Amino acid self-forming coagulum and preparation method and application thereof

Abstract

This invention relates to the field of biomaterials technology, specifically to an amino acid-autogenic aggregate, its preparation method, and its applications. The invention provides a method for preparing an amino acid-autogenic aggregate, comprising the following steps: dissolving the amino acid in water, adjusting the pH to neutral or weakly alkaline using an acid-base adjusting agent, preparing an amino acid buffer solution, adding an organic solvent, and mixing thoroughly. The amino acid-autogenic aggregate obtained by this invention can serve as a high-performance drug delivery system, especially providing highly efficient protection for easily degraded biopharmaceuticals such as RNA and proteases, significantly improving their in vivo stability. It can also be applied to the development of cell cryopreservation solutions, organ transplantation preservation solutions, enzyme reactors, cosmetic emulsions, and poorly soluble drug delivery / sustained-release systems.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to an amino acid-autogenous aggregate, its preparation method, and its application. Background Technology

[0002] Bionics, as a core interdisciplinary field in modern biomedicine, is not only a key technological support for expanding disease treatment methods, but also an important pathway to reveal the essential laws of life activities and break through the bottlenecks of traditional diagnosis and treatment. Its core principle lies in using the characteristics of endogenous biomolecules to accurately simulate the physiological microenvironment and biological structure formation mechanisms in vitro, exploring innovative treatment solutions at the molecular level, and providing a brand-new approach to the innovation of biomedical technology.

[0003] Biomolecule condensates are a class of intracellular membrane-free assemblies formed through liquid-liquid phase separation (LLPS), primarily composed of biomolecules such as peptides, proteins, and nucleic acids assembled through non-covalent interactions. These condensates play a crucial role in the life activities of eukaryotic cells, participating in a series of key physiological processes including organelle localization, chromatin organization, maintenance of genome stability, DNA damage response and repair, gene transcription regulation, and cell signal transduction. Notably, the regulation of the dynamic phase transition between liquid and solid states in biomolecule condensates is a core prerequisite for ensuring the normal functioning of many cellular biological activities. Liquid-like condensates, due to their dynamic characteristics, can achieve biochemical functions such as RNA localization regulation and translation process regulation, while solid-like condensates can serve as dormant storage sites for biomolecules, effectively resisting external environmental interference and achieving the enrichment of specific proteins. In addition to irreversible phase transitions, the phase oscillation behavior of biomolecular condensates is also indispensable for the precise regulation of certain physiological functions. For example, in the regulation of circadian rhythms, ATXN2 and ATXN2L proteins can regulate oscillatory translation processes through spatiotemporally specific condensation, thereby achieving precise expression regulation of circadian rhythm-related genes. Based on these characteristics, manipulating the phase transition behavior of biomolecular condensates has become a highly promising technological toolkit for regulating biological functions and developing novel therapeutic strategies.

[0004] Recent studies have confirmed that membraneless organelle-like biomolecule condensates formed by LLPS can achieve highly efficient and selective aggregation of various biomolecules and efficiently perform physiological functions based on their unique microenvironment characteristics. These early membrane-like compartmentalized structures, formed through liquid-liquid phase separation, can precisely divide biomolecules into independent spaces, providing a stable microenvironment for material storage, efficient biochemical reactions, and targeted delivery. Amino acids, as the basic structural units of polypeptides and proteins, through programmed encoding and precise assembly, form bioactive substances with diverse life functions, permeating the entire life process, including information transmission, metabolism, disease development, and aging. More importantly, as endogenous biomolecules, amino acids possess extremely high biocompatibility, well-defined in vivo metabolic pathways, and good biodegradability. These natural advantages make them ideal candidate units for constructing biomimetic materials. Therefore, constructing biomolecule condensates using amino acids as basic building blocks opens up a new direction for the development of biomimetic materials, and is expected to solve key problems such as insufficient biocompatibility and unclear metabolic mechanisms in traditional biomimetic materials.

[0005] Currently, there are no publicly reported technologies related to amino acids independently forming biomolecular condensates. In particular, there is a lack of technical solutions that rely solely on small-molecule amino acids as the only building blocks to form condensates through simple preparation processes, and to apply these condensates to drug delivery carriers, cell storage, or organ storage. Furthermore, there is a lack of publicly disclosed applications of such amino acid-autogenously formed condensates in the fields of biomedicine and chemical engineering. Based on this, this application provides an amino acid-autogenously formed condensate, its preparation method, and its applications. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing amino acid-autogenous aggregates, comprising protective delivery of active biomolecules, as well as cell / tissue / organ storage solutions and poorly soluble drug delivery / sustained-release systems. These aggregates exhibit strong biocompatibility and stability and can be mass-produced.

[0007] In a first aspect, the present invention provides a method for preparing amino acid self-formed aggregates, the steps of which include: dissolving amino acids in water, adjusting the pH to neutral or weakly alkaline using an acid-base adjusting substance, preparing an amino acid buffer solution, adding an organic solvent, and mixing thoroughly to obtain amino acid self-formed aggregates.

[0008] The aggregates are dispersed in a mixture of water and hydrophilic organic solvent in a defined ratio. Amino acids of different concentrations can form aggregates of regular size in the dispersed phase. These aggregates can slowly and spontaneously agglomerate into droplets after standing and disperse with a gentle shake.

[0009] Furthermore, the amino acid includes at least one of arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), glycine (G), and serine (S).

[0010] Furthermore, the volume ratio of water to organic solvent is between 1:100 and 100:1; the concentration of amino acids is between 1 mg / mL and 1000 mg / mL.

[0011] Further, the organic solvent includes at least one of dimethyl sulfoxide, polyethylene glycol PEG200, polyethylene glycol PEG300, polyethylene glycol PEG400, polyethylene glycol PEG500, polyethylene glycol PEG600, polyethylene glycol PEG800, polyethylene glycol PEG1000, N-methylpyrrolidone, methanol, ethanol, isopropanol, and N,N-dimethylformamide.

[0012] Furthermore, the hydrophilic solvent can be used alone or in combination.

[0013] Furthermore, the acid-base regulating substance includes at least one of phosphoric acid, citric acid, sodium hydroxide, and ammonia water.

[0014] Furthermore, the adjustment of pH to neutral or weakly alkaline includes pH 6.5-8.5, preferably pH 6.5-7.5.

[0015] Furthermore, the size of the amino acid-independent aggregates is between 0.05 and 100 μm, preferably between 0.05 and 10 μm.

[0016] Furthermore, the phrase "disperses easily with a light shake" means that after the amino acid aggregates have completely fused, shaking can cause them to redisperse, with their size remaining between 0.05-10 μm.

[0017] The amino acid aggregates obtained by this invention can be synthesized using suitable hydrophilic organic solvents depending on the specific requirements. This preparation method is simple, uses inexpensive and readily available materials, allows for large-scale production, and exhibits good biocompatibility and recyclability. It can be used as a biomimetic drug delivery system, effectively protecting the biological activity of RNA and proteases. Furthermore, it can be applied to the research and development of cell cryopreservation solutions, organ transplantation preservation solutions, enzyme reactors, cosmetic emulsions, and delivery / sustained-release systems for poorly soluble drugs, demonstrating broad application prospects.

[0018] In a preferred embodiment of the present invention, the mass ratio of the amino acid in water is between 0.1% and 90%, and further, the mass ratio of the amino acid raw material is between 30% and 90%, until the amino acid aqueous solution is supersaturated.

[0019] Table 1 shows the preparation schemes for arginine (R) condensates. Table 1 .

[0020] Secondly, the application of self-formed amino acid condensates in the protective delivery system of bioactive macromolecules. Self-formed amino acid condensates can serve as a protective delivery system for bioactive macromolecules without the need for cryopreservation. The bioactive macromolecules are encapsulated or adsorbed within or onto the surface of the condensate. Bioactive macromolecules include, but are not limited to, proteins, enzymes, RNA, DNA, and hormones.

[0021] Protein drugs include, but are not limited to, recombinant proteins such as insulin, interferon, and growth factors, as well as monoclonal antibodies such as trastuzumab, rituximab, bevacizumab, cetuximab, PD-1 / PD-L1 inhibitors, and TNF-α inhibitors.

[0022] Biological enzyme drugs include, but are not limited to, digestive enzymes (including but not limited to trypsin, pancreatic lipase, cellulase, etc.); thrombolytic enzymes and thrombins (including but not limited to streptokinase, urokinase, alteplase, tenecteplase, heparin, coagulation factors, etc.); anti-inflammatory and repair-promoting enzymes (including but not limited to superoxide dismutase (SOD), catalase and cyclooxygenase-2 (COX-2), chymotrypsin, collagenase, etc.); and metabolic coenzymes (including but not limited to coenzyme Q10, etc.).

[0023] RNA-based drugs include, but are not limited to, antisense nucleotide (ASO) drugs (such as Nusinersen / Spinraza, Mipomersen, Volanesorsen, Eteplirsen, Golodirsen, Casimersen, Inotersen / Tegsedi, etc.); interfering RNA drugs (such as Patisiran / Onpattro, Givosiran, Lumasiran, Inclisiran, Vutrisiran, Fitusiran, etc.); mRNA drugs (such as mRNA vaccines, therapeutic mRNAs); circular RNA drugs; tRNA drugs; and self-replicating RNA drugs.

