Method for inducing amino acid and polypeptide to be self-assembled into nanostructure material and application

Abstract

The invention provides a method for inducing amino acid and polypeptide to be self-assembled into a nano-structure material and application, and belongs to the technical field of nano-materials and biomolecule self-assembled materials. According to the method, amino acid and / or polypeptide are / is taken as a substrate, self-assembly is effectively realized in a system containing periodate ions to form a stable nano-structure material, the nano-structure material is in the shape of spherical nano-particles, filamentous nano-fibers or flaky nano-structures and the like, and the product is uniform in size and relatively high in quality. Wherein the diameter of the spherical nanoparticles of the nanostructure material obtained under the condition of neutral periodate is 5-200 nm, and the diameter of the filamentous nanofibers is 10-50 nm. The periodate ion induced self-assembly method is adopted, the process is simple, the condition is mild, the application range is wide, and the nano-structure material obtained through self-assembly has special physicochemical properties, excellent biocompatibility and biodegradability and can be widely applied to the fields of industry, agriculture, energy, medicine and the like.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials and biomolecular self-assembly materials technology, specifically to a method and application for inducing amino acids and peptides to self-assemble into nanostructure materials. Background Technology

[0002] Various natural molecules, including proteins, glycans, and nucleic acids, can self-assemble into different nanostructured materials, which exhibit high biocompatibility and biodegradability. However, the self-assembly of nanostructured materials from natural molecules such as proteins, glycans, and nucleic acids faces limitations such as high preparation costs, polydispersity, and difficulty in purification.

[0003] Peptides and amino acids exhibit remarkable self-assembly capabilities, and their nanostructured materials also possess potential applications due to their high biocompatibility and biodegradability. Amino acids and their derivatives can spontaneously assemble into ordered nanostructures through non-covalent interactions, including hydrogen bonds, hydrophobic interactions, van der Waals forces, ionic bonds, π-π stacking, and electrostatic interactions. The synergistic effects of these forces and bonds promote the spontaneous formation of complex and ordered polypeptide sequences and higher-order structures, resulting in novel nanostructures with multiple functions. The self-assembly of peptides and amino acids offers advantages such as low synthesis cost, simple modeling, excellent biocompatibility, and in vivo biodegradability, making it more environmentally friendly and industrially friendly. Furthermore, the potential for amino acid functionalization and the rich diversity of side chains allow for a wide variety of structure types obtained through self-assembly, providing abundant options for further development.

[0004] Research on induced amino acid self-assembly has shown a significant upward trend in overall development, especially in applications at the intersection of materials science and biochemistry. Various methods exist for preparing amino acid nanomaterials, mainly including solvent-induced methods, pH-controlled methods, template methods, and enzymatic methods. Solvent-induced methods drive the assembly of amino acid molecules by changing the polarity or solubility of the solvent, but often require large amounts of organic solvents, posing environmental pollution problems. pH-controlled methods utilize the change in the charge state of amino acids at different pH values ​​to achieve assembly, but the reaction conditions are harsh and controllability is poor. Template methods guide amino acid assembly by introducing template molecules (such as nanoparticles or polymers), but template removal is complex and may destroy the assembled structure. Enzymatic methods utilize the catalytic action of enzymes to achieve the directed assembly of amino acids, offering advantages such as mild conditions and high selectivity, but are costly and have limited efficiency. In practical applications, how to precisely control the size, morphology, and function of these nanostructures, and how to achieve the stability and consistency of these self-assembly processes on an industrial scale, remain pressing challenges. Therefore, developing a green, efficient, and controllable method for amino acid self-assembly has become an important research direction.

[0005] Periodate oxidation is a green chemistry method with high selectivity and controllability, showing great potential in biomolecular modification and functionalization research. Periodate can selectively oxidize specific functional groups (such as hydroxyl and amino groups) in amino acids to generate aldehyde or carboxyl groups, thereby altering the chemical properties and interactions of the amino acids. The periodate oxidation reaction is carried out under mild conditions in aqueous solution, avoiding the use of organic solvents and aligning with the principles of green chemistry. Therefore, periodate oxidation provides a new pathway for amino acid self-assembly. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a method and application for inducing the self-assembly of amino acids and peptides into nanostructured materials by periodate ion oxidation. This provides a novel approach and pathway for the self-assembly of amino acids and peptides, further promoting their application in the preparation of functional materials.

[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for inducing amino acids and peptides to self-assemble into nanostructured materials, comprising the following steps: reacting amino acids and / or peptides in a system containing periodate ions to obtain nanostructured materials.

