. The average particle size of the nanoparticles is 83.25 nm, with a uniform particle size distribution. Their absolute Zeta potential is 32.46 mV, indicating strong electrostatic repulsion between particles, effectively preventing aggregation and precipitation during storage, thus forming a highly stable colloidal dispersion system.

[0012] 3. Significant sustained-release performance: In vitro simulated release experiments demonstrated that the soybean protein-cannabidiol nanoparticles prepared in this invention possess excellent sustained-release characteristics. During a 32-hour release cycle, free cannabidiol was rapidly released within 2 hours and reached a plateau, with a cumulative release of only 8.12%. In contrast, the soybean protein-cannabidiol nanoparticles exhibited continuous and gradual release throughout the entire cycle, with a cumulative release of 71.16% over 32 hours, displaying typical controlled-release kinetics. This characteristic helps maintain effective blood drug concentrations for an extended period in vivo, thereby improving therapeutic efficacy and reducing dosing frequency.

[0013] 4. Enhanced antioxidant activity: After encapsulation with this invention, the in vitro antioxidant capacity of cannabidiol is significantly enhanced. At the same cannabidiol content (100 μg / mL), the scavenging rate of DPPH free radicals by nanoparticles is increased by 51.37% compared with unencapsulated cannabidiol, and the scavenging rate of ABTS+ free radicals is increased by 63.84%, greatly improving the inhibitory ability against oxidative stress.

[0014] 5. High Industrial Adaptability: The entire system of this invention uses food-grade soy protein as a carrier, eliminating the need for any organic solvents or chemical surfactants throughout the process. This fundamentally eliminates the risk of biotoxicity from solvent residues, ensuring high product safety. The encapsulation efficiency of this method not only far exceeds that of common protein carriers such as whey protein (encapsulation rate <50%), but also achieves a significant improvement of approximately 13% compared to traditional soy protein encapsulation technology (encapsulation rate approximately 85%), thereby greatly enhancing the utilization efficiency of cannabidiol. The preparation process involves mild conditions, mainly including conventional unit operations such as physical mixing, rotary evaporation, and freeze-drying. It requires low equipment investment, has a short production cycle, and its process parameters are easy to control and scale up, demonstrating excellent prospects for industrial production and laying a solid foundation for large-scale application in the pharmaceutical field. Attached Figure Description

[0015] Figure 1 This diagram illustrates a comparison of the encapsulation efficiency of cannabidiol (CBD) by refolded and non-refolded soy protein at different mass ratios in an embodiment of the present invention. The diagram visually demonstrates that the refolding process described in this invention can significantly improve the encapsulation efficiency of soy protein-CBD nanoparticles.

[0016] Figure 2 This image shows a sample of soybean protein-cannabidiol nanoparticles prepared according to the optimal mass ratio (soybean protein:cannabidiol = 10:1) according to the present invention. The image illustrates the morphology of the nano-dispersion, demonstrating its excellent colloidal state and water dispersibility.

[0017] Figure 3This is a graph evaluating the stability and release characteristics of the refolded soybean protein-cannabidiol nanoparticles prepared in this invention. Figure 3 A is a comparison of the thermal stability of nanoparticles and free cannabidiol, showing the excellent thermal stability of nanoparticles under high temperature conditions. Figure 3 B represents the in vitro simulated release curve of the nanoparticles, revealing their significant sustained-release properties.

[0018] Figure 4 Schematic diagram of the nanoparticle preparation process. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of the invention. Example

[0020] This embodiment provides a method for preparing cannabidiol nanoparticles loaded with refolded soybean protein, the specific steps of which are as follows: 1. Raw material preparation (1) Accurately weigh 1.25 g of soy protein isolate (purity ≥98%), dissolve it in 100 mL of ultrapure water, and magnetically stir at 1000 rpm for 1 h to fully dissolve it, so as to prepare a soy protein solution with a concentration of 12.5 mg / mL.

[0021] (2) Accurately weigh 125 mg of cannabidiol (purity ≥99.99%), dissolve it in 25 mL of anhydrous ethanol (purity ≥99%), and prepare a cannabidiol ethanol stock solution with a concentration of 5 mg / mL.

[0022] (3) Prepare a 1 mol / L NaOH solution: accurately weigh 40.0 g of sodium hydroxide granules, dissolve them in an appropriate amount of ultrapure water, stir until completely dissolved, and make up to 1000 mL. Shake well and set aside.

[0023] (4) Prepare a 1 mol / L HCl solution: Measure 85 mL of concentrated hydrochloric acid and slowly add it to about 900 mL of ultrapure water under continuous stirring and ventilation. After cooling, make up to 1000 mL and shake well for later use.

[0024] 2. Preparation process (1) Add 1 M NaOH solution dropwise to the prepared soybean protein solution using a burette while continuously stirring magnetically (500 rpm). Simultaneously, monitor the pH change of the solution in real time using a pH meter and precisely adjust the pH of the solution to 12.0. During this process, the solution can be observed to change from a pale yellow semi-transparent state to a homogeneous pale yellow transparent solution, indicating that the soybean protein structure has unfolded and the internal hydrophobic regions have been fully exposed.

[0025] (2) While maintaining the system at 500 rpm, slowly and dropwise add 5 mg / mL cannabidiol ethanol stock solution to the above alkaline soy protein solution (the volume ratio of cannabidiol solution to soy protein solution is 1:4). After the addition is complete, continue stirring for 30 min to ensure that the unfolded soy protein and cannabidiol molecules are fully pre-bound and self-assembled through hydrophobic interactions.

[0026] (3) While continuously stirring (500 rpm), 1 M HCl solution was added dropwise to the resulting mixture, and the pH of the solution was slowly adjusted back to 7.0 using a pH meter for precise control. During this process, the solution was observed to change from a uniform transparent state to a semi-transparent state, indicating that the soybean protein underwent refolding and self-assembly to form composite nanoparticles encapsulating cannabidiol.

[0027] (4) The obtained nanoparticle dispersion was transferred to a rotary evaporator and rotary evaporated at a water bath temperature of 45℃ and a rotation speed of 60 rpm. The vacuum was drawn to -0.09 MPa and continued for 5 min to completely remove the ethanol in the system. Then, the concentrated nanoparticle dispersion was poured into a clean beaker and allowed to stand at room temperature (25℃) for 8 h to further stabilize the nanoparticle structure.

[0028] (5) Centrifuge the dispersion at 5000 rpm for 15 min and carefully collect the supernatant (i.e., the purified nanoparticle dispersion). Pre-freeze the supernatant in an ultra-low temperature freezer at -80℃ for 4 h, and then transfer it to a freeze dryer. Freeze-dry the supernatant for 16 h at a cold trap temperature of -50℃ and a vacuum degree of <10 Pa to finally obtain a white, loose powder of refolded soybean protein-cannabidiol nanoparticles.

[0029] Comparative Example 1 Simple mixing to prepare soybean protein-cannabidiol nanoparticles Objective: This comparative example aims to illustrate that the encapsulation efficiency and stability of soybean protein-cannabidiol nanoparticles prepared by simple mixing without pH shift-induced refolding are significantly lower than those prepared by the method of this invention, thus demonstrating the key role of the protein refolding step in the system of this invention.

[0030] Preparation: Accurately weigh 1.25 g of soy protein and disperse it in 100 mL of ultrapure water. Stir magnetically at 1000 rpm for 1 h until completely dissolved. Adjust the pH to 7.0 to prepare a protein solution of 12.5 mg / mL. Subsequently, add 10 mL of a 5 mg / mL cannabidiol ethanol stock solution dropwise to 40 mL of the above protein solution while stirring, and continue stirring for 30 min. The mixture was then processed using the same rotary evaporation, settling, centrifugation, and freeze-drying steps as described in this invention.

[0031] Results: The encapsulation efficiency of the nanoparticles obtained in this comparative example was only 85.12% (when the mass ratio of soybean protein to cannabidiol was 10:1), significantly lower than that of the refolding method of this invention (96.01%). Furthermore, the obtained nanoparticles exhibited a wide particle size distribution and poor physical stability of the dispersion system, easily leading to aggregation after standing.

[0032] Proof: This comparative result demonstrates that without a controllable protein unfolding and refolding process, soy protein cannot effectively expose sufficient hydrophobic regions and complete precise structural reorganization, thus making it difficult to form stable nanostructures that can efficiently encapsulate cannabidiol. Therefore, pH-shift-induced protein refolding is the core technology for achieving high encapsulation efficiency and high stability, and cannot be replaced by simple physical mixing.

[0033] Comparative Example 2: Objective: This comparative example aims to verify the unique advantage of soy protein in encapsulating cannabidiol by comparing it with two common food proteins (sodium caseinate and whey protein) under the same preparation conditions, thereby demonstrating the rationality and superiority of choosing soy protein as the carrier material in this invention.

[0034] Preparation: 1.25g of sodium caseinate (SC) and whey protein (WP) were accurately weighed to replace soy protein isolate and dispersed in 100mL of ultrapure water respectively. Parallel experiments were conducted according to the complete process of the technical solution of this invention (including pH shift to 12.0, addition of cannabidiol ethanol solution, pH adjustment to 7.0, rotary evaporation, centrifugation and freeze drying). All key parameters (such as concentration, pH, stirring time, etc.) were consistent with those in the embodiments of this invention.

[0035] Results: Under the same 10:1 mass ratio, the encapsulation efficiency of sodium caseinate-cannabidiol nanoparticles was 71.5%, while that of whey protein-cannabidiol nanoparticles was only 48.3%. Both are far lower than the 96.01% encapsulation efficiency achieved by the soybean protein system of this invention.

[0036] Evidence: The comparative results clearly demonstrate that, despite employing the same advanced preparation process as this invention, different protein carriers exhibit significant differences in their encapsulation capabilities for cannabidiol due to their unique molecular structural differences. Soy protein, in the refolding system described in this invention, demonstrates encapsulation efficiency far superior to sodium caseinate and whey protein, proving it to be an ideal and irreplaceable carrier material for achieving efficient nanodelivery of cannabidiol.

Claims

1. A method for preparing soybean protein-cannabidiol composite nanoparticles, characterized in that, Includes the following steps: (1) Soy protein unfolding: Soy protein is dissolved in water, and the pH is adjusted to a strongly alkaline range of 10.0-12.5 to induce the unfolding of the soy protein structure and obtain unfolded soy protein solution; (2) Protein-polyphenol self-assembly: Cannabidiol was dissolved in ethanol to prepare a cannabidiol ethanol solution, which was then added to the unfolded soy protein solution obtained in step (1) under stirring and mixed and self-assembled in an alkaline environment. (3) Soy protein refolding and nanoparticle formation: Adjust the pH of the mixture obtained in step (2) to neutral to induce soybean protein refolding, thereby encapsulating cannabidiol and forming a composite nanoparticle dispersion; (4) Post-processing: Remove ethanol from the dispersion obtained in step (3) and separate and purify to obtain the soybean protein-cannabidiol composite nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step (1), the soybean protein is soybean protein isolate; the concentration of the soybean protein solution is 5-20 mg / mL; and the pH is adjusted using a 1 M alkali solution.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the alkali used to adjust the pH is sodium hydroxide; the pH value is 10.0-12.

5.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the cannabidiol ethanol solution is 2-10 mg / mL; the mass ratio of the soy protein to cannabidiol is (5:1) to (20:1); and the volume ratio of the cannabidiol ethanol solution to the unfolded soy protein solution is 1:

4.

5. The preparation method according to claim 1, characterized in that, In step (3), the pH is adjusted to 7.0 using an acid solution with a concentration of 1 M; the acid is hydrochloric acid.

6. The preparation method according to claim 1, characterized in that, In step (4), ethanol is removed by rotary evaporation; separation and purification are carried out by low-speed centrifugation at a speed of 3000-6000 rpm for 10-20 min.

7. The preparation method according to claim 1, characterized in that, In step (4), after removing ethanol and before separation and purification, the dispersion is allowed to stand at room temperature for 4-12 h; and / or, after separation and purification, the obtained nanoparticle dispersion is freeze-dried to obtain a solid powder.

8. A soybean protein-cannabidiol composite nanoparticle prepared by any one of claims 1 to 7.

9. The soybean protein-cannabidiol composite nanoparticles according to claim 8, characterized in that, The nanoparticles have a particle size range of 50-200 nm and an encapsulation efficiency of not less than 90% for cannabidiol.

10. The use of the soybean protein-cannabidiol composite nanoparticles according to claim 8 or 9 in the preparation of pharmaceuticals.

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