[0024] DNA drugs include, but are not limited to, plasmid DNA (pDNA, such as Zolgensma, Luxturna, etc.); DNA vaccines (such as Papzimeos™, etc.); and DNA aptamers (such as Macugen pilgatani sodium, Izervay, etc.).

[0025] Hormonal drugs include, but are not limited to, adrenocortical hormones (such as glucocorticoids prednisone, dexamethasone, etc., and mineralocorticoids deoxycorticosterone, fludrocortisone); growth hormones (such as growth factors); sex hormones (such as estrogens estradiol, ethinylestradiol, norethindrone, diethylstilbestrol, androgen testosterone, methyltestosterone, and progestins progesterone, dydrogesterone, norethindrone, levonorgestrel); thyroid hormones; and pituitary and hypothalamic hormones (such as recombinant human growth hormone, human chorionic gonadotropin, desmopressin, posterior pituitary extract, etc.).

[0026] Furthermore, the mass ratio of amino acids in the solution of the self-formed amino acid aggregates is between 1% and 90%, preferably between 10% and 50%.

[0027] Furthermore, the mass ratio of the bioactive macromolecular component to the amino acid is between 0.1% and 20%, preferably between 1% and 10%.

[0028] Thirdly, the application of amino acid-derived aggregates in cell cryopreservation solutions. Specifically, isotonicity is adjusted by adding 0.9% NaCl or 5% glucose for cell storage.

[0029] Furthermore, the mass ratio of amino acids in the solution to the solution is between 5% and 20%, preferably between 10% and 15%; the volume ratio of cell suspension to amino acid aggregates is between 1% and 20%.

[0030] Furthermore, the cryopreserved cells include at least one of embryonic stem cells, bone marrow stem cells, primary cells, neutrophils, macrophages, oocytes, sperm, and T cells.

[0031] Fourthly, the application of amino acid-autogenous aggregates in micro-enzyme-catalyzed reaction generators.

[0032] Fifthly, the application of amino acid-independently formed aggregates in the dissolution of poorly soluble drugs involves the following steps: thoroughly grinding and mixing the amino acid and the poorly soluble drug components, adding a certain amount of water, ultrasonically dispersing, adjusting the pH to neutral or weakly alkaline using acid-base adjusting substances (phosphoric acid, citric acid, or sodium hydroxide, ammonia), adding a hydrophilic organic solvent, and thoroughly mixing to obtain amino acid-independently formed aggregates for dissolving poorly soluble drugs.

[0033] The poorly soluble drugs include traditional Chinese medicine monomers, poorly soluble small molecule drugs, and biological pigments.

[0034] Furthermore, this includes single-molecule drugs of traditional Chinese medicine, including but not limited to rhein, emodin, baicalin, tanshinone, paclitaxel, vincristine, ursolic acid, shikonin, aloe-emodin, atractylodes, turmeric alcohol, paeonol, curcumin, resveratrol, etc. Poorly soluble small-molecule drugs (drugs with low oral bioavailability or those that cannot be taken orally), including but not limited to cisplatin, doxorubicin, etc. Biopigments, including but not limited to porphyrin, protoporphyrin, bilirubin, biliverdin, heparin.

[0035] Furthermore, the mass ratio of amino acids in the solution in the self-formed aggregates of amino acids is 1%-90%, preferably 20%-60%.

[0036] Further, the mass ratio of the poorly soluble drug to the amino acid in the solution is 0.1%-40%. Preferably, it is 1%-20%. In a preferred embodiment, the mass ratio of the poorly soluble drug to the amino acid is 1:100.

[0037] Sixthly, the application of amino acid-autogenously formed aggregates in the preservation solution of biological enzymes: An amino acid buffer solution is prepared according to the first aspect. The protease is mixed with the amino acid buffer solution, and then the hydrophilic organic solvent described in the first aspect is added. This solution can be used for short-term storage of the protease at room temperature or long-term storage at 4°C. It can effectively protect the enzyme's activity from environmental interference.

[0038] Furthermore, the mass ratio of amino acids in the solution relative to the solution is 1%-90%, preferably 10%-50%.

[0039] Furthermore, the mass ratio of protease to amino acids is 0.1%-30%, preferably 1%-10%.

[0040] Furthermore, the concentration of the protein preserved in the amino acid-autogenous aggregates is between 100 ng and 4 mg / mL, preferably between 0.1 mg and 2 mg / mL.

[0041] Seventhly, the application of amino acid-autogenously formed aggregates in organ storage. After adjusting to isotonicity according to the method described in the third aspect, tissues and organs such as liver tissue, kidneys, lungs, spleen, skin, and corneas are immersed in prepared amino acid aggregate droplets and placed in an environment of 4°C, which can effectively prolong the in vitro survival time of tissues and organs.

[0042] Furthermore, the organs and tissues include, but are not limited to, liver tissue, kidneys, lungs, spleen, skin, cornea, etc., preferably liver tissue, spleen, skin, and cornea.

[0043] Eighthly, the application of the amino acid-autogenous aggregates in this invention in atomized formulations or subcutaneous injections for medical aesthetics and cosmetics.

[0044] The amino acid condensate described in this invention can form a biomimetic, flowable membrane structure, which effectively promotes the absorption and endocytic uptake of the carried active ingredients by cells. When this amino acid condensate is used as a delivery carrier in transdermal delivery systems for skin repair and skin elasticity-enhancing active ingredients, it can significantly improve the delivery efficiency and skin absorption and utilization of the active ingredients. Simultaneously, some of the amino acids constituting the condensate possess natural antioxidant activity, which can further enhance the anti-aging biological activity of skin cells. Based on these multiple technical advantages, this amino acid condensate can be widely used in the preparation and production of cosmetics.

[0045] Further, the amino acids include, but are not limited to, phenylalanine (F), glutamic acid (E), methionine (M), histidine (H), tyrosine (Y), tryptophan (W), and asparagine (N). Preferably, the amino acids are histidine (H), glutamic acid (E), tryptophan (W), and phenylalanine (F).

[0046] These amino acids can autonomously form aggregates or be added to a buffer solution containing aggregates formed autonomously by the amino acids described in the first aspect.

[0047] Furthermore, the skin repair substances and skin elasticity-enhancing substances include, but are not limited to: collagen, hyaluronic acid, squalene, cholesterol, trehalose, dextran, plant proteins, etc.

[0048] Ninthly, the method for preparing amino acid-autogenous aggregates of the present invention has the following advantages: (1) It has a single component and is easy to prepare.

[0049] (2) The materials are widely available, cheap and easy to obtain.

[0050] (3) The aggregates are stable and can be stored for a long time.

[0051] (4) Amino acids have extremely high biocompatibility and high safety.

[0052] (5) It has a wide range of uses and high potential application value.

[0053] Therefore, the amino acid-autogenous aggregates of the present invention have the following applications: for the protective delivery of easily degraded and inactivated bioactive macromolecular enzymes, RNA, and DNA; for storage solutions for cells / tissues and organs; for room temperature storage of enzyme drugs; as delivery carriers for hydrophobic drugs; for the preparation of cosmetics; and for microenzymatic reactions, etc.

[0054] The present invention has the following beneficial effects: This invention discloses a novel discovery of amino acids capable of autonomously forming aggregates, along with their preparation method and practical applications. Specifically, it relates to a multifunctional amino acid-autogenously formed aggregate / droplet, its preparation method, and its applications, belonging to the field of novel biomaterials. This technology can be applied to the active protective delivery of biopharmaceutical drugs such as DNA, RNA, and enzymes, effectively enhancing drug uptake by cells. It can be applied in drug delivery, microenvironment construction for microenzyme reactions, cell preservation, tissue and organ protection, and medical aesthetics and cosmetics.

[0055] This invention enables the successful preparation of self-formed amino acid condensates / droplets at room temperature through simple control of key parameters such as the solvent system and amino acid concentration. Based on this breakthrough discovery, this invention was completed. The self-formed amino acid condensates / droplets prepared by this invention have broad application prospects. For example, they can serve as high-performance drug delivery systems, especially providing efficient protection for easily degraded biopharmaceuticals such as RNA and proteases, significantly improving their in vivo stability. Simultaneously, they can also be applied to the research and development of cell cryopreservation solutions, organ transplantation preservation solutions, enzyme reactors, cosmetic emulsions, and poorly soluble drug delivery / sustained-release systems. Their application fields cover multiple important areas such as biomedicine, food industry, chemical engineering, and defense technology, possessing extremely high industrialization value. Attached Figure Description

[0056] Figure 1 The image shows the physical structure and microstructure of the arginine aggregates prepared in Example 1.

[0057] Figure 2 This refers to the fusion process of the arginine condensate prepared in Example 1.

[0058] Figure 3 This is a comparison of the arginine aggregates prepared in Example 1 before and after the formation of the arginine solution.

[0059] Figure 4 The particle size of the arginine aggregates prepared in Example 1 is shown.

[0060] Figure 5 The image shows the physical sample and microstructure of the arginine aggregates prepared in Example 2.

[0061] Figure 6 The image shows the physical structure and microstructure of the lysine aggregates prepared in Example 3.

[0062] Figure 7 The image shows the physical sample and microstructure of the aspartic lysine aggregate prepared in Example 4.

[0063] Figure 8 The image shows the physical sample and microstructure of the glutamate aggregates prepared in Example 5.

[0064] Figure 9 The image shows the physical specimen and microstructure of the RU-COA prepared in Example 6.

[0065] Figure 10 This refers to the RU-COA fusion process prepared in Example 6.

[0066] Figure 11 The graph shows a comparison of enzyme activity and urokinase activity in RU-COA prepared in Example 6, as well as a graph showing the enzyme activity reaction rate.

[0067] Figure 12 The activity of RU-COA prepared in Example 6 was compared with that of unprotected urokinase after being stored for up to 10 days, and the enzyme activity reaction rate was plotted.

[0068] Figure 13 The images show the physical sample and microscopic image of the resveratrol aggregate (2 mg / mL) prepared in Example 7, as well as a comparison image in aqueous solution.

[0069] Figure 14 The images show the physical sample and microscopic image of the hesperidin aggregate (2 mg / mL) prepared in Example 7, as well as a comparison image in aqueous solution.

[0070] Figure 15 The images show actual photos of rhein-COA prepared in Example 7 (1, 2, 4 mg / mL) and a comparison image of rhein in water.

[0071] Figure 16 This is a comparison of cell viability before and after cryopreservation in Example 8.

[0072] Figure 17 The images show the physical and microscopic details of the anti-aging medical aesthetic cosmetic subcutaneous injection prepared in Example 10.

[0073] Figure 18 The graph shows the rate of the microenzyme-catalyzed reaction prepared in Example 11.

[0074] Figure 19 Electron micrograph of the nano-needled formulation for pneumonia treatment prepared in Example 12. Detailed Implementation

[0075] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be described in detail below through embodiments. The described solutions are only some embodiments, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the protection scope of the present invention.

[0076] The present invention will be further described below with reference to specific embodiments. The following embodiments are intended to illustrate the present invention and not to further limit the present invention.

[0077] Example 1 This embodiment provides a self-formed amino acid aggregate, specifically an arginine aggregate (R-COA), and the preparation method includes the following steps: Weigh 50, 100, 150, 200, and 300 mg of arginine and add them to 1 mL of deionized water, respectively. Dissolve the arginine by sonication at 20 kHz for 5 min. Adjust the pH to neutral using 100 mM phosphate to prepare amino acid buffers. Add 45%, 40%, 30%, 25%, and 15% PEG300 solvent (by volume) to each buffer solution, respectively. Mix thoroughly to prepare aggregates with different amino acid concentrations.

[0078] Take 10 μL of agglomerate solution containing 15% PEG300, place it on a glass slide, and observe the droplet morphology and fusion characteristics using a phase contrast microscope. Take 1 mL of agglomerate solution, place it in a 1 cm * 1 cm cuvette, and detect the agglomerate particle size using dynamic light scattering (DLS).

[0079] Figure 1 The image shows the physical structure and microstructure of the arginine aggregates prepared in Example 1.

[0080] Figure 2 This refers to the fusion process of the arginine condensate prepared in Example 1.

[0081] Figure 3 This is a comparison of the arginine aggregates prepared in Example 1 before and after the formation of the arginine solution.

[0082] Figure 4 The particle size of the arginine aggregates prepared in Example 1 is shown.

[0083] The results showed that by adjusting the concentration of arginine and the ratio of solvent in the solution, arginine could undergo liquid-liquid phase separation to form aggregates. The size of these aggregates could reach the nanometer scale.

[0084] Example 2 This embodiment provides a self-formed amino acid aggregate, specifically an arginine aggregate (R-COA), and the preparation method includes the following steps: Weigh 200 mg of arginine and add it to 1 mL of deionized water. Dissolve the arginine by sonication at 20 kHz for 5 min. Adjust the pH to neutral using 100 mM phosphoric acid. Add 20%, 15%, 10%, and 5% DMSO solvent (by volume) to the solution, respectively. Mix thoroughly to prepare aggregates with different amino acid concentrations. Take 10 μL of the aggregate solution containing 15% DMSO, place it on a glass slide, and observe the droplet morphology and fusion characteristics using a phase-contrast microscope.

[0085] Figure 5 The image shows the physical sample and microstructure of the arginine aggregates prepared in Example 2.

[0086] Example 3 This embodiment provides a self-formed amino acid aggregate, specifically a lysine aggregate (K-COA), and the preparation method includes the following steps: Weigh 100 mg of lysine and add it to 1 mL of deionized water. Dissolve the lysine by sonication at 20 kHz for 5 min. Adjust the pH to neutral using 100 mM phosphoric acid. Add 25% PEG300 solvent relative to the solution. Mix thoroughly to prepare amino acid-autogenic aggregates. Take 10 μL of the aggregate solution and observe the droplet morphology and fusion characteristics using a phase-contrast microscope.

