Silk fibroin nanocage and application thereof

Abstract

The application discloses a silk fibroin nanocage and application thereof. The silk fibroin nanocage is self-assembled from silk fibroin fragments with a specific molecular weight distribution, and the size of the nanocage can be regulated by selecting different molecular weight fragments. The nanocage is used as a template to guide the reaction of metal ions and a sulfur source in a strong alkali environment, so that metal sulfide nanoparticles with a core-shell structure can be prepared, and the particle size can be controlled in the range of 5-50 nm. By selecting nanocage templates with different molecular weights, the functions of the nanoparticles can be directionally optimized, so that the nanoparticles are respectively applicable to efficient photothermal therapy, high-contrast medical imaging, or photothermal immune synergistic therapy and tissue repair. The application realizes universal and accurate regulation of the size and function of nanomaterials from a template source, and the nanoparticle products have good biocompatibility and excellent performance, and have wide application prospects in the fields of tumor diagnosis and treatment, antibacterial and anti-inflammatory, and regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to a silk fibroin nanocage and its applications. Background Technology

[0002] Metal sulfide nanoparticles (such as CuS, Ag2S, Bi2S3, and CdS) exhibit great potential for application in the biomedical field due to their unique optical, electrical, and catalytic properties, particularly in photothermal therapy (PTT), photoacoustic imaging (PAI), drug delivery, antibacterial activity, and immunomodulation. However, the effectiveness of these applications is highly dependent on the size, morphology, dispersibility, and biocompatibility of the nanoparticles. Traditional chemical synthesis methods (such as hydrothermal methods and high-temperature pyrolysis methods) typically have the following inherent drawbacks: First, the reaction conditions are harsh, requiring high temperatures, high pressures, or the use of toxic organic solvents and external reducing agents, making the process environmentally unfriendly and costly. Second, particle size and morphology are difficult to control precisely, and the products are prone to aggregation, resulting in a wide particle size distribution, which seriously affects the uniformity of their optical properties and the reliability of their biological functions. Third, they have poor biocompatibility; residual organic ligands on the surface or leakage of heavy metal ions due to inadequate encapsulation may cause cytotoxicity, limiting their clinical translation prospects. Fourth, they lack universality; different metal sulfides often require differentiated synthetic strategies, and there is a lack of a unified and mild control method.

[0003] To overcome the aforementioned problems, utilizing natural proteins as templates for green and biomimetic synthesis has become an important strategy. For example, albumins (such as bovine serum albumin, BSA), gelatin, and ferritin are widely used as nanoreactors due to their excellent biocompatibility and abundant functional groups. These proteins unfold their structures under alkaline conditions, and their internal hydrophobic cavities or surface carboxyl and amino groups can capture metal ions, achieving in-situ nucleation and growth. However, these protein templates still have significant limitations: albumin has few free thiol (Cys) sites, limiting its coordination ability and regulatory precision; gelatin's amino acid sequence is relatively disordered, resulting in insufficient stability of the template structure; and ferritin has a fixed and narrow internal lumen size (approximately 8 nm), greatly limiting the range of nanoparticle sizes it can support and its application flexibility. Therefore, developing a protein template that combines excellent biocompatibility, structural tunability, broad applicability, and precise size control is a key problem urgently needing to be solved in this field.

[0004] Silk fibroin (SF), a natural polymer extracted from silkworm silk, is characterized by its wide availability, biodegradability, low immunogenicity, and excellent mechanical properties. In existing technologies, silk fibroin has been explored for improving the properties of nanocomposites. For example, Memoona Akhtar et al. (Biomed. Mater. 19 (2024)035016) introduced a small amount of silk fibroin into a sodium alginate-gelatin matrix to improve the dispersibility and hydration stability of Cu-Ag-doped sulfide glass nanoparticles. Parisa Heydari Foroushani et al. (Nanomaterials 2022,12, 3426) utilized the carboxyl groups of silk fibroin to react with Ag... + Coordination and stabilization of silver nanoparticles were used to prepare composite nanofibers. Guomei Zhang et al. (Sensors & Actuators: B. Chemical 279 (2019) 361-368) reported the synthesis of copper nanoclusters (F@CuNCs) with an average particle size of about 8.468 nm using silk fibroin as a template.