[0009] This invention uses amino acids and / or peptides as raw materials. In a system containing periodate ions, the oxidative functional groups of the amino acids and / or peptides are modified, thereby inducing their self-assembly to form nanostructured materials. The morphologies of these nanostructured materials include spherical nanoparticles, filamentous nanofibers, or sheet-like nanostructures, expanding the diversity of morphological forms of self-assembled nanostructured materials. The method of this invention is simple, mild, and widely applicable. The self-assembled nanostructured materials possess unique physicochemical properties, excellent biocompatibility, and biodegradability, enabling their widespread application in various fields such as industry, agriculture, energy, and medicine.

[0010] Furthermore, the system containing periodate ions is obtained by dissolving periodic acid and / or periodate in water.

[0011] Furthermore, after dissolving periodic acid and / or periodate in water, the pH of the system is adjusted using an alkaline solution, including but not limited to lithium hydroxide (LiOH), sodium hydroxide (NaOH), or potassium hydroxide (KOH).

[0012] Furthermore, the pH of the system is adjusted to neutral using lithium hydroxide solution; or the pH of the system is adjusted to less than 5 using sodium hydroxide solution; or the pH of the system is adjusted to greater than 9 using potassium hydroxide solution.

[0013] Furthermore, amino acids and / or peptides are reacted in a neutral periodate system to obtain nanostructured materials; the neutral periodate system is formed by dissolving periodic acid and / or periodate in water and adjusting the pH of the system to neutral using lithium hydroxide.

[0014] Furthermore, the periodate includes, but is not limited to, sodium periodate and / or potassium periodate.

[0015] Further, the mass ratio of the amino acids and / or polypeptides, periodic acid and / or periodate, and water is (1~4):(1~4):(25~120).

[0016] Furthermore, the reaction is carried out in a light-protected environment at a temperature of 15-35 °C for a time of 24-72 h.

[0017] Furthermore, the amino acids include, but are not limited to, natural amino acids and / or their isomers, wherein the natural amino acids include, but are not limited to, at least one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; and the polypeptide is a polypeptide composed of multiple natural amino acids and / or their isomers.

[0018] Furthermore, after the reaction was completed, a small amount of ethylene glycol was added to neutralize the unreacted periodate ions, and the precipitate was removed by centrifugation, dialyzed, and dried to obtain the nanostructured material.

[0019] Secondly, the present invention provides nanostructured materials prepared by the method described above.

[0020] Furthermore, the morphology of the nanostructured materials includes, but is not limited to, spherical nanoparticles, filamentous nanofibers, or sheet-like nanostructures. Depending on the type of amino acid and the reaction conditions, the resulting nanostructured materials exhibit different structural morphologies and sizes; among them, nanostructured materials prepared under neutral periodate conditions have spherical nanoparticles with diameters of 5–200 nm and filamentous nanofibers with diameters of 10–50 nm. For example, the morphology of nanostructured materials formed by the self-assembly of amino acids containing benzene rings (such as phenylalanine and tryptophan) and amino acids containing hydroxyl groups (such as serine) induced by neutral periodate is relatively stable, exhibiting spherical nanostructures with a diameter of 50-100 nm. The self-assembly products of amino acids containing alkyl groups (such as alanine and proline) induced by neutral periodate can be controlled by reaction conditions, and the products can be controlled according to applicability to be filamentous nanofibers with a diameter of 10-50 nm or spherical nanoparticles with a diameter of 20-200 nm. The morphology of nanostructured materials formed by the self-assembly of asparagine or cystine induced by neutral periodate is filamentous nanofibers with a diameter of 20-50 nm, which are less affected by conditions and have a stable structure. The morphology of nanostructures formed by the self-assembly of amino acids or peptides containing thiol groups (such as cysteine ​​and glutathione) induced by neutral periodate is mostly irregular sheet-like nanostructures with obvious internal lattice structures and β-sheets with a spacing of about 0.36 nm.

[0021] Thirdly, this invention provides applications of the aforementioned nanostructured materials in the fields of biomedicine, food, daily chemicals, agriculture, or energy storage. For example, by selecting different amino acids and / or peptides to induce self-assembly into targeted nanostructured materials in a system containing periodate ions, without the use of toxic reagents in the preparation process and products, medical nanostructured materials with specific chemical activities (such as targeting and drug loading capacity) can be obtained, suitable for the biomedical field; by selecting targeted nanostructured materials to replace traditional emulsifiers for encapsulating active substances such as vitamins, polyphenols, and hyaluronic acid, stable Pickering emulsions can be prepared, improving the utilization rate of these active substances in food or cosmetics; by selecting targeted nanostructured materials to encapsulate pesticides, the sustained-release effect of pesticides can be improved, reducing environmental runoff and toxicity.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] 1. This invention provides a method for oxidatively inducing the self-assembly of amino acids and / or peptides to form nanostructured materials in a system containing periodate ions. The preparation process is simple, the conditions are mild, and it is widely applicable. The self-assembled nanostructured materials have special physicochemical properties, excellent biocompatibility and biodegradability, and can be widely used in various fields such as industry, agriculture, energy and medicine.

[0024] 2. The method provided by this invention is highly efficient and controllable. Depending on the requirements, different amino acids / peptides can be used for self-assembly in different systems containing periodate ions to obtain products with different structural morphologies and sizes, suitable for various scenarios. Furthermore, the method of this invention also has functional designability. Depending on the applicable scenario, by selecting different amino acids and / or peptides for self-assembly into targeted nanostructure materials, medical nanomaterials with specific chemical activities (such as targeting and drug loading capacity), Pickering emulsions encapsulating active substances in micelles, or sustained-release pesticides encapsulated in nanocapsules can be obtained. Attached Figure Description

[0025] Figure 1 SEM images of nanostructured materials obtained by self-assembly of different amino acids / peptides induced by neutral periodate oxidation;

[0026] Figure 2 TEM image of alanine nanostructured material obtained by self-assembly induced by neutral periodate oxidation;

[0027] Figure 3 TEM image of nanostructured materials obtained by phenylalanine through periodate oxidation-induced self-assembly;

[0028] Figure 4 TEM image of nanostructured materials obtained by self-assembly of tryptophan induced by oxidation of neutral periodate;

[0029] Figure 5 TEM image of nanostructured materials obtained by proline self-assembly induced by neutral periodate oxidation;

[0030] Figure 6 TEM image of nanostructured materials obtained by serine through neutral periodate oxidation-induced self-assembly;

[0031] Figure 7 High-resolution TEM image of nanostructured materials obtained by self-assembly of cysteine ​​and glutathione via neutral periodate oxidation;

[0032] Figure 8 TEM image of nanostructured materials obtained by self-assembly of asparagine and cystine via neutral periodate oxidation;

[0033] Figure 9 FTIR images of nanostructured materials obtained by self-assembly induced by neutral periodate oxidation of different amino acids / peptides;

[0034] Figure 10 Image of a Pickering emulsion containing nanostructured materials obtained by self-assembly of different amino acids / peptides through neutral periodate oxidation;

[0035] Figure 11 The results of cytotoxicity testing of the nanostructured materials of this invention;

[0036] Figure 12 Comparison of particle size distribution of the products obtained from Examples 3, 5, 6 and Examples 10, 11, 12 after 1 day of reaction;

[0037] Figure 13 The particle size distribution of the products obtained from phenylalanine as a raw material in Examples 10, 11, and 12 at different reaction times is shown in the comparison diagram.

[0038] Figure 14 The particle size distribution of the products obtained from the reaction of tryptophan as a raw material at different times in Examples 10, 11 and 12 is compared.

[0039] Figure 15 The image shows a comparison of the particle size distribution of the products obtained from the reaction of serine as a raw material at different times in Examples 10, 11, and 12. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] This invention provides a method for inducing the self-assembly of amino acids and peptides into nanostructured materials, comprising the following steps: reacting amino acids and / or peptides in a system containing periodate ions to obtain nanostructured materials.

[0042] In some examples, the system containing periodate ions is obtained by dissolving periodic acid and / or periodate in water, or by dissolving periodic acid and / or periodate in water and then adjusting the pH of the system with an alkaline solution, including but not limited to lithium hydroxide (LiOH), sodium hydroxide (NaOH) or potassium hydroxide (KOH).

[0043] In some examples, lithium hydroxide solution is used to adjust the pH of the system to neutral; or sodium hydroxide solution is used to adjust the pH of the system to less than 5; or potassium hydroxide solution is used to adjust the pH of the system to greater than 9.

[0044] In some examples, the periodate includes, but is not limited to, sodium periodate and / or potassium periodate.

[0045] In some examples, the mass ratio of the amino acids and / or polypeptides, periodic acid, and water is (1~4):(1~4):(25~120).

[0046] In some examples, the reaction is carried out in the dark at a temperature of 15–35 °C for a time of 24–72 h.

[0047] In some examples, the amino acid includes, but is not limited to, natural amino acids and / or their isomers, wherein the natural amino acid includes, but is not limited to, at least one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; and the polypeptide is a polypeptide composed of multiple natural amino acids and / or their isomers.

[0048] In the following specific embodiments, the amino acids used—alanine (Ala), proline (Pro), cysteine ​​(Cys), phenylalanine (Phe), tryptophan (Trp), serine (Ser), asparagine (Asn) and the polypeptides used—cystine ((Cys)2) and glutathione (GSH), as well as the periodic acid, lithium hydroxide, and ethylene glycol used—were all purchased from Maclean's Reagent Company.

[0049] Examples 1-9

[0050] Nanostructured materials formed by the self-assembly of amino acids or peptides

[0051] Weigh 1-4 g of amino acids / peptides (see Table 1 for details) and 1-4 g of periodic acid and dissolve them in 25 mL of deionized water. Adjust the pH to 7 using LiOH solution. Stir continuously at 800 rpm for 1-3 days at room temperature in the dark. After the reaction is complete, add a small amount of ethylene glycol to neutralize any unreacted periodate. Centrifuge the reaction solution at 7000×g for 10 min to remove incompletely reacted substrate. Transfer the supernatant to a 3500 Da dialysis membrane for dialyzing, changing the water multiple times until the conductivity is close to that of pure water. Freeze-dry to obtain the nanostructured material.

[0052] Table 1: Amino acids / peptides used in Examples 1-9

[0053]

[0054] In the embodiments of this invention, the self-assembly characteristics of nine different amino acids or peptides under neutral periodate oxidation conditions were studied in detail. The subjects of the study included alanine (Ala), proline (Pro), cysteine ​​(Cys), phenylalanine (Phe), tryptophan (Trp), serine (Ser), asparagine (Asn), cystine ((Cys)2), and glutathione (GSH).

[0055] The morphology of the nanostructured materials obtained in Examples 1-9 was characterized by scanning electron microscopy (SEM), and the results are as follows: Figure 1 As shown, most amino acid self-assembled nanostructures exhibit typical spherical particle morphology, with significant differences in their size distribution. Specifically, the self-assembled products of alanine, proline, cysteine, tryptophan, asparagine, cystine, and glutathione are relatively small, mainly concentrated in the 20–50 nm range. This nanoscale size characteristic may be related to the weak intermolecular interactions of these amino acids. Asparagine, in addition to its spherical particle morphology, also exhibits lamellar and filamentous structures, showing a more complex morphology. In contrast, the nanostructures obtained by self-assembly of phenylalanine or serine not only maintain a regular spherical morphology but also have significantly larger sizes than other amino acids, with average diameters of 90.4 ± 31.4 nm and 79.9 ± 24.4 nm, respectively. This size difference may be related to the π-π stacking of the benzene ring in the phenylalanine molecule and the hydrogen bonding interaction of the hydroxyl group in the serine molecule; these strong intermolecular forces promote the formation of larger self-assembled structures.

[0056] The morphology of the nanostructured material obtained in Example 1 was characterized by transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown, the self-assembly behavior of alanine is highly dependent on experimental conditions. Under a fixed reaction time of 3 days, the mass ratio of alanine to periodic acid exhibits a significant concentration-dependent effect on morphology regulation. Specifically, when the mass ratio of alanine to periodic acid is 1 g:1 g, the resulting nanostructured materials are mainly filamentous with a diameter of 20–50 nm; when the mass ratio of alanine to periodic acid is increased to 1 g:2 g to 1 g:4 g, the resulting nanostructured materials are mainly spherical with a diameter of 20–100 nm.

[0057] When the amino acid is phenylalanine, the effects of the amount of periodic acid and the reaction time on the morphology of the self-assembled nanostructured materials are as follows: Figure 3As shown in the figure, under different experimental conditions, neutral periodate can effectively induce phenylalanine to self-assemble into spherical nanoparticles with a "shell-core" structure. Under fixed reaction time conditions, with changes in the mass ratio of phenylalanine to periodate (1 g:2 g ~ 1 g:4 g), phenylalanine can self-assemble into spherical particles, and these spherical particles are coated with a ring-like morphology, forming a typical "shell-core" structure. Similarly, under fixed periodate dosage conditions, similar morphological characteristics were observed when the reaction time was changed (1-3 days). The resulting nanostructured particles all exhibited a spherical "shell-core" structure, but their size distribution varied, mainly in the range of 50-250 nm. This unique "shell-core" structure may originate from the fact that during the self-assembly of phenylalanine, the fully reacted molecules form the nanosphere "core" under the oxidation of periodate, and the partially oxidized phenylalanine molecules further assemble on the surface of the sphere "core" to form the outer shell structure.

[0058] When the amino acid is tryptophan, the effects of the amount of periodic acid and reaction time on the morphology of the self-assembled nanostructured materials are as follows: Figure 4 As shown in the figure, periodate effectively induced tryptophan self-assembly into spherical nanoparticles under different experimental conditions. Specifically, tryptophan self-assembled into spherical structures when the mass ratio of tryptophan to periodate varied from 1 g:2 g to 1 g:4 g; similarly, the same morphological characteristics were observed within a reaction time range of 1–3 days. Notably, despite the variations in experimental conditions, the size distribution of all samples was relatively uniform, mainly within the range of 50–80 nm. This stable spherical morphology and narrow size distribution may be related to the unique structural features of the tryptophan molecule. The indole ring structure in its molecule may provide directional guidance for the self-assembly process through π-π stacking interactions, while the amino and carboxyl groups stabilize the nanoparticle structure through hydrogen bonding and electrostatic interactions.