[0087] Figure 6 The image shows the physical structure and microstructure of the lysine aggregates prepared in Example 3.

[0088] Example 4 This embodiment provides a self-formed amino acid aggregate, specifically an aspartic acid aggregate (D-COA), and the preparation method includes the following steps: Weigh 200 mg of aspartic acid and add it to 1 mL of deionized water. Dissolve the aspartic acid by sonication at 20 kHz for 5 min. Adjust the pH to neutral using 0.1 M sodium hydroxide. Add 10% (by volume) of Tween 20 solvent and mix thoroughly to prepare amino acid-autogenic aggregates. Take 10 μL of the aggregate solution and observe the droplet morphology and fusion characteristics under a microscope.

[0089] Figure 7 The image shows the physical sample and microstructure of the aspartic acid condensate prepared in Example 4.

[0090] Example 5 This embodiment provides a self-formed amino acid aggregate, specifically a glutamic acid aggregate (E-COA), and the preparation method includes the following steps: Weigh 200 mg of glutamic acid and add it to 1 mL of deionized water. Sonicate at 20 kHz for 5 min to dissolve the amino acid. Adjust the pH to neutral using 0.1 M sodium hydroxide. Add 10% (by volume) of Tween 20 solvent. Mix thoroughly to prepare amino acid-autogenic aggregates. Take 10 μL of the aggregate solution and observe the droplet morphology and fusion characteristics under a microscope.

[0091] Figure 8 The image shows the physical sample and microstructure of the glutamic acid aggregate prepared in Example 5.

[0092] Example 6 The preparation method of urokinase loaded onto arginine condensates in this embodiment includes the following steps: According to Example 1, 200 mg of arginine was weighed, the pH was adjusted to neutral with 100 mM phosphate, 2 mg of urokinase was added, and after dissolution, PEG300 at a volume ratio of 20% was added and mixed thoroughly to prepare arginine-urokinase-coagulate (RU-COA).

[0093] As a control, we prepared liposome-loaded urokinase using the following methods: 3.8 g of phospholipids and 1 g of cholesterol were dissolved and mixed in 5 mL of methanol. The methanol was removed in a rotary evaporator to prepare a lipid film. 5 mL of 2 mg / mL urokinase was added to the lipid film. The mixture was stirred thoroughly at 37 °C and then subjected to ultrasonic hydration. Finally, the mixture was homogenized in an ultrasonic homogenizer at 30% power for 5 min to prepare urokinase liposomes (U-LPS).

[0094] 25 μL of S-2251 (3 mmol / L) was used as the fibrinogen chromogenic substrate and added to 100 μL of Tris-HCl buffer (0.05 mmol, pH 7.4) with 20 μL of UPA (0.01 mg / mL). The mixture was incubated at 37°C for 15 min. Then, 25 μL of fibrinogen (0.75 mg / mL) was added, and the optical density (OD) value was immediately recorded minute by minute using a microplate reader for a total of 25 min. The results were then analyzed using the formula... Calculate the rate of enzyme-catalyzed reaction.

[0095] Figure 9 The image shows the physical specimen and microstructure of the RU-COA prepared in Example 6.

[0096] Figure 10 This refers to the RU-COA fusion process prepared in Example 6.

[0097] Figure 11The graph shows a comparison of enzyme activity and urokinase activity in RU-COA prepared in Example 6, as well as a graph showing the enzyme activity reaction rate.

[0098] Figure 12 The activity of RU-COA prepared in Example 6 was compared with that of unprotected urokinase after being stored for up to 10 days, and the enzyme activity reaction rate was plotted.

[0099] Experimental results confirm that amino acid condensates can effectively load urokinase. The compartments formed by LLPS not only effectively isolate external environmental interference and maintain the conformational and activity stability of enzyme molecules, but also provide a dedicated microreaction site for enzyme catalysis, thereby improving overall catalytic efficiency. Compared with traditional liposome-based enzyme delivery carriers, this amino acid condensate system can protect enzyme activity for up to 10 days, and its activity retention effect is significantly better than that of liposomes.