[0005] However, according to the search and analysis, the existing applications of silk fibroin mainly focus on: (1) as an auxiliary dispersant or mechanical reinforcing phase incorporated into composite materials; (2) using some of its functional groups to stabilize zero-valent metal nanoparticles (such as Ag, Au) or extremely small metal nanoclusters (usually less than 2 nm). Currently, there are no reports on the systematic synthesis and precise control of various "depositional" metal sulfide nanoparticles (such as CuS, Ag2S, Bi2S3, CdS) under mild conditions by using the silk fibroin molecule itself as a designable and controllable core template, through its rich amino acid coordination sites (such as the carboxyl groups of Asp and Glu, the hydroxyl groups of Ser and Tyr, and the thiol groups of Cys) and controllable molecular chain length. The existing technology has failed to reveal the intrinsic law between the molecular weight of silk fibroin and its template spatial confinement ability, and it is also impossible to achieve universal control of the size and function of different types of metal sulfide nanoparticles by regulating protein precursors. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a silk fibroin nanocage and its application. Based on the core strategy of silk fibroin molecular weight classification, a series of metal sulfide nanoparticles are prepared universally, precisely, and controllably under mild conditions. The resulting products have both excellent photothermal / imaging properties and biocompatibility, thus constructing a highly efficient and multifunctional biomedical application platform.

[0007] This invention is achieved through the following technical solution:

[0008] A silk fibroin nanocage is formed by the self-assembly of silk fibroin fragments with a molecular weight distribution selected from any of the following groups, and its size is controlled by the molecular weight of the selected silk fibroin fragments:

[0009] (a) 10~66 kDa, 34~135 kDa or 65~220 kDa;

[0010] (b) 10~60 kDa, 60~115 kDa or 120~250 kDa;

[0011] (c) 10~45 kDa, 50~110 kDa or 100~300 kDa;

[0012] The nanocage is formed and maintained in its unfolded conformation by the self-assembly of silk fibroin fragments in a strongly alkaline environment with a pH value of not less than 11.

[0013] Preferably, the surface or inner cavity of the nanocage has a main coordination network composed of aspartic acid, glutamic acid, serine, and tyrosine, as well as auxiliary reduction sites provided by trace amounts of cysteine ​​residues.

[0014] Preferably, the molecular weight of the silk fibroin fragment determines the confined space topology of the nanocage, specifically:

[0015] (1) When the molecular weight distribution is in the range of 10~66 kDa, the nanocage forms a multi-molecular aggregate structure with elastic deformation capability to adapt to crystal growth;

[0016] (2) When the molecular weight is distributed in the range of 34~135 kDa, the nanocage forms a single-molecule dense spherical structure with a rigid confined cavity;

[0017] (3) When the molecular weight distribution is in the range of 65~220 kDa, the nanocage forms a three-dimensional steric structure with a cage-like core and abundant chain segments on the surface.

[0018] Preferably, the confined space size of the nanocage is significantly correlated with the amino acid and molecular weight of the silk fibroin fragment, such that:

[0019] (1) Nanocages with molecular weight distribution in the range of 10~66 kDa are suitable for guiding the formation of metal sulfide nanoparticles with an average particle size of 6~50 nm.

[0020] (2) Nanocages with a molecular weight distribution in the range of 34~135 kDa are suitable for guiding the formation of metal sulfide nanoparticles with an average particle size of 2~10 nm.

[0021] (3) Nanocages with a molecular weight distribution in the range of 65~220 kDa are suitable for guiding the formation of metal sulfide nanoparticles with an average particle size in the range of 4~15 nm.

[0022] A type of metal sulfide nanoparticle coated with silk fibroin is prepared from the above-mentioned silk fibroin nanocage; the nanoparticle has a core-shell structure, with the core being a metal sulfide and the shell being silk fibroin; the particle size of the nanoparticle is 5~50 nm.

[0023] Preferably, the metal sulfide is Ag2S, CuS, Bi2S3, CdS, ZnS, PbS, or MoS2.

[0024] A method for preparing the above-mentioned silk fