A regenerated silk fibroin solution imitating mulberry silkworm gland protein, and a preparation method and application thereof

Abstract

This invention discloses a regenerated silk fibroin solution mimicking silkworm gland protein, its preparation method, and its application. The method involves degumming natural silk and dissolving it in a salt solution system, followed by dialysis and purification to obtain an RSF aqueous solution. Based on pre-defined material performance requirements, the Na... + K + Ca 2+ Mg 2+ and Fe 3+ The amount, order of addition, and mode of action of Na are determined; according to the set amount, order of addition, and mode of action, Na is introduced into the RSF aqueous solution. + K + Ca 2+ Mg 2+ and Fe 3+ Finally, by adjusting the pH value, an RSF solution mimicking silkworm gland protein was obtained. This invention simulates the physiological ionic environment within silkworm glands and utilizes the synergistic effect of trace amounts of multi-metal ions to reconstruct the microstructure of RSF, thereby obtaining a high-viscosity, high-stability RSF precursor solution, which is beneficial for improving the mechanical properties of RSF fibers.

Description

Technical Field

[0001] This invention belongs to the field of biopolymer materials technology, specifically relating to a regenerated silk fibroin solution that mimics silkworm gland protein, its preparation method, and its application. Background Technology

[0002] Regenerated silk fibroin (RSF), as a highly promising biopolymer material, has attracted much attention in the fields of medical sutures and tissue engineering. However, in the process of extracting RSF from natural silk, regeneration treatments such as strong alkali degumming or high-concentration salt solution dissolution inevitably damage the original multi-level ordered structure of the silk fibroin. This damage to the microstructure leads to a significant decrease in the mechanical properties of the regenerated material, especially the regenerated silk fibroin fibers, which show a significant gap in strength and toughness compared to natural silk. To improve the mechanical properties of regenerated silk fibroin fibers, existing technologies have explored various improvement pathways. For example, strength can be increased by introducing inorganic reinforcing components such as carbide particles into the regenerated silk fibroin spinning solution, or the interfacial interaction of fillers such as nanocellulose whiskers can be used to enhance intermolecular bonds, or the ductility can be improved by blending with polymers such as polyvinyl alcohol and by using post-drawing orientation methods. The relevant technologies are reflected in Chinese invention patent applications with application numbers CN202510380641.2, CN201310369250.8, and CN202110312949.5. Although these solutions improve the mechanical properties of materials to some extent, their inherent limitations remain obvious. These methods are highly dependent on exogenous reinforcing components. The introduction of non-silk protein components can easily reduce the purity of the system and may affect biocompatibility. Moreover, the regulation is mostly focused on structural repair after fiber formation, lacking precise intervention in the molecular conformational evolution process in the solution stage, resulting in significant differences in the rheological properties of the spinning solution compared to natural silk proteins.

[0003] In fact, thanks to the structural pre-assembly induced by the physiological ionic environment within the glands, natural silk fibroin exhibits excellent spinnability, making it easier to transform into high-performance fiber materials. Silkworm glands utilize sodium (Na₂O₃) for spinnability. + ), potassium (K) + ), calcium (Ca 2 + ), magnesium (Mg) 2+ The types and concentration gradients of metal ions drive the ordered pre-assembly of natural silk fibroin molecules. The coordination and charge shielding effects between metal ions and natural silk fibroin molecules maintain the solution in a stable state of high concentration and high viscosity, facilitating the formation of ordered microstructures and high-strength, high-toughness fiber materials during the spinning process. Although current research has focused on the regulatory role of metal ions, existing ion regulation methods are generally relatively singular, typically focusing only on single Ca2+ ions. 2+The inductive effect of ions (as described in Chinese invention patent application CN102220661A) or by employing a simple component mixing method. For example, in the English paper "Effects of environment parameters on sol-gel transition and dry-spinnability of regenerated silk fibroin aqueous solution", a high concentration of K is directly added to the solution. + Mg 2+ Ca 2+ Plasma can be used to regulate the properties of a system. However, this method neglects the synergistic effect between metal ions of different valence states and types, and does not take into account the influence of the order and content of ion addition on the conformational transformation of protein molecular chains. As a result, the prepared solutions often have problems such as low viscosity and excessive β-sheet content and insufficient α-helix content in the secondary structure, making it difficult to achieve a balance between strength and toughness in mechanical properties. Summary of the Invention

[0004] To address the problem that the microstructure of regenerated silk fibroin is severely damaged during its preparation, resulting in materials with significant mechanical brittleness and insufficient strength and toughness, and that existing methods for improving performance often rely on the introduction of exogenous reinforcing materials and struggle to maintain material purity, this invention provides a regenerated silk fibroin solution mimicking silkworm gland protein, along with its preparation method and applications. This method aims to reconstruct the microstructure of regenerated silk fibroin by simulating the physiological ionic environment within silkworm glands and utilizing the synergistic effect of trace amounts of multi-metal ions. This results in a high-viscosity, high-stability regenerated silk fibroin precursor solution, which is beneficial for improving the mechanical properties of regenerated silk fibroin fibers.