[0059] When the amino acid is proline, the effects of the amount of periodic acid and the reaction time on the morphology of the self-assembled nanostructured materials are as follows: Figure 5As shown in the figure, the self-assembly behavior of proline is highly dependent on experimental conditions. Proline and periodic acid can only effectively form spherical nanoparticles under appropriate mass ratios and reaction times. Specifically, when the mass ratio of proline to periodic acid is 1 g:3 g, the reaction time significantly affects the morphology evolution. During the reaction period of 1-2 days, the products are mainly filamentous and irregular substances; when the reaction time is extended to 3 days, uniformly shaped spherical nanoparticles with a size of 39.6 ± 7.9 nm can be formed. This transformation process may be due to the intermolecular interactions not yet reaching equilibrium within a short time, leading to the formation of irregular intermediate structures. Sufficient reaction time is beneficial for molecular rearrangement and energy minimization, thus forming a more stable spherical structure. Under the condition of a fixed reaction time of 3 days, the amount of periodic acid shows a significant concentration dependence on the morphology regulation of the nanostructured materials. When the mass ratio of proline to periodic acid varies within the range of 1 g:2 g to 1 g:4 g, although the formation of spherical structures can be induced in all cases, the morphological purity varies significantly. Specifically, when the mass ratio of proline to periodic acid was 1 g:2 g, the products were mainly filamentous, irregular spherical, and disordered. When the mass ratio of proline to periodic acid increased to 1 g:3 g and 1 g:4 g, spherical nanoparticles became the main products. Notably, the size of the spherical particles decreased with increasing proline to periodic acid mass ratio. At a mass ratio of 1 g:3 g, the particle size was relatively large (39.6 ± 7.9 nm), while at a mass ratio of 1 g:4 g, the particle size decreased significantly to 5.2 ± 1.9 nm. This size change may be due to the faster nucleation at higher oxidant inputs, resulting in the formation of more but smaller nanoparticles.

[0060] When the amino acid is serine, the effect of periodic acid dosage on the morphology of the self-assembled nanostructure material is as follows: Figure 6 As shown in the figure, under different periodsic acid concentrations, serine can be effectively induced to self-assemble into regularly shaped spherical nanoparticles. Specifically, with a fixed reaction time of 1 day, when the mass ratio of serine to periodic acid varied within the range of 1 g:1 g to 1 g:4 g, serine could self-assemble into spherical structures with a relatively uniform particle size distribution, mainly in the range of 50–100 nm. This morphological characteristic, which is relatively insensitive to periodate concentration, may be related to the unique structural characteristics of the serine molecule. The hydroxyl groups (-OH) in its molecule may provide stable interaction forces for the self-assembly process through an intermolecular hydrogen bond network, thus maintaining a similar assembly path under different oxidant concentrations.

[0061] The microstructure of the self-assembled nanostructured material of cysteine ​​and glutathione was characterized in detail by high-resolution transmission electron microscopy (HR-TEM), and the results are as follows: Figure 7 As shown, the nanostructured material formed by cysteine ​​self-assembly exhibits distinct lattice fringes with a lattice spacing of 0.36 ± 0.03 nm. Similarly, the nanostructured material formed by glutathione self-assembly also shows a clear lattice structure under high magnification, with a lattice spacing of 0.36 ± 0.02 nm, consistent with the lattice of cysteine. This consistent lattice spacing characteristic suggests that the self-assembly products of cysteine ​​and glutathione may have similar ordered structures. Notably, the observed 0.36 nm lattice spacing closely matches the characteristic spacing of β-sheet structures, indicating that the core region of the nanostructured material formed by cysteine ​​or glutathione self-assembly may be mainly composed of β-sheet structures. The formation of this structural feature may be closely related to the thiol groups (-SH) and disulfide bonds (-SS-) in the cysteine ​​and glutathione molecules. Under the oxidation of neutral periodate, stable β-sheet structures may form between molecules, thus producing regular lattice fringes.

[0062] When the amino acid / peptide is asparagine / cystine, the effect of reaction time on its self-assembly behavior is as follows: Figure 8 As shown in the figure, within a reaction time range of 1–3 days, both asparagine and cysteine ​​can be effectively induced by neutral periodate to self-assemble into filamentous nanofiber structures with diameters of 10–50 nm. It is noteworthy that although variations in reaction time affect the size and morphological integrity of the filaments, they do not alter the fundamental characteristics of their one-dimensional structure.

[0063] The nanostructured materials of Examples 1-9 were analyzed by Fourier transform infrared spectroscopy (FTIR), and the results are as follows: Figure 9 (The control group in the figure refers to the amino acids / peptides before the reaction.) The figure shows that the oxidative self-assembly of neutral periodate significantly alters the chemical structure of amino acids or peptides. Taking alanine as an example, the infrared spectrum of alanine is in the range of 600-3100 cm⁻¹. -1 Multiple characteristic absorption peaks were observed within the range, corresponding to CH bonds (2800-3100 cm⁻¹). -1 C=O bond (1700 cm) -1 ), NH bond (1500-1600 cm) -1 The vibrations of functional groups such as ) and ). After self-assembly into nanostructured materials, their spectral characteristics change significantly, 1392 cm -1 1062 cm -1 and 852 cm -1The new peak at 1600 cm⁻¹ indicates the breaking of the CH bond and the formation of new CO or CN bonds, suggesting that the oxidative self-assembly of neutral periodate involves decarboxylation, deamination, and hydroxylation reactions. Phenylalanine and tryptophan, as representative amino acids containing benzene rings, show in their infrared spectroscopy that the oxidative self-assembly of neutral periodate mainly affects the alkyl chain, leading to the breaking of the CH bond and the formation of the CO bond, while the benzene ring and indole ring structures remain relatively stable. After the oxidative self-assembly of phenylalanine via neutral periodate, the peak at 1600 cm⁻¹... -1 and 700 cm -1 The characteristic peaks at these locations correspond to newly formed conjugated double bonds or amide bonds and the retention of CH bonds in the benzene ring, respectively. After the self-assembly of tryptophan via neutral periodate oxidation, the peak at 1610 cm⁻¹... -1 and 740 cm -1 The characteristic peaks at this location further support the stability of the aromatic ring and the oxidation reaction of the alkyl chain. Serine, as a representative of hydroxyl-containing amino acids, exhibits an infrared spectrum at 1635 cm⁻¹ after its self-assembly via neutral periodate oxidation. -1 and 1095 cm -1 The appearance of a new peak indicates the formation of C=O or CN bonds, suggesting that the oxidative self-assembly of neutral periodate involves the oxidation, decarboxylation, and dehydration of hydroxyl groups. Cysteine, cysteine, and glutathione, as representatives containing thiol groups and disulfide bonds, showed in their infrared spectroscopy that the oxidative self-assembly of neutral periodate preferentially involves sulfur atoms, leading to the breaking of CS and SS bonds and the formation of new CO or CN bonds. After the oxidative self-assembly of neutral periodate, cysteine ​​showed a peak at 1608 cm⁻¹. -1 and 1180 cm -1 The characteristic peak at 1652 cm⁻¹ indicates the formation of C=C or C=O bonds; after cystine undergoes self-assembly via neutral periodate oxidation, the peak at 1652 cm⁻¹... -1 and 1161 cm -1 The characteristic peak at 1650 cm⁻¹ further supports the preferential oxidation of sulfur atoms; after glutathione undergoes self-assembly via neutral periodate oxidation, the peak at 1650 cm⁻¹... -1 and 1539 cm -1 The characteristic peak at that location indicates the formation of amide bonds.

[0064] In summary, the neutral periodate oxidation self-assembly process of this invention significantly alters the chemical structure of the original amino acids and peptides. Under the action of neutral periodate, the amino and carboxyl groups in amino acid and peptide molecules are converted into aldehyde groups, thereby promoting the self-assembly of amino acids and peptides to form stable nanostructure materials. In addition, the CS and SS bonds in amino acids or peptides will break and form new CO, CN, or C=C bonds under the action of neutral periodate, thereby promoting the self-assembly of amino acids and peptides to form stable nanostructure materials.

[0065] Performance testing of nanostructured materials in Examples 1-9

[0066] The nanostructured materials in Examples 1-9 were prepared into solutions of 10 g / L, and mixed with n-dodecane at a volume fraction of 70%-75%. The mixtures were homogenized at 9000 rpm for 1 min using a high-speed mixer, and then homogenized at 11550 rpm for 1 min to obtain Pickering emulsions. The total volume of each sample was set to 6 mL.

[0067] Figure 10 The image shows a physical representation of the resulting Pickering emulsion. As can be seen from the image, the nanostructured materials in Examples 1-9 can all homogeneously form stable emulsions with the oil phase, indicating that the nanostructured materials of this invention possess amphiphilic (hydrophilic and lipophilic) properties of both amino acids and peptides. This provides a theoretical basis for their application in the emulsion field. In subsequent practical applications, the target nanostructured materials can be selected to replace traditional synthetic emulsifiers, encapsulating active substances such as vitamins, polyphenols, and hyaluronic acid to form stable Pickering emulsions for use in the food or cosmetic fields.

[0068] The nanostructured materials of Examples 5, 7, and 9 were subjected to cytotoxicity analysis. Normal HUVEC (venous endothelial cells) were selected as the research subject, and the cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured at 37 °C and 5% CO2 for 24 h until adherence. During the culture, the cells were divided into an experimental group and a control group. The culture medium used in the experimental group contained the nanostructured material of this invention (the final concentration of the nanostructured material in the culture medium was 1 μg / mL), while the culture medium used in the control group did not contain the nanostructured material of this invention. After the culture was completed, the cells were stained with Calcein-AM (green, live cells), observed, and photographed.

[0069] The results are as follows Figure 11 As shown, compared with the control group, the survival rate of HUVECs in the experimental group was higher (greater than 90%), indicating that the nanostructured material of the present invention has good biocompatibility and no significant biotoxicity, which provides a basis for the application of the nanostructured material in the field of biomedicine.

[0070] Example 10

[0071] Weigh 1 g of phenylalanine / tryptophan / serine and 3 g of periodic acid and dissolve them in 50 mL of deionized water (pH 1.5-2). React at 800 rpm for 1-7 days under dark conditions at room temperature. Add a small amount of ethylene glycol to neutralize any unreacted periodic acid. Centrifuge the reaction solution at 7000×g for 10 min to remove incompletely reacted substrate. Transfer the supernatant to a 3500 Da dialysis membrane for dialyzing, changing the water multiple times until the conductivity approaches that of pure water. Freeze-dry to obtain the nanostructured material.

[0072] Example 11

[0073] Weigh 1 g of phenylalanine / tryptophan / serine and 3 g of sodium periodate and dissolve them in 50 mL of deionized water (pH 4-5). React at 800 rpm for 1-7 days under dark conditions at room temperature. Add a small amount of ethylene glycol to neutralize any unreacted periodic acid. Centrifuge the reaction solution at 7000×g for 10 min to remove incompletely reacted substrate. Transfer the supernatant to a 3500 Da dialysis membrane for dialyzing, changing the water multiple times until the conductivity approaches that of pure water. Freeze-dry to obtain the nanostructured material.

[0074] Example 12

[0075] Weigh 1 g of phenylalanine / tryptophan / serine and 3 g of periodic acid and dissolve them in 50 mL of deionized water. Add a small amount of KOH solution to adjust the pH to 10. React at 800 rpm for 1-7 days under dark conditions at room temperature. Add a small amount of ethylene glycol to neutralize any unreacted periodic acid. Centrifuge the reaction solution at 7000×g for 10 min to remove incompletely reacted substrate. Transfer the supernatant to a 3500 Da dialysis membrane for dialyzing, changing the water multiple times until the conductivity is close to that of pure water. Freeze-dry to obtain the nanostructured material.

[0076] The results showed that, compared to the neutral periodate systems of Examples 1-9, the efficiency of inducing self-assembly of amino acids and / or peptides using pure periodic acid (Example 10), pure sodium periodate (Example 11), or adjusting the pH of the system to 10 with potassium hydroxide (Example 12) was lower. Under the same conditions, the overall reaction efficiency was: Li + >K + ≈Na + >H + .

[0077] This invention uses the amino acids (phenylalanine, tryptophan, and serine) of spherical products as examples, and uses DLS to determine the particle size distribution of products obtained under different cation periodate conditions. Examples 3, 5, and 6 can be considered as using phenylalanine, tryptophan, and serine as raw materials respectively in Li... + / Periodate conditions (Li + The preparation of nanostructured materials, as described in Example 10, can be considered as using phenylalanine, tryptophan, or serine as raw materials in H... + / Periodate conditions (H + The preparation of nanostructured materials, as described in Example 11, can be considered as using phenylalanine, tryptophan, or serine as raw materials in Na+. + / under periodate conditions (Na) + The preparation of nanostructured materials, as described in Example 12, can be considered as using phenylalanine, tryptophan, or serine as raw materials in K... + / under periodate conditions (K + Preparation of nanostructured materials; particle size distribution of products obtained from phenylalanine, tryptophan, and serine under different cation periodate conditions as shown in the figure. Figure 12 As shown (reaction time 1 day), the particle size distributions of the products obtained by reacting phenylalanine, tryptophan, or serine under different cation periodate conditions for different times are as follows: Figure 13-15 As shown.

[0078] Depend on Figure 12 It can be seen that the particle size of the product obtained by phenylalanine under different cation periodate conditions is: K + >Li + ≈Na + >H + The particle size of the product obtained from tryptophan under different cation periodate conditions is: Na + >H + ≈K + >Li + The particle size of the serine product obtained under different cation periodate conditions is: Li + >Na + >K + >H + .

[0079] Depend on Figure 13 It can be seen that phenylalanine in H + After reacting for 2–7 days under periodate conditions, the particle size tends to stabilize at approximately 100–800 nm; under K... + Under periodate conditions, the particle size gradually decreased with increasing reaction time, reaching approximately 50–100 nm after 7 days; in Na+... + Under periodate conditions, changes in reaction time have little effect on particle size, which ranges from 80 to 600 nm.

[0080] Depend on Figure 14 It can be seen that tryptophan in H +Under periodate conditions, reaction time has little effect on particle size, and the particle size tends to stabilize, distributed in the range of 800–700 nm; under K... + Under periodate conditions, the particle size did not change significantly with increasing reaction time, remaining within the range of 50–600 nm; under Na+ conditions… + Under periodate conditions, the product particle size is small in the early stage of the reaction, about 20~600 nm. After 3~7 days of reaction, the particle size gradually increases. This may be because the excessive reaction destroys the stability of the original surface groups, leading to particle aggregation.