[0100] Example 7 The preparation method of arginine aggregate loaded with hydrophobic traditional Chinese medicine monomers in this embodiment includes the following steps: An amino acid buffer solution was prepared according to Example 1. 2 mg of rhein was added to the buffer solution, and the mixture was ultrasonically broken up using an ultrasonic homogenizer at 30% power. After thorough mixing, PEG300 was added at a volume ratio of 25% relative to the solution. The mixture was then thoroughly mixed to prepare rhein-coagulated aggregates (rhein-COA), which can effectively enhance the solubility of poorly soluble drugs (as shown in Table 2). Solubility determination method: Excess rhein was added to 1 mL of water, and the solution was shaken and incubated at room temperature. The concentration of rhein in the solution was measured using a UV spectrometer at 2 h, 4 h, 8 h, 12 h, and 24 h. The concentration that no longer changed was the solubility concentration of rhein in water.

[0101] Table 2 Solubility of rhein in different solvents .

[0102] In a further embodiment, rhein can be replaced with resveratrol or hesperidin.

[0103] Aggregates of resveratrol and hesperidin were prepared according to the method in Example 7 to enhance their solubility and thus improve their bioavailability.

[0104] Figure 13 The images show the physical sample and microscopic image of the resveratrol aggregate (2 mg / mL) prepared in Example 7, as well as a comparison image in aqueous solution.

[0105] Figure 14 The images show the physical sample and microscopic image of the hesperidin aggregate (2 mg / mL) prepared in Example 7, as well as a comparison image in aqueous solution.

[0106] Figure 15 The images show actual photos of rhein-COA prepared in Example 7 (1, 2, 4 mg / mL) and a comparison image of rhein in water.

[0107] The results showed that rhein had a water solubility of 3.89 μg / mL and a solubility of only 0.1 mg / mL in PEG300. Amino acid aggregates could increase the solubility of rhein to 4 mg / mL. Resveratrol had a water solubility of 0.03 mg / mL, but amino acid aggregates could increase its solubility to 2 mg / mL. Similarly, hesperidin had a water solubility of 6.32 μg / mL, but amino acid aggregates could increase its solubility to 2 mg / mL. These results indicate that amino acid aggregates can significantly improve the water solubility of poorly soluble traditional Chinese medicine monomers, thereby enhancing drug bioavailability.

[0108] Example 8 A method for storing cells using an amino acid-autogenous aggregate as a cell cryopreservation solution includes the following steps: Accurately weigh 100 mg of arginine, adjust the pH to neutral according to the method in Example 1, and prepare arginine condensate cell cryopreservation solution.

[0109] The liver fibroblast suspension was prepared at a ratio of 1×10 6 Cells were added to the cryopreservation solution at the specified concentration, carefully pipetted until homogeneous, and quickly placed in -80°C for cryopreservation. A control experiment was conducted using commercial cell cryopreservation solution (Beyotime C0210). After 2 hours, the cells were removed and revived in 37°C water. Adherent cells were then cultured, and cell viability was assessed using the CCK8 assay kit.

[0110] Figure 16 This is a comparison of cell viability before and after cryopreservation in Example 8. The results show no significant difference in cell viability between the two cell types after thawing. This indicates that amino acid aggregates have the potential to replace traditional cryopreservation solutions.

[0111] Example 9 An amino acid-autogenous aggregate is used as a tissue and organ storage preservative solution, which includes the following steps for tissue and organ storage: After adjusting to isotonicity according to Example 8, PEG300 was added at a volume ratio of 15% relative to the solution to prepare amino acid-independent aggregates. The aggregates were placed at 4°C for 24 hours. After the aggregates formed aggregate droplets, freshly extracted liver tissue was immersed in the droplets and placed at 4°C for transplantation within 4 hours.

[0112] Example 10 An amino acid-independently formed aggregate is used in the preparation of a subcutaneous injection for anti-aging cosmetic medical aesthetics, specifically including the following scheme: An amino acid buffer solution was prepared according to Example 1, and 10 mg / mL hyaluronic acid was added. The solution was adjusted to be isotonic according to the method in Example 4 to prepare agglomerates for subcutaneous injection.

[0113] Figure 17 The images show the physical and microscopic details of the anti-aging medical aesthetic cosmetic subcutaneous injection prepared in Example 10.

[0114] This type of amino acid condensate-based drug delivery system not only significantly enhances the cells' ability to absorb hyaluronic acid, but also protects hyaluronic acid from enzyme-mediated degradation, effectively prolonging the duration of drug action. Simultaneously, the antioxidant activity of the amino acids themselves can synergistically antagonize oxidative stress damage to epidermal cells, thereby inhibiting skin aging.

[0115] Example 11 An amino acid-autogenous condensate is used in a microenzyme reactor, specifically comprising: Arginine aggregates with a concentration of 150 mg / mL were prepared according to Example 1. Oxaloacetic acid (0.25 mM) and reduced coenzyme I (NADH, 0.2 mM) were mixed thoroughly and preheated at 37°C for 5 minutes. Malate dehydrogenase (0.05 U / mL) was added to initiate the reaction, and the mixture was immediately transferred to a quartz cuvette. The absorbance was recorded at 340 nm every 15–30 seconds for 5 minutes. The rate of change of absorbance (ΔA / min) during the initial linear phase of the reaction was used to calculate enzyme activity. The above steps were repeated using Tris-HCl buffer as a control group.

[0116] Figure 18 The graph shows the rate of the microenzyme-catalyzed reaction prepared in Example 11.

[0117] Experimental results show that, compared with the free enzyme system in Tris-HCl buffer, the microenzyme reactor constructed based on amino acid condensates exhibits significantly improved catalytic reaction kinetics, confirming that this microenzyme reactor can effectively enhance enzyme catalytic efficiency. This phenomenon can be attributed to the fact that amino acid condensates provide relatively independent and advantageous microcompartments for enzyme catalytic reactions, reducing the interference of external environmental factors on enzyme molecular conformation and activity through spatial confinement effects, thereby enhancing enzyme catalytic performance.

[0118] Example 12 A method for preparing a nano-nebulized formulation of amino acid-autogenous aggregates for pneumonia treatment, specifically comprising: Prepare 2 mL of 100 mg lysine buffer according to Example 1, add 5 nmol of HOXB7 siRNA, and mix thoroughly. Then prepare lysine aggregates according to the method in Example 1. Check the particle size of the mixture using a nanoparticle size analyzer; it should be ≤10 μm. Prepare a nano-atomized formulation, and shake thoroughly before use to ensure complete dispersion.

[0119] Figure 19 Electron micrograph of the nano-needled formulation for pneumonia treatment prepared in Example 12.

[0120] This amino acid condensate-based nano-atomized formulation can encapsulate and protect interfering RNA molecules from nuclease degradation through self-assembled nanocompartments, achieving stable delivery to the lungs. At the same time, the nanoscale condensates can significantly improve the efficiency of intracellular RNA delivery and release through endocytosis, thereby enhancing the therapeutic effect of the drug.

[0121] The applicant declares that the present invention is illustrated by the above embodiments to demonstrate the product and detailed preparation method of the present invention, but the present invention is not limited to the above product and detailed preparation method, that is, it does not mean that the present invention must rely on the above product and detailed preparation method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of excipients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0122] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention. These simple modifications all fall within the protection scope of the present invention.

[0123] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0124] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for preparing amino acid-autogenic aggregates, characterized in that, The steps include: After dissolving amino acids in water, the pH is adjusted to neutral or weakly alkaline using an acid-base regulator to prepare an amino acid buffer solution. An organic solvent is then added and thoroughly mixed to obtain amino acid aggregates that form autonomously.

2. The method for preparing an amino acid self-formed aggregate according to claim 1, characterized in that, The amino acid includes at least one of arginine, histidine, lysine, aspartic acid, glutamic acid, glycine, and serine.

3. The method for preparing an amino acid self-formed aggregate according to claim 1, characterized in that, The volume ratio of water to organic solvent is between 1:100 and 100:1; the concentration of amino acids is between 1 mg / mL and 1000 mg / mL.

4. The method for preparing an amino acid self-formed aggregate according to claim 1, characterized in that, The acid-base regulating substance includes at least one of phosphoric acid, citric acid, sodium hydroxide, and ammonia water.

5. The method for preparing an amino acid self-formed aggregate according to claim 1, characterized in that, The organic solvent includes at least one of dimethyl sulfoxide, polyethylene glycol PEG200, polyethylene glycol PEG300, polyethylene glycol PEG400, polyethylene glycol PEG500, polyethylene glycol PEG600, polyethylene glycol PEG800, polyethylene glycol PEG1000, N-methylpyrrolidone, methanol, ethanol, isopropanol, and N,N-dimethylformamide.

6. The application of the amino acid autonomously formed aggregates prepared by the preparation method according to any one of claims 1-5 in a protective delivery system of bioactive macromolecules.

7. The application according to claim 6, characterized in that, The bioactive macromolecules include at least one of the following: proteins, enzymes, RNA, DNA, and hormones.

8. The application of the amino acid self-formed aggregates prepared by the preparation method according to any one of claims 1-5 in the dissolution of poorly soluble drugs.

9. The application according to claim 8, characterized in that, The poorly soluble drugs include at least one of the following: traditional Chinese medicine monomers, poorly soluble small molecule drugs, and biopigments.

10. The use of amino acid-independently formed aggregates prepared by the preparation method according to any one of claims 1-5 in cell cryopreservation solutions, microenzyme reactors, tissue cryopreservation solutions, atomized preparations, or subcutaneous injections in medical aesthetic cosmetics.

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