[0005] To solve the above-mentioned technical problems and achieve the above-mentioned technical effects, the present invention is implemented through the following technical solution: A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein includes the following steps: Step 1: Natural silk is degummed, dissolved, dialyzed, and purified to obtain a regenerated silk fibroin water (RSF) solution; Step 2: Based on the preset material performance requirements, set the sodium ion (Na) ratio. + ), potassium ions (K) + ), calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ) and iron ions (Fe 3+ The amount, order of addition, and method of action of ( ); The order of addition can be an order of addition based on increasing valence state, an order of addition based on decreasing valence state, an order of addition based on synchronous competitive coordination, an order of addition based on alternating monovalent and divalent ions, or an order of addition based on the difference in radius of ions with the same valence. Step 3: According to the set addition amount, addition order, and mode of action, introduce sodium ions (Na+) into the RSF aqueous solution. + ), potassium ions (K) + ), calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ) and iron ions (Fe 3+ ), to obtain an aqueous solution of multi-metal ion RSF; Step 4: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 to obtain a regenerated silk fibroin solution that mimics silkworm gland protein.

[0006] Furthermore, the concentration of the RSF aqueous solution is 36 wt.%.

[0007] Furthermore, the specific method for adding items in ascending order according to valence state gradient is as follows: First, potassium ions (K) are simultaneously introduced into the RSF aqueous solution. + ) and sodium ions (Na + These two monovalent metal ions were then simultaneously introduced with magnesium ions (Mg). 2+ ) and calcium ions (Ca 2+ These two divalent metal ions, and finally the introduction of iron ions (Fe) 3 + This is a trivalent metal ion.

[0008] Furthermore, the specific method for adding items in descending order of valence state gradient is as follows: First, iron ions (Fe) are introduced into the RSF aqueous solution. 3+ This trivalent metal ion, followed by the simultaneous introduction of magnesium ions (Mg). 2+ ) and calcium ions (Ca 2+ These two divalent metal ions are then simultaneously introduced with potassium ions (K). + ) and sodium ions (Na + These are two monovalent metal ions.

[0009] Furthermore, the specific method for adding positions according to the synchronous competition coordination order is as follows: Add the preset amount of potassium ions (K) + ) and sodium ions (Na + These two monovalent metal ions, magnesium ions (Mg 2+ ) and calcium ions (Ca 2+ These two divalent metal ions and iron ions (Fe)3+ These trivalent metal ions are first premixed and then added to the RSF aqueous solution all at once.

[0010] Furthermore, the specific method for adding monovalent and divalent ions in an alternating order is as follows: First, potassium ions (K) are introduced into the RSF aqueous solution. + This monovalent metal ion, then the introduction of magnesium ions (Mg) 2+ This is a divalent metal ion, which is then followed by the introduction of sodium ions (Na). + This monovalent metal ion, followed by the introduction of calcium ions (Ca). 2+ This divalent metal ion, ultimately introducing iron ions (Fe). 3+ This is a trivalent metal ion.

[0011] Furthermore, the specific method for adding ions according to their radius differences is as follows: Potassium ions (K ions) are introduced sequentially into the RSF aqueous solution according to the order of radii of metal ions of the same valence from smallest to largest or a specific spatial occupancy logic. + Sodium ions (Na) + ), magnesium ions (Mg 2+ ), calcium ions (Ca 2+ ) and iron ions (Fe 3+ ).

[0012] Furthermore, the introduction of sodium ions (Na+) + ), potassium ions (K) + ), calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ) and iron ions (Fe 3+ After that, the sodium ions (Na+) in the multi-metal ion regenerated silk fibroin aqueous solution are... + The content of ) is 0.5~1.5mg / g, potassium ions (K) + The content of ) is 5~14mg / g, and the calcium ion (Ca) content is 5~14mg / g. 2+ The content of magnesium ions (Mg) is 0.3~1.2 mg / g. 2+ The content of ) is 0.8~1.6 mg / g, and the iron ion (Fe) content is 0.8~1.6 mg / g. 3+ The content of ) is 0.01~0.03mg / g.