[0081] Depend on Figure 15 It can be seen that serine in H + Under periodate conditions, particle size is significantly affected by reaction time. In the first three days, the particle size is small, approximately 5-80 nm. After three days of reaction, the particle size increases to approximately 400-1000 nm, which may be due to over-reaction leading to particle aggregation. Under K... + Under periodate conditions, the particle size gradually increases with increasing reaction time, with the overall particle size distribution ranging from 80 to 800 nm; in Na... + Under periodate conditions, the reaction time has a significant impact on particle size as the reaction time increases, with the overall particle size distribution ranging from 80 to 900 nm.

[0082] Comparative Example 1

[0083] 1 g of phenylalanine / tryptophan / serine was dissolved in 50 mL of phosphate buffer (pH=7). The mixture was stirred continuously at 800 rpm for 3 days at room temperature in the dark. The reaction solution was then centrifuged at 7000×g for 10 min to remove incompletely reacted substrate. The supernatant was transferred to a 3500 Da dialysis membrane for dialyzing, with water changed multiple times until the conductivity approached that of pure water. The solution was then freeze-dried. The results showed that almost no product was collected, i.e., the yield was close to 0. This indicates that neutral buffer conditions cannot induce amino acid self-assembly. Therefore, the most important factor in the neutral periodate-induced self-assembly of this invention is the oxidizing power of the periodate ion.

[0084] In summary, the particle size of products obtained from different amino acids varies significantly in different systems containing periodate ions. This method, using amino acids and / or peptides as substrates, effectively achieves self-assembly in systems containing periodate ions to form stable nanostructured materials. These nanostructured materials exhibit morphologies such as spherical nanoparticles, filamentous nanofibers, or sheet-like nanostructures, with uniform size and high quality. Different amino acids / peptides can be used for self-assembly in different systems containing periodate ions to obtain products with different structural morphologies and sizes, suitable for various applications. Specifically, the nanostructured materials prepared under neutral periodate conditions have spherical nanoparticles with diameters of 5–200 nm and filamentous nanofibers with diameters of 10–50 nm. This invention provides a method for the self-assembly of amino acids and / or peptides into nanostructured materials in systems containing periodate ions. The preparation process is simple, the conditions are mild, and the application is wide-ranging. The self-assembled nanostructured materials possess unique physicochemical properties, excellent biocompatibility, and biodegradability, making them widely applicable in various fields such as industry, agriculture, energy, and medicine.

[0085] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for inducing the self-assembly of amino acids and polypeptides into nanostructured materials, characterized in that, The process includes the following steps: reacting amino acids and / or peptides in a system containing periodate ions to obtain nanostructured materials.

2. The method for inducing the self-assembly of amino acids and polypeptides into nanostructured materials according to claim 1, characterized in that, The system containing periodate ions is obtained by dissolving periodic acid and / or periodate in water.

3. The method for inducing the self-assembly of amino acids and peptides into nanostructured materials according to claim 2, characterized in that, After dissolving periodic acid and / or periodate in water, the pH of the system is adjusted using an alkaline solution.

4. The method for inducing the self-assembly of amino acids and polypeptides into nanostructured materials according to claim 2, characterized in that, The mass ratio of the amino acids and / or polypeptides, periodic acid and / or periodate, and water is (1~4):(1~4):(25~120).

5. The method for inducing the self-assembly of amino acids and peptides into nanostructured materials according to claim 1, characterized in that, The reaction was carried out in a light-protected environment at a temperature of 15–35 °C for 24–72 h.

6. The method for inducing the self-assembly of amino acids and polypeptides into nanostructured materials according to claim 1, characterized in that, The amino acids include, but are not limited to, natural amino acids and / or their isomers. The natural amino acids include, but are not limited to, at least one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The polypeptide is a polypeptide composed of multiple natural amino acids and / or their isomers.

7. The method for inducing the self-assembly of amino acids and polypeptides into nanostructured materials according to claim 1, characterized in that, After the reaction was completed, a small amount of ethylene glycol was added to neutralize the unreacted periodate ions, and the nanostructured material was obtained by centrifugation, dialysis and drying.

8. Nanostructured materials prepared by the method according to any one of claims 1 to 7.

9. The nanostructured material according to claim 8, characterized in that, The morphology of the nanostructured material includes, but is not limited to, spherical nanoparticles, filamentous nanofibers, or sheet-like nanostructures. In the nanostructured material prepared under neutral periodate conditions, the diameter of the spherical nanoparticles is 5-200 nm, and the diameter of the filamentous nanofibers is 10-50 nm.

10. The application of the nanostructured material of claim 8 in the fields of biomedicine, food, daily chemicals, agriculture or energy storage.

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