Protein protective agent with supramolecular action and application of protein protective agent

Abstract

The invention relates to the technical field of biological agents and material science, and discloses a supramolecular action protein protective agent and application thereof. The supramolecular interaction protein protective agent is prepared from a deep eutectic solvent and water, and the mass content of the water is 10-30 wt% of the total mass of the deep eutectic solvent and the water; the eutectic solvent is a hydrophilic eutectic solvent. The invention discloses a supramolecular action protein protective agent and application thereof. The moisture content in the deep eutectic solvent is accurately controlled to be about 13-19 wt%, preferably about 16 wt%, on one hand, protein aggregation and precipitation can be effectively inhibited, and the natural conformation of the protein can be maintained; on the other hand, the stability of the protein under the conditions of illumination, high temperature and long-term storage can be remarkably improved, and the method is suitable for protection and storage of various proteins.

Description

Technical Field

[0001] This invention relates to the fields of biopharmaceuticals and materials science and technology, and in particular to a supramolecular protein protectant and its applications. Background Technology

[0002] Proteins are the primary carriers of life activities, and their physiological functions depend heavily on their accurate and precise three-dimensional conformation. However, in many industrial fields such as biopharmaceuticals, diagnostic reagents, cosmetics, and functional foods, proteins are highly susceptible to exposure to various external stresses during production, purification, formulation, storage, and transportation. These stresses include, but are not limited to, high temperatures, freeze-thaw cycles, mechanical shearing, gas-liquid or solid-liquid interface adsorption, dehydration, and oxidation, all of which can lead to reversible or irreversible denaturation, misfolding, oligomerization, and even the formation of insoluble aggregates in protein molecules. Such stability issues not only cause a significant loss of protein biological activity but may also trigger immunogenic reactions or deterioration of the physicochemical properties of products, severely restricting the development efficiency, shelf life, and clinical application safety of protein-based biopharmaceuticals.

[0003] To address the critical technical bottleneck of protein stability, several traditional methods have been developed in this field. Among the most representative are: 1) Low-temperature freeze-drying: While effectively inhibiting chemical degradation in solution, the ice crystal formation, phase separation, and dehydration stress during freeze-drying can damage protein structure; 2) Adding stabilizers: Sugars such as sucrose and trehalose, or polyols such as sorbitol and mannitol, which thermodynamically stabilize the native state of proteins through a "preferential exclusion" mechanism. However, these typically require high concentrations to be effective, which often significantly increases the viscosity of the formulation system, hindering injection administration or downstream processing. Furthermore, the hypertonic environment may negatively impact cell viability; 3) Optimizing the solution environment: Such as using buffer salts to maintain pH or adding specific salts. However, high ionic strength salt solutions may actually promote protein aggregation and precipitation through the "salting out" effect, and increased formulation complexity may introduce new compatibility risks.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a supramolecular protein protectant and its application, aiming to solve the problem that existing protein stabilization strategies still cannot effectively maintain the conformational integrity and functional activity of proteins.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a supramolecular protein protectant, the supramolecular protein protectant being prepared from a eutectic solvent and water, wherein the mass content of the water is 10-30 wt% of the total mass of the eutectic solvent and water; and the eutectic solvent is a hydrophilic eutectic solvent.

[0007] Preferably, the water content is 13-19 wt% of the total mass of the eutectic solvent and water.

[0008] Preferably, the water content is 16 wt% of the total mass of the eutectic solvent and water.

[0009] Optionally, the eutectic solvent includes hydrogen bond acceptors and hydrogen bond donors.

[0010] Optionally, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-4).

[0011] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2.

[0012] Optionally, the hydrogen bond acceptor includes at least one of quaternary ammonium salts, amino acids, and betaine compounds.

[0013] Preferably, the hydrogen bond acceptor includes at least one of choline chloride, betaine, L-carnitine, acetamide, and urea.

[0014] Optionally, the hydrogen bond donor includes at least one of polyols, carboxylic acids, and amides.

[0015] Preferably, the hydrogen bond donor includes at least one of glycerol, ethylene glycol, 1,3-butanediol, xylitol, sorbitol, lactic acid, citric acid, malic acid, and urea.

[0016] Optionally, the hydrogen bond acceptor and hydrogen bond donor include at least one of the following combinations: choline chloride: glycerol (CG), betaine: glycerol (BG), L-carnitine: glycerol (LG), choline chloride: urea (CU), and betaine: sorbitol (BS).

[0017] Optionally, the supramolecular protein protectant may also be prepared from pharmaceutically or food-grade excipients.

[0018] A second aspect of the present invention provides a complex consisting of a protein and a supramolecularly acting protein protectant, comprising: a. An effective amount of at least one protein or polypeptide; and b. The protein protectants mentioned above.

[0019] Optionally, the protein may include plant-derived protein, animal-derived protein, hormone protein, structural protein, or enzyme.

[0020] Preferably, the proteins include, but are not limited to: whey protein isolate (WPI), soy protein isolate (SPI), insulin, lysozyme, bovine serum albumin (BSA), recombinant human collagen (such as type III collagen rhCol III), antibody fragments, enzymes (such as lipase, cellulase), etc.

[0021] Optionally, in the complex formed by the protein and the supramolecular protein protectant, the concentration of the protein is 0.1-100 mg / mL.

[0022] A third aspect of the present invention provides a method for preparing a complex composed of a protein and a supramolecularly acting protein protectant, the method comprising the following steps: S1. Mix the hydrogen bond acceptor and hydrogen bond donor in a predetermined molar ratio and stir at 60-90°C to obtain a eutectic solvent; S2. Add a predetermined mass content of water to the eutectic solvent and stir at room temperature for 24 hours to obtain a supramolecular protein protectant. S3. Add the protein powder or concentrate to the supramolecular protein protectant and stir to obtain the complex composed of the protein and the supramolecular protein protectant.

[0023] In a fourth aspect, the present invention provides the application of the above-described supramolecular protein protectant in stabilizing proteins.

[0024] Optionally, the applications of the stable protein include its use in the preparation of stable liquid protein pharmaceutical formulations, in the preparation of cosmetic compositions, in the preparation of food or health products, in the preparation of biomaterials, and in the field of biocatalysis.

[0025] A fifth aspect of the present invention provides a method for improving protein stability, the method comprising: The protein was mixed with the supramolecular protein protectant described above and stored at 4°C.

[0026] The present invention has the following beneficial effects: This invention provides a supramolecular protein protectant and its application. Compared with existing protein protectants, it has the following beneficial effects: 1. Excellent anti-aggregation and structural stabilization effects: By combining a hydrophilic eutectic solvent (DES) with water within a specific range (10-30 wt%), the resulting hydrated DES system can effectively inhibit protein aggregation and precipitation under storage and stress conditions, maintaining its native spatial conformation. Preferably, when the water content is controlled at approximately 13-19 wt%, especially approximately 16 wt%, under this water content condition, hydrogen bonds undergo nonlinear changes, and water participates in the reconstruction of the hydrogen bond network of DES, simultaneously achieving the dual functions of stabilizing the protein's secondary structure and preventing aggregation, with a particularly significant stabilizing effect. 2. High long-term storage stability: In the hydrated DES protein protectant of this invention, after accelerated storage for up to 28 days, the core secondary structure retention rate of the target protein can still reach over 95%, demonstrating excellent long-term conformational stability. 3. Strong resistance to environmental stress: Under accelerated testing conditions simulating strong light irradiation (equivalent to 6 months of natural light aging), compared with traditional buffer salt or sugar protection systems, proteins using the protective agent of this invention exhibit significant advantages in structural integrity, demonstrating their excellent resistance to environmental stresses such as photo-oxidation. 4. Broad applicability: Based on the above characteristics, the hydrated eutectic solvent protein protective agent proposed in this invention has broad applicability and can be effectively used for the protection and long-term storage of various types of proteins, providing an efficient and universal solution to the stability problems of protein drugs, biological agents, and enzyme preparations. Attached Figure Description

[0027] Figure 1 Different DES optical photographs prepared for embodiments of the present invention; Figure 2 Characterization of the DES structure prepared for an embodiment of the present invention (attenuated total reflectance-Fourier transform infrared spectroscopy). Figure 3-5 The fitting results of SPI, WPI and INS in PBS and CG-3 after 0 days and 7 days, respectively; Figure 6 The UV-Vis spectra of WPI in PBS and DES are shown in the range of 250 to 310 nm. Detailed Implementation

[0028] This invention provides a supramolecular protein protectant and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0029] In recent years, deep eutectic solvents (DES) have attracted widespread attention in the field of biotechnology, especially in protein stabilization, due to their low toxicity, biodegradability, designable physicochemical properties, and unique solubility. DES are typically formed by mixing hydrogen bond acceptors (such as quaternary ammonium salts) and hydrogen bond donors (such as polyols, carboxylic acids, or amides) in a certain molar ratio, thereby forming a eutectic mixture through intermolecular interactions.

[0030] Existing research has confirmed that certain specific DES systems can provide proteins with microenvironments different from traditional aqueous solutions. For example, through "molecular confinement" or interactions with specific groups on the protein surface, they can inhibit thermal denaturation or chemical degradation to some extent. However, existing technologies mainly have the following limitations and shortcomings: 1. Limited applicability of pure DES systems: Most pure DES have high viscosity, which not only restricts the diffusion and conformational movement of protein molecules within them, affecting their functional activity, but excessively high microenvironment viscosity may even induce proteins to form unnatural folded intermediates or mis-aggregated pathways, thus threatening their stability. 2. Unclear mechanism of action of water and lack of universal guidance: Introducing water is an effective means of regulating the physical properties of DES (such as viscosity, polarity, and hydrogen bond network), but the role of water in the DES-protein ternary system is complex and non-monotonic. Too high a water content will cause the system to approach that of a traditional buffer solution, losing the unique stabilizing effect of DES; too low a water content cannot overcome the adverse effects of high viscosity. Although existing technologies widely recognize water content as a key parameter, related research has largely focused on exploring a broad range of water content (e.g., below 50 wt%) or observing phenomena only with single, stable model proteins (such as lysozyme and bovine serum albumin). 3. Lack of broad-spectrum, quantitative design principles: Currently, the field lacks a clear, quantitative, and universally applicable guiding principle to clarify whether, for different types of proteins with vastly different structures, sizes, and surface properties, a universally "optimal" or "critical" water content point can be found by precisely controlling the water content in DES to maximize its stabilizing efficacy while also considering solution processing performance. This lack of crucial knowledge limits the development of DES from a promising laboratory tool into a reliable and universal protein protection technology platform.

[0031] Based on this, embodiments of the present invention provide a supramolecular protein protectant, which is prepared from a eutectic solvent and water, wherein the mass content of the water is 10-30 wt% of the total mass of the eutectic solvent and water; the eutectic solvent is a hydrophilic eutectic solvent; wherein all formulations and subsequent analyses are based on the defined molar ratios and molecular weights of the components, rather than the calculated average molecular weight of the DES mixture based on the molar ratios of the components.

[0032] The embodiments provided by this invention reveal the synergistic mechanism and key technical effects behind the significant protective effect of hydrated eutectic solvents on proteins at specific water contents, as detailed below: Technical Effects: 1. Superior Anti-aging and Long-Term Stability: Under optimized water content (especially critical hydration point) conditions, the hydrated eutectic solvent of this invention effectively maintains the structural integrity of proteins. Experiments show that after accelerated photoaging tests (equivalent to 6 months of natural light irradiation), the secondary structure retention rate of proteins in the solvent is significantly higher than that of the traditional phosphate-buffered saline (PBS) system. Simultaneously, the proteins remain stable during 28 days of cold storage, with no observable aggregation. 2. Safe, Green, and Flexible Components: The components of the eutectic solvent (such as choline salts, glycerol, betaine, etc.) possess low toxicity, biodegradability, and good biocompatibility, complying with green chemistry principles and relevant product safety regulations. The composition of this system can be flexibly designed and adjusted according to the characteristics of the target protein or the needs of the application scenario, demonstrating high applicability and customizability. 3. Simplified Process and Significant Cost-Effectiveness: The core of the protection method of this invention lies in the precise control of the water content of the hydrated eutectic solvent, eliminating the need for complex formulations of multiple auxiliary stabilizers and special processing techniques. This feature significantly simplifies the production process, reduces raw material and process costs, and ensures stable and reliable protective effects.

[0033] The specified water content range (10-30 wt%) is key to achieving efficient protection, and its mechanism of action is mainly reflected in the following two aspects: 1. Active regulation of the bulk structure of the eutectic solvent: At the optimal water content (e.g., approximately 16 wt%), water molecules transcend the role of a simple diluent, actively participating in and reorganizing the hydrogen bond network of the eutectic solvent. This reorganization does not completely destroy the inherent nanoscale structure of the eutectic solvent, but rather induces the formation of unique "hydrated nanodomains." This structural transformation leads to significant nonlinear abrupt changes in a series of bulk properties of the solvent (such as viscosity, conductivity, refractive index, density change rate, etc.), thereby creating a stable bulk phase environment with suitable microviscosity and molecular mobility for the protein. 2. Optimization of protein-solvent interface interactions: The reorganization of the bulk structure directly regulates the interfacial properties between the eutectic solvent-water system and the protein surface. Near the critical hydration point, the wettability of the system on the protein surface is enhanced (contact angle decreases), while the interfacial tension exhibits nonlinear changes. This indicates that a thermodynamically stable, moderately hydrated dynamic interface layer is formed in situ on the protein surface. This interface layer can cleverly balance the protein’s need for solvation with the spatial confinement effect of the eutectic solvent, thereby effectively inhibiting protein unfolding, aggregation and non-specific adsorption at the molecular level, and achieving dynamic stability protection of protein conformation.

[0034] In some preferred embodiments, the water content is 13-19 wt% of the total mass of the eutectic solvent and water.

[0035] In some preferred embodiments, the water content is 16 wt% of the total mass of the eutectic solvent and water.

[0036] At a moisture content of 16 wt%, the physicochemical properties of various hydrophilic DESs (such as CG, BG, and LG) undergo a synergistic leap, forming a "critical microenvironment" that is extremely favorable for protein stability. This critical point is effective for a variety of proteins, including soy protein (SPI), whey protein (WPI), hormone protein (insulin), and structural protein (collagen), overcoming the limitations of existing technologies that are only effective for single proteins or have unclear optimal moisture contents.

[0037] Water molecules actively modulate the supramolecular interaction network at approximately 16 wt%. At this hydration level, water and DES components jointly participate in solvation of the protein surface, forming a robust shell of hydrogen bonds and electrostatic interactions. This protective layer maintains the native folding of peptides and proteins, induces clustering that reduces hydrophobic core exposure, decreases surface hydrophobicity, and thus inhibits intermolecular hydrophobic interactions and aggregation within the hydrophilic DES.

[0038] In some embodiments, the eutectic solvent includes hydrogen bond acceptors and hydrogen bond donors.

[0039] In some preferred embodiments, the eutectic solvent is a glycerol-based eutectic solvent.

[0040] In some embodiments, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-4).

[0041] In some preferred embodiments, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2.

[0042] When the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:2, DES forms a highly ordered supramolecular network, which facilitates the formation of more hydrogen bonds between the DES components and proteins (such as WPI), resulting in a stable "solvent-bridged" structure between the protein and the DES protein protectant. Simultaneously, the DES network at the 1:2 ratio exhibits structural universality, adapting to the surface characteristics of different proteins and providing non-specific protection.

[0043] In some embodiments, the hydrogen bond acceptor includes, but is not limited to, at least one of quaternary ammonium salts, amino acids, and betaine compounds.

[0044] In some preferred embodiments, the hydrogen bond acceptor includes, but is not limited to, at least one of choline chloride, betaine, L-carnitine, acetamide, and urea.

[0045] In some embodiments, the hydrogen bond donor includes, but is not limited to, at least one of polyols, carboxylic acids, and amides.

[0046] In some preferred embodiments, the hydrogen bond donor includes, but is not limited to, at least one of glycerol, ethylene glycol, 1,3-butanediol, xylitol, sorbitol, lactic acid, citric acid, malic acid, and urea.

[0047] In some embodiments, the hydrogen bond acceptor and hydrogen bond donor include at least one of the following combinations: choline chloride: glycerol (CG), betaine: glycerol (BG), L-carnitine: glycerol (LG), choline chloride: urea (CU), and betaine: sorbitol (BS).

[0048] Among the above combinations, choline chloride:glycerol (CG), betaine:glycerol (BG), and L-carnitine:glycerol (LG) are preferred. Urea is a known protein denaturant. Even when bound within a DES network, it can release free urea molecules at certain water contents, directly attacking the protein's hydrogen bond network and causing denaturation or aggregation. Glycerol, on the other hand, is a mild, biocompatible polyol that can form stable hydrogen bond networks without causing protein denaturation. The DES formed by these hydrogen bond acceptors and glycerol in a 1:2 ratio has viscosity, polarity, and hydrogen bond density suitable for protein stabilization.

[0049] In some embodiments, the supramolecular protein protectant is further prepared from pharmaceutically or food-grade excipients.

[0050] This invention provides a complex composed of a protein and a supramolecularly acting protein protectant, comprising: a. An effective amount of at least one protein or polypeptide; and b. The supramolecular protein protectants mentioned above.

[0051] The complex maintains a stable protein conformation and activity during storage.

[0052] In some embodiments, the protein includes plant-derived protein, animal-derived protein, hormone protein, structural protein, or enzyme.

[0053] In some embodiments, the proteins include, but are not limited to: whey protein isolate (WPI), soy protein isolate (SPI), insulin, lysozyme, bovine serum albumin (BSA), recombinant human collagen (such as type III collagen rhCol III), antibody fragments, enzymes (such as lipase, cellulase), etc.

[0054] In some embodiments, the concentration of the protein in the complex formed by the protein and the supramolecularly acting protein protectant is 0.1-100 mg / mL.

[0055] This invention provides a method for preparing a complex composed of a protein and a supramolecular protein protectant, the method comprising the following steps: S1. Mix the hydrogen bond acceptor and hydrogen bond donor in a predetermined molar ratio and stir at 60-90°C to obtain a eutectic solvent; S2. Add a predetermined mass content of water to the eutectic solvent and stir at room temperature for 24 hours to obtain a supramolecular protein protectant. S3. Add the protein powder or concentrate to the supramolecular protein protectant and stir to obtain the complex composed of the protein and the supramolecular protein protectant.

[0056] This invention provides the application of the supramolecular protein protectant described above in stabilizing proteins.

[0057] In some embodiments, the applications of the stable protein include its use in the preparation of stable liquid protein pharmaceutical formulations, in the preparation of cosmetic compositions, in the preparation of food or health products, in the preparation of biomaterials, and in the field of biocatalysis.

[0058] This invention provides a method for improving protein stability (or protein preservation), the method comprising: The protein was mixed with the supramolecular protein protectant described above and stored at 4°C.

[0059] The method is used to inhibit protein aggregation, prevent protein precipitation, maintain the native secondary structure of proteins, and enhance the thermal stability or light stability of proteins.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0061] The following detailed description uses specific examples.

[0062] Example 1 The preparation of supramolecular protein protectants includes the following steps: Synthesis of pure DES: Choline chloride (ChCl, >98%) and glycerol (Gly, >99%) were dried separately in a vacuum oven at 60°C for 24 hours. ChCl and Gly were weighed at a molar ratio of 1:2 and placed in a sealed glass bottle. The mixture was magnetically stirred (500 rpm) in an 80°C water bath until completely dissolved and a homogeneous, transparent liquid was formed. Figure 1 Mark it as CG (pure), seal it immediately, and store it in a desiccator after cooling.

[0063] Preparation of gradient-content DES: Ultrapure water (18.2 MΩ·cm) was added to pure CG by precise weighing. After vortex mixing, the mixture was stirred in a constant temperature oven at 25℃ for 24 hours to obtain a sample with a water content of about 1.5%, which was labeled as CG-1.

[0064] Example 2 The preparation of the supramolecular protein protectant in this embodiment is basically the same as that in Example 1, except that: ultrapure water (18.2 MΩ·cm) was added to pure CG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 9%, which was labeled as CG-2.

[0065] Example 3 The preparation of the supramolecular protein protectant in this embodiment is basically the same as that in Example 1, except that: ultrapure water (18.2 MΩ·cm) was added to pure CG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 16%, which was labeled as CG-3.

[0066] Example 4 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 1, except that ultrapure water (18.2 MΩ·cm) was added to pure CG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 23%, which was labeled as CG-4.

[0067] Example 5 The preparation of the supramolecular protein protectant in this embodiment is basically the same as that in Example 1, except that: ultrapure water (18.2 MΩ·cm) was added to pure CG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 29%, which was labeled as CG-5.

[0068] Example 6 The preparation of supramolecular protein protectants in this embodiment is basically the same as in Example 1, except that choline chloride (ChCl, >98%) and glycerol (Gly, >99%) are dried in a vacuum oven at 60°C for 24 hours, and the final sample is labeled as BG-1.

[0069] Example 7 The preparation of the supramolecular protein protectant in this embodiment is basically the same as that in Example 6, except that: ultrapure water (18.2 MΩ·cm) was added to pure BG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 9%, which was labeled as BG-2.

[0070] Example 8 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 6, except that ultrapure water (18.2 MΩ·cm) was added to pure BG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 16%, which was labeled as BG-3.

[0071] Example 9 The preparation of the supramolecular protein protectant in this embodiment is basically the same as that in Example 6, except that: ultrapure water (18.2 MΩ·cm) was added to pure BG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 23%, which was labeled as BG-4.

[0072] Example 10 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 6, except that ultrapure water (18.2 MΩ·cm) was added to pure BG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 26%, which was labeled as BG-5.

[0073] Example 11 The preparation of supramolecular protein protectants in this embodiment is basically the same as that in Example 1, except that: L-carnitine (L-car, >98%) and glycerol (Gly, >99%) are dried in a vacuum oven at 60°C for 24 hours, and the final sample is labeled as LG-1.

[0074] Example 12 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 11, except that ultrapure water (18.2 MΩ·cm) was added to pure LG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 9%, which was labeled as LG-2.

[0075] Example 13 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 11, except that ultrapure water (18.2 MΩ·cm) was added to pure LG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 16%, which was labeled as LG-3.

[0076] Example 14 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 11, except that: ultrapure water (18.2 MΩ·cm) was added to pure LG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 23%, which was labeled as LG-4.

[0077] Example 15 The preparation of the supramolecular protein protectant was carried out in the same way as in Example 11, except that: ultrapure water (18.2 MΩ·cm) was added to pure LG by precise weighing, and after vortex mixing, it was stirred in a constant temperature oven at 25°C for 24 hours to obtain a sample with a water content of about 29%, which was labeled as LG-5.

[0078] Optical photographs of the supramolecular protein protectants prepared in the above embodiments are shown below. Figure 1 .

[0079] The supramolecular protein protectants prepared in the above examples were characterized by physical properties: their structures were detected using attenuated total reflectance-Fourier transform infrared spectroscopy at 25 °C. The results are as follows... Figure 2 As shown, the ATR-FTIR spectra of the three eutectic solvents (LG, CG, BG) reveal that all three have wavelengths between 3600 and 3000 cm⁻¹. - The system exhibits a typical broad and strong OH stretching vibration absorption band within the range, indicating the presence of a distinct hydrogen bond network, which is a key structural feature for the formation of eutectic solvents.

[0080] Performance testing 1. Evaluation of the effect on protein stability Sample preparation: Whey protein isolate powder (WPI) (>99%) was dispersed at a concentration of 2 mg / mL in PBS (pH 7.4) and the above-prepared CG-3, BG-3, and LG-3 (water content ~16%). The samples were then sonicated in an ice bath for 10 minutes to aid dissolution.

[0081] UV-Vis spectroscopy: Place the above sample in a cuvette and measure its spectrum between 250 and 310 nm. Figure 3 The figures show the relative exposure of Tyr and Trp amino acid residues in different solvent environments after storage at 4°C for one day. No significant difference was observed in Trp of WPI between PBS and DES, indicating that the secondary structure of WPI retained its native fold after storage at 4°C for one day at approximately 16 wt% moisture content.

[0082] 2. Studies on the stabilizing effect of proteins from multiple sources on secondary structure Sample preparation: SPI, WPI, and insulin were dissolved in PBS and CG-3 at a concentration of 1 mg / mL, respectively. A circular dichroism spectroscopy system was used to scan the samples in the wavelength range of 190–260 nm.

[0083] Conformational stability: Figure 4-6 The figure shows the fitting results of the amide I region of the three proteins after storage in PBS and CG-3 for 0 days and 7 days.

[0084] Compared to CG-3, WPI showed significant structural rearrangement after 7 days of storage in PBS, with a 4% decrease in α-helices and an 11% increase in β-sheets, while in CG-3, the α-helix content remained unchanged and the β-sheets increased by only 1%. Figure 4 ).

[0085] In insulin, PBS induces a 7% decrease in α-helices and a 9% increase in β-sheets, while in CG-3, α-helix content remains stable and β-sheets increase by only 2%. Figure 5 ).

[0086] FTIR was used to monitor the changes in the secondary structure of SPI over time (0, 7 days). The contents of α-helices and β-sheets were quantified by amide I band segmentation fitting. The results showed that after 7 days of storage in PBS, the relative content of α-helices in SPI decreased by approximately 6%, while the content of β-sheets increased by approximately 6%; however, in CG-3, the contents of both secondary structures fluctuated by less than 1% over 7 days, indicating that the DES system effectively maintains the conformational stability of SPI. Figure 6 ).

[0087] From day 0 to day 7, the data showed that the secondary structure of proteins in PBS changed non-linearly over time, while in CG-3, the secondary structures of proteins from all sources remained stable, which may be attributed to supramolecular interactions present in DES.

[0088] WPIs were dispersed at a concentration of 2 mg / mL using CG-1 (water content ~1.5%) prepared in Example 1 as a solvent. The sample viscosity was found to be too high, making protein dispersion difficult. After 24 hours of storage, PSD showed the presence of large micron-sized particles, and TEM observation revealed significant protein aggregation. UV-Vis second derivative spectroscopy showed an abnormal Tyr / Trp ratio, indicating conformational perturbation of the protein. This demonstrates that when the water content is too low, the DES viscosity is too high, failing to provide sufficient motility and hydration for the proteins, and instead promoting aggregation.

[0089] WPI was dispersed at a concentration of 2 mg / mL using CG-5 (water content ~29%) prepared in Example 5 as a solvent. The sample exhibited good flowability. Short-term storage (1 day) showed that the PSD particle size was smaller than that of the PBS group but larger than that of the CG-3 group. After heat treatment at 60°C for 30 minutes, its inhibitory effect on aggregation was significantly weaker than that of the CG-3 group, and more aggregates were formed. FTIR analysis showed that after UV accelerated aging, its protective effect on rhCol III was not as significant as that of CG-3. This demonstrates that when the water content exceeds the critical point significantly, the inherent hydrogen bond network of DES is over-diluted, its unique "protective microenvironment" is weakened, and the stabilizing effect decreases.

[0090] A 20 wt% trehalose aqueous solution (a commonly used protein lyophilization protectant) was prepared and used to disperse WPI at a concentration of 2 mg / mL. The stability was acceptable at 4°C, but after 7 days of storage at 25°C, the PSD showed a significant increase in particle size (>300 nm), and the sample became slightly turbid. Under heat stress at 60°C, its anti-aggregation effect was far inferior to the CG-3 system. Furthermore, the high-concentration trehalose solution had a high viscosity, which was unfavorable for injection or filtration.

[0091] As shown above, both bulk and interfacial properties exhibit a nonlinear transition at a critical water content (approximately 16 wt%) in the three types of hydrophilic DES. Accompanying the DES-protein secondary structure stability experiments, FTIR and CD analyses confirmed that at this critical point, DES can stabilize a variety of proteins, maintaining their native structure even under continuous UV irradiation for up to six months.

[0092] In summary, this invention discloses a supramolecular protein protectant and its applications. By precisely controlling the moisture content in a eutectic solvent at approximately 13–19 wt%, preferably approximately 16 wt%, it effectively inhibits protein aggregation and precipitation, maintaining its native conformation. Furthermore, in this supramolecular protein protectant, the secondary structure retention rate of proteins stored for 28 days is ≥95%, and the structural integrity after simulating 6 months of photoaging is significantly better than that of traditional buffer systems, making it suitable for the protection and storage of various types of proteins.

[0093] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A supramolecular protein protectant, characterized in that, The supramolecular protein protectant is prepared from a eutectic solvent and water, wherein the water content is 10-30 wt% of the total mass of the eutectic solvent and water; and the eutectic solvent is a hydrophilic eutectic solvent.

2. The supramolecular protein protectant according to claim 1, characterized in that, The water content is 16 wt% of the total mass of the eutectic solvent and water.

3. The supramolecular protein protectant according to claim 1, characterized in that, The eutectic solvent includes hydrogen bond acceptors and hydrogen bond donors; The hydrogen bond acceptor includes at least one of quaternary ammonium salts, amino acids, and betaine compounds, and the hydrogen bond donor includes at least one of polyols, carboxylic acids, and amide compounds.

4. The supramolecular protein protectant according to claim 3, characterized in that, The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-4).

5. The supramolecular protein protectant according to claim 3, characterized in that, The hydrogen bond acceptor and hydrogen bond donor include at least one of the following combinations: choline chloride: glycerol, betaine: glycerol, L-carnitine: glycerol, choline chloride: urea, betaine: sorbitol.

6. A complex consisting of a protein and a supramolecularly acting protein protectant, comprising: a. An effective amount of at least one protein or polypeptide; as well as b. The supramolecular protein protectant according to any one of claims 1-5.

7. The complex of protein and supramolecular protein protectant according to claim 6, characterized in that, The proteins include plant-derived proteins, animal-derived proteins, hormone proteins, structural proteins, or enzymes.

8. The method for preparing the complex of protein and supramolecular protein protectant according to claim 6 or 7, characterized in that, The preparation method includes the following steps: S1. Mix the hydrogen bond acceptor and hydrogen bond donor in a predetermined molar ratio and stir at 60-90°C to obtain a eutectic solvent; S2. Add a predetermined mass content of water to the eutectic solvent and stir at room temperature to obtain a supramolecular protein protectant. S3. Add the protein powder or concentrate to the supramolecular protein protectant and stir to obtain the complex composed of the protein and the supramolecular protein protectant.

9. The use of the supramolecular protein protectant according to any one of claims 1-5 in stabilizing proteins.

10. A method for improving protein stability, characterized in that, The method includes: The protein is mixed with the supramolecular protein protectant according to any one of claims 1-5 and stored.

Images