[0013] Furthermore, sodium ions (Na) + Introduced in the form of sodium chloride (NaCl), potassium ions (K) + Introduced in the form of potassium chloride (KCl), calcium ions (Ca) 2+ It is introduced in the form of calcium chloride (CaCl2), magnesium ions (Mg) 2+It is introduced in the form of magnesium chloride (MgCl2), iron ions (Fe) 3+ It is introduced in the form of ferric chloride (FeCl3).

[0014] Based on the above-described online preparation method, this invention provides a regenerated silk fibroin solution that mimics silkworm gland protein and is prepared using the above method.

[0015] This invention provides an application of the above-mentioned regenerated silk fibroin solution, which mimics silkworm gland protein, in fiber spinning.

[0016] This invention provides an application of the above-mentioned regenerated silk fibroin solution, which mimics silkworm gland protein, as a high-performance reinforcing matrix in the preparation of various biomaterials such as membranes, hydrogels, and scaffolds.

[0017] The beneficial effects of this invention are as follows: 1. This invention simulates the physiological ionic environment within the silkworm glands, introduces different types and valence states of metal ions into the regenerated silk fibroin solution, and strictly controls the order in which the ions are added, thereby achieving effective regulation of the intermolecular interactions and microstructure of the regenerated silk fibroin.

[0018] 2. This invention deeply simulates the physiological ionic environment within the silkworm glands, eliminating the dependence on exogenous polymers or inorganic reinforcing components in traditional methods. While maintaining the high purity of the regenerated silk fibroin system, it achieves the pre-assembly of the solution microstructure, ensuring the excellent biocompatibility of the material.

[0019] 3. The regenerated silk fibroin solution obtained by this invention has stable properties, with an initial viscosity adjustable within the range of 100-5000 Pa·s, exhibiting extremely high viscosity and rheological stability. Its zeta potential value is close to the zero potential corresponding to the isoelectric point of silk fibroin, and the content of secondary structures is controllable. This overcomes the bottleneck of the difficulty in processing existing aqueous phase regenerated silk fibroin solutions, laying the foundation for the continuous and stable production of high-performance fibers.

[0020] 4. This invention reveals the intrinsic relationship between the action mode of metal ions and fiber properties, enabling the prepared fibers to exhibit highly controllable mechanical properties. Using the regenerated silk fibroin solution of this invention, continuous and stable spinning of high-performance regenerated silk fibroin fibers can be achieved. Furthermore, depending on the specific application scenario, by changing the action mode of the metal ions, the regenerated silk fibroin fibers can be endowed with excellent and adjustable mechanical properties. This results in high-strength fibers with a breaking strength of 100-300 MPa and highly elastic fibers with a breaking elongation of 20%-130%, overcoming the limitation of simultaneously achieving both strength and toughness in regenerated silk fibroin materials.

[0021] 5. The regenerated silk fibroin solution prepared by this invention has good versatility. It is not only suitable for fiber spinning, but can also be used as a high-performance reinforcing matrix for the preparation of various biomaterials such as membranes, hydrogels and scaffolds. It can significantly improve the comprehensive mechanical strength and toughness of these materials and has broad industrialization prospects.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0023] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a comparison chart of the fiber-forming properties of the regenerated silk fibroin solution with simulated silkworm gland protein prepared in Example 1 of the present invention and the pure regenerated silk fibroin solution.

[0024] Figure 2 The results show the effect of the order of metal ion addition on the structure and properties of the regenerated silk fibroin solution prepared in this invention. Figure 2 (a) in the figure shows the viscosity test results. Figure 2 (b) in the figure shows the potential test results. Figure 2 (c) in the figure shows the results of the circular dichroism test. Figure 2 (d) in the figure represents the results of quantitative analysis of the secondary structure. Detailed Implementation

[0025] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to provide a clearer understanding of the invention's purpose, features, and advantages. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the invention's technical solution.

[0026] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments may be practiced without one or more of these specific details. In other instances, well-known apparatuses, structures, and techniques associated with this application may not have been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0027] Unless the context requires otherwise, throughout the specification and claims, the word “comprising” and its variations, such as “including” and “having”, shall be understood to have an open, inclusive meaning, that is, to be interpreted as “including, but not limited to”.

[0028] Throughout this specification, references to "an embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

[0029] The singular forms “a” and “the” used in this specification and the appended claims include plural references unless otherwise expressly stated herein. It should be noted that the term “or” is generally used to mean “and / or” unless otherwise expressly stated herein.

[0030] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0031] This invention provides a method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein, comprising the following steps: Step 1: Degumming the natural silk.

[0032] Step 2: Dissolve the degummed natural silk in a salt solution system to obtain a degummed silk salt solution.

[0033] Step 3: Dialyze and purify the degummed silk salt solution to obtain a regenerated silk fibroin (RSF) aqueous solution with a concentration of 36 wt.%.

[0034] Step 4: Based on the preset material performance requirements, set Na... + K + Ca 2+ Mg 2+ and Fe 3+ The amount, order of addition, and method of action of the substance.

[0035] Na + Introduced in the form of NaCl, K + Introduced in the form of KCl, Ca 2+ Introduced in the form of CaCl2, Mg 2+ Introduced in the form of MgCl2, Fe 3+ It is introduced in the form of FeCl3.

[0036] The order of addition can be an increasing order of valence, a decreasing order of valence, an order of simultaneous competitive coordination, an order of alternating monovalent and divalent ions, or an order of addition based on the difference in radius of ions with the same valence.

[0037] (1) The specific method of adding items in ascending order of valence state gradient is as follows: First, K is simultaneously introduced into the RSF aqueous solution. + and Na + These two monovalent metal ions were subsequently introduced into Mg. 2 + and Ca 2+ These two divalent metal ions were finally introduced with Fe. 3+ This is a trivalent metal ion.

[0038] (2) The specific method of adding items in descending order of valence gradient is as follows: First, Fe is introduced into the RSF aqueous solution. 3+ This trivalent metal ion was subsequently introduced simultaneously with Mg. 2+ and Ca 2+ These two divalent metal ions were then simultaneously introduced with K. + and Na + These are two monovalent metal ions.

[0039] (3) The specific method for adding synchronous competing coordination is as follows: Add the preset amount of K + and Na + These two monovalent metal ions, Mg 2+ and Ca 2+ These two divalent metal ions and Fe 3+ These trivalent metal ions are first premixed and then added to the RSF aqueous solution all at once.

[0040] (4) The specific method of adding monovalent and divalent ions in alternating order is as follows: First, K is introduced into the RSF aqueous solution. + This monovalent metal ion, then introduced with Mg 2+ This divalent metal ion was subsequently introduced with Na + This monovalent metal ion, followed by the introduction of Ca... 2+ This divalent metal ion was finally introduced with Fe. 3+ This is a trivalent metal ion.

[0041] (5) The specific method for adding ions according to the difference in radius of the same valence is as follows: K is introduced sequentially into the RSF aqueous solution according to the order of the radii of metal ions of the same valence from smallest to largest or a specific spatial occupancy logic. +Na + Mg 2+ Ca 2+ and Fe 3+ .

[0042] Step 5: According to the set addition amount, addition order, and mode of action, introduce Na into the RSF aqueous solution. + K + Ca 2+ Mg 2+ and Fe 3+ An aqueous solution of multi-metal ion RSF was obtained.

[0043] Introducing Na + K + Ca 2+ Mg 2+ and Fe 3+ Subsequently, the Na in the multi-metal ion regenerated silk fibroin aqueous solution... + The content is 0.5~1.5mg / g, K + The content is 5~14mg / g, Ca 2+ The content is 0.3~1.2 mg / g, Mg 2+ The content is 0.8~1.6 mg / g, Fe 3+ The content is 0.01~0.03mg / g.

[0044] Step 6: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 using dilute hydrochloric acid to obtain a regenerated silk fibroin solution that mimics silkworm gland protein.

[0045] In this invention, the mechanism of action of metal ions has a decisive influence on the microstructural evolution of RSF solutions. By selecting specific combinations of monovalent and polyvalent metal ions and utilizing the coordination and charge shielding effects between ions and protein molecular chains, this invention can induce ordered pre-assembly of RSF molecules. Specifically, the order of metal ion addition is designed as a key variable to regulate the degree of molecular chain coiling and entanglement, effectively controlling the secondary structure content of the solution and bringing the solution's zeta potential closer to zero, indicating that the solution pH is near the isoelectric point of silk fibroin (pI ~ 4). The regenerated silk fibroin solution mimicking silkworm gland protein obtained by this invention exhibits good rheological stability, with its viscosity adjustable across orders of magnitude from 100 to 5000 Pa·s. Using this regenerated silk fibroin solution mimicking silkworm gland protein as a spinning solution, continuous steady-state spinning of RSF fibers can be achieved in a pure water system. By changing the metal ion regulation scheme, this invention can directly affect the final molecular arrangement and orientation of the fiber, thereby endowing the fiber with significant mechanical plasticity.

[0046] It is important to note that if only a single metal ion is added to the RSF solution (e.g., only monovalent or divalent ions), the effect often depends on high concentrations of high-valence metal ions (e.g., high concentrations of Ca). 2+ Only under these conditions can the change of solution state be barely achieved. However, under such single-component metal ion addition conditions, the regularity of the internal microstructure of the RSF solution is poor, and when the addition is within the trace amount required by this invention, the prepared RSF solution has extremely poor spinnability, making it difficult to achieve a continuous, stable, and long-lasting spinning process in subsequent processing.

[0047] Based on the above-described online preparation method, the present invention also provides a regenerated silk fibroin solution that mimics silkworm gland protein and is prepared using the above-described preparation method.

[0048] The present invention also provides the application of the above-mentioned regenerated silk fibroin solution, which is a mimicking silkworm gland protein, in fiber spinning.

[0049] The present invention also provides the application of the above-mentioned regenerated silk fibroin solution, which is a mimicking silkworm gland protein, as a high-performance reinforcing matrix in the preparation of various biomaterials such as membranes, hydrogels and scaffolds, so as to improve the strength and toughness of these biomaterials.

[0050] The following details several specific embodiments of the present invention and their related test results.

[0051] Example 1 (added in ascending order of valence gradient) A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein includes the following steps: Step 1: Degumming the natural silk.

[0052] Step 2: Dissolve the degummed natural silk in a salt solution system to obtain a degummed silk salt solution.

[0053] Step 3: Dialyze, purify and centrifuge the degummed silk salt solution to obtain an RSF aqueous solution with a concentration of 36 wt.%.

[0054] Step 4: Add Na to the RSF aqueous solution in order of increasing valence gradient. + K + Ca 2+ Mg 2+ and Fe 3+ Specifically: First, KCl and NaCl solutions were added to a 36 wt.% RSF solution to make KCl and NaCl solutions... + and Na + The contents were 11 mg / g and 0.7 mg / g, respectively, to reduce electrostatic repulsion between molecular chains and induce chain segment prefolding; Then MgCl2 and CaCl2 were added, so that Mg 2+ and Ca 2+ The contents were 1.2 mg / g and 0.4 mg / g, respectively, and a bridging structure was formed between molecular chains through coordination. Finally, FeCl3 is added to make Fe 3+ The concentration is 0.02 mg / g to enhance multi-point coordination and cross-linking, thereby obtaining a multi-metal ion RSF aqueous solution.

[0055] Step 5: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 using 0.1 mol / L dilute hydrochloric acid to finally obtain a regenerated silk fibroin solution that mimics silkworm gland protein, denoted as R+MDT.

[0056] A 36 wt.% RSF aqueous solution was used as the control group, denoted as PR. See also Figure 2 As shown in (a) of this example, the apparent viscosity of the R+MDT solution prepared in this embodiment is approximately 4900 Pa·s. See also Figure 2 As shown in (b), the zeta potential of the R+MDT solution system prepared in this embodiment is -3.34 mV, close to the zero potential point, indicating that the solution pH is near the isoelectric point of silk fibroin (pI ~4). Compared with the control group PR solution (zeta potential of -9.31 mV), the absolute value of the zeta potential is significantly increased. At this point, the electrostatic repulsion of the system is weakened, making it easier to induce structural changes. See also Figure 2 As shown in (c), the PR solution exhibits a predominantly negative peak at 195 nm, indicating a predominantly random coil structure. In contrast, the R+MDT solution shows distinct positive and negative peaks at 193 nm and 203 nm, respectively, corresponding to the characteristic peaks of α-helical and β-sheet structures, demonstrating that metal ion regulation significantly promotes the transformation of molecular conformation from random coil to ordered structure. See also Figure 2 As shown in (d), under the sequential synergistic regulation of multivalent metal ions, the contents of α-helical and β-sheet structures in the R+MDT solution system reached 27% and 26%, respectively, indicating that the molecular chain conformation was effectively regulated. Meanwhile, see also... Figure 1 As shown, the R+MDT solution prepared in this embodiment forms a stable pre-assembled network structure inside, which can significantly improve the structural stability of the system without the introduction of external reinforcing components, and can stably draw out longer and more uniform fiber streams.

[0057] Step 6: Using the R+MDT solution as the spinning solution, fibers were prepared using a wet spinning process, and the pH of the coagulation bath was adjusted to 3 by adding citric acid. The resulting fibers had a breaking stress of 282 MPa and a breaking strain of 25%.

[0058] Example 2 (Added in descending order of valence gradient) A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein includes the following steps: Step 1: Degumming the natural silk.

[0059] Step 2: Dissolve the degummed natural silk in a salt solution system to obtain a degummed silk salt solution.

[0060] Step 3: Dialyze, purify and centrifuge the degummed silk salt solution to obtain an RSF aqueous solution with a concentration of 36 wt.%.

[0061] Step 4: Add Na to the RSF aqueous solution in descending order of valence gradient. + K + Ca 2+ Mg 2+ and Fe 3+ Specifically: First, FeCl3 was added to a 36 wt.% RSF solution to make Fe... 3+ The content is 0.03 mg / g; Subsequently, divalent ions MgCl2 and CaCl2 were added, causing Mg... 2+ and Ca 2+ The contents were 1.5 mg / g and 0.8 mg / g, respectively; Finally, KCl and NaCl are added to make K + and Na + The contents were 14 mg / g and 1.2 mg / g, respectively, thus obtaining an aqueous solution of multi-metal ion RSF.

[0062] Step 5: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 using 0.1 mol / L dilute hydrochloric acid to obtain the regenerated silk fibroin solution that mimics silkworm gland protein, denoted as R+TDM.

[0063] See Figure 2 As shown in (a) of this example, the apparent viscosity of the R+TDM solution prepared in this embodiment is approximately 1335 Pa·s. The results indicate that under conditions of preferential introduction of high-valence ions, the lack of effective electrostatic shielding in the initial stage of the system leads to local coordination and contraction of the molecular chains, resulting in a higher solution viscosity. See also... Figure 2 As shown in (b) of this embodiment, the zeta potential of the R+TDM solution system is -4.17 mV. See also... Figure 2 As shown in (d), the contents of α-helices and β-sheets in the R+TDM solution system are 6% and 26%, respectively, and the system is still dominated by random coil structures (about 37%). This conformational distribution results in poor structural uniformity within the solution, making it difficult to form a stable pre-assembled network structure.

[0064] Step 6: Using the R+TDM solution as the spinning solution, fibers were prepared using a wet spinning process, and the pH of the coagulation bath was adjusted to 3 by adding citric acid. The resulting fibers had a breaking stress of 208 MPa and a breaking strain of 52%.

[0065] Example 3 (Added according to synchronous competitive coordination) A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein includes the following steps: Step 1: Degumming the natural silk.

[0066] Step 2: Dissolve the degummed natural silk in a salt solution system to obtain a degummed silk salt solution.

[0067] Step 3: Dialyze, purify and centrifuge the degummed silk salt solution to obtain an RSF aqueous solution with a concentration of 36 wt.%.

[0068] Step 4: Introduce Na into the RSF aqueous solution according to the order of simultaneous competitive coordination. + K + Ca 2+ Mg 2+ and Fe 3+ Specifically: KCl, NaCl, MgCl2, CaCl2, and FeCl3 were simultaneously added to a 36 wt.% RSF solution at concentrations of 8 mg / g, 0.5 mg / g, 0.8 mg / g, 0.3 mg / g, and 0.01 mg / g, respectively, to simulate the natural environment of free competitive coordination of ions, thereby obtaining a multi-metal ion RSF aqueous solution.

[0069] Step 5: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 using 0.1 mol / L dilute hydrochloric acid to obtain the regenerated silk fibroin solution that mimics silkworm gland protein, denoted as RS.

[0070] See Figure 2 As shown in (a) of this example, the apparent viscosity of the RS solution prepared in this embodiment is approximately 112 Pa·s. See also Figure 2 As shown in (b) of this example, the zeta potential of the RS solution system prepared in this embodiment is -4.36 mV. See also Figure 2 As shown in (d), the contents of α-helix and β-sheet in the RS solution system are 3% and 16%, respectively. The random coil (57%) conformation still dominates in the system, indicating that only limited conformational changes occur under these conditions and a stable and ordered molecular chain assembly structure has not yet been formed.

[0071] Step 6: Using the RS solution as the spinning solution, fibers were prepared using a wet spinning process, and the pH of the coagulation bath was adjusted to 3 by adding citric acid. The resulting fibers had a breaking stress of 153 MPa and a breaking strain of 111%.

[0072] Example 4 (Added in alternating order of monovalent and divalent ions) A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein includes the following steps: Step 1: Degumming the natural silk.

[0073] Step 2: Dissolve the degummed natural silk in a salt solution system to obtain a degummed silk salt solution.

[0074] Step 3: Dialyze, purify and centrifuge the degummed silk salt solution to obtain an RSF aqueous solution with a concentration of 36 wt.%.

[0075] Step 4: Introduce Na+ into the RSF aqueous solution in an alternating order of monovalent and divalent ions. + K + Ca 2+ Mg 2+ and Fe 3+ Specifically: First, add KCl to a 36 wt.% RSF solution to make K + The content is 10 mg / g; Next, MgCl2 is added to make Mg 2+ The content is 1.3 mg / g; Then add NaCl to make Na + The content is 0.9 mg / g; Then add CaCl2 to make Ca 2+ The content is 1.0 mg / g; Finally, add FeCl3, Fe 3+ The content of ions was 0.03 mg / g, thus obtaining a multi-metal ion RSF aqueous solution.

[0076] Step 5: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 using 0.1 mol / L dilute hydrochloric acid to finally obtain a regenerated silk fibroin solution that mimics silkworm gland protein, denoted as R-KMNCF.

[0077] See Figure 2 As shown in (a) of this example, the apparent viscosity of the R-KMNCF solution prepared in this embodiment is approximately 2538 Pa·s. See also... Figure 2 As shown in (b) of this embodiment, the zeta potential of the R-KMNCF solution system is -3.87 mV. See also... Figure 2As shown in (d), secondary structure analysis revealed that the contents of α-helices and β-sheets in the R-KMNCF solution system were 23% and 32%, respectively, indicating that the ordered microstructure dominated under these conditions.

[0078] Step 6: Using R-KMNCF solution as the spinning solution, fibers were prepared using a wet spinning process, and the pH of the coagulation bath was adjusted to 3 by adding citric acid. The resulting fibers had a breaking stress of 298 MPa and a breaking strain of 18%.

[0079] Example 5 (Added in order of difference in radius of ions with the same valence) A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein includes the following steps: Step 1: Degumming the natural silk.

[0080] Step 2: Dissolve the degummed natural silk in a salt solution system to obtain a degummed silk salt solution.

[0081] Step 3: Dialyze, purify and centrifuge the degummed silk salt solution to obtain an RSF aqueous solution with a concentration of 36 wt.%.

[0082] Step 4: Add Na to the RSF aqueous solution according to the order of addition based on the difference in the radius of ions with the same valence, that is, according to the order of the radius of metal ions with the same valence from smallest to largest or a specific space occupancy logic. + K + Ca 2+ Mg 2+ and Fe 3+ Specifically: First, add KCl to a 36 wt.% RSF solution to make K + The content is 5 mg / g; Next, add NaCl to make Na + The content is 1.5 mg / g; Then add MgCl2 to make Mg 2+ The content is 1.0 mg / g; Then add CaCl2 to make Ca 2+ The content is 1.1 mg / g; Finally, add FeCl3, Fe 3+ The content of is 0.02 mg / g, thus obtaining a multi-metal ion RSF aqueous solution.

[0083] Step 5: Adjust the pH of the multi-metal ion RSF aqueous solution to 6 using 0.1 mol / L dilute hydrochloric acid to finally obtain a regenerated silk fibroin solution that mimics silkworm gland protein, denoted as R-NKMCF.

[0084] See Figure 2 As shown in (a) of this example, the apparent viscosity of the R-NKMCF solution prepared in this embodiment is approximately 3338 Pa·s. See also... Figure 2 As shown in (b) of this embodiment, the zeta potential of the R-NKMCF solution system is -2.85 mV. See also... Figure 2 As shown in (d), secondary structure analysis revealed that the contents of α-helices and β-sheets in the R-NKMCF solution system were 28% and 32%, respectively. The system was still dominated by the random coil (40%) conformation, indicating that only limited conformational changes occurred under these conditions and a stable and ordered molecular chain assembly structure had not yet been formed.

[0085] Step 6: Using the R-NKMCF solution as the spinning solution, fibers were prepared using a wet spinning process, and the pH of the coagulation bath was adjusted to 3 by adding citric acid. The resulting fibers had a breaking stress of 233 MPa and a breaking strain of 79%.

[0086] This invention simulates the physiological ionic environment within the silkworm gland and introduces trace amounts of multi-metal ions for synergistic regulation. The key focus is on controlling the order of ion addition to effectively construct the intermolecular interactions and microstructure of RSF molecules. This method not only eliminates dependence on exogenous reinforcing components, ensuring material purity and biocompatibility, but also achieves fundamental control over the rheological properties and molecular conformation of RSF solutions. The resulting solution exhibits good stability and processing performance, and the prepared fibers can demonstrate high strength or high elasticity mechanical properties according to practical application requirements, thus providing a new approach to obtaining high-performance RSF materials with controllable properties.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a regenerated silk fibroin solution that mimics silkworm gland protein, characterized in that, Includes the following steps: Step 1: Natural silk is degummed, dissolved, dialyzed, and purified to obtain a regenerated silk fibroin aqueous solution; Step 2: Based on the preset material performance requirements, set the amount, order of addition, and mode of action of sodium ions, potassium ions, calcium ions, magnesium ions, and iron ions. The order of addition can be an order of addition based on increasing valence state, an order of addition based on decreasing valence state, an order of addition based on synchronous competitive coordination, an order of addition based on alternating monovalent and divalent ions, or an order of addition based on the difference in radius of ions with the same valence. Step 3: According to the set addition amount, addition order and action method, sodium ions, potassium ions, calcium ions, magnesium ions and iron ions are introduced into the regenerated silk fibroin aqueous solution to obtain a multi-metal ion regenerated silk fibroin aqueous solution. Step 4: Adjust the pH of the multi-metal ion regenerated silk fibroin aqueous solution to 6, thereby obtaining a regenerated silk fibroin solution that mimics silkworm gland protein.

2. The preparation method according to claim 1, characterized in that, The concentration of the regenerated silk fibroin aqueous solution is 36 wt.%.

3. The preparation method according to claim 1, characterized in that, The specific method for adding items in ascending order according to valence state gradient is as follows: First, potassium and sodium ions, two monovalent metal ions, are simultaneously introduced into the regenerated silk fibroin aqueous solution. Then, magnesium and calcium ions, two divalent metal ions, are simultaneously introduced. Finally, iron ions, a trivalent metal ion, are introduced.

4. The preparation method according to claim 1, characterized in that, The specific method for adding items in descending order of valence state gradient is as follows: First, iron ions, a trivalent metal ion, are introduced into the aqueous solution of the regenerated silk fibroin. Then, magnesium ions and calcium ions, two divalent metal ions, are introduced simultaneously. Finally, potassium ions and sodium ions, two monovalent metal ions, are introduced simultaneously.

5. The preparation method according to claim 1, characterized in that, The specific method for adding positions according to the synchronous competitive coordination order is as follows: The pre-mixed amounts of potassium and sodium ions (monovalent metal ions), magnesium and calcium ions (divalent metal ions), and iron ions (trivalent metal ions) are then added simultaneously to the regenerated silk fibroin aqueous solution.

6. The preparation method according to claim 1, characterized in that, The specific method for adding monovalent and divalent ions in alternating order is as follows: First, potassium ions, a monovalent metal ion, are introduced into the regenerated silk fibroin aqueous solution. Then, magnesium ions, a divalent metal ion, are introduced. Next, sodium ions, a monovalent metal ion, are introduced. Then, calcium ions, a divalent metal ion, are introduced. Finally, iron ions, a trivalent metal ion, are introduced.

7. The preparation method according to claim 1, characterized in that, The specific method for adding ions according to their radii of the same valence is as follows: Potassium ions, sodium ions, magnesium ions, calcium ions, and iron ions are introduced sequentially into the regenerated silk fibroin aqueous solution according to the order of the radii of metal ions of the same valence from smallest to largest or a specific spatial occupancy logic.

8. The preparation method according to claim 1, characterized in that, After introducing sodium ions, potassium ions, calcium ions, magnesium ions, and iron ions, the sodium ion content in the multi-metal ion regenerated silk fibroin aqueous solution is 0.5~1.5 mg / g, the potassium ion content is 5~14 mg / g, the calcium ion content is 0.3~1.2 mg / g, the magnesium ion content is 0.8~1.6 mg / g, and the iron ion content is 0.01~0.03 mg / g.

9. A regenerated silk fibroin solution of silkworm gland protein prepared by the preparation method according to any one of claims 1-8.

10. The application of the regenerated silk fibroin solution of the silkworm gland protein as described in claim 9 in fiber spinning and / or preparation of biomaterials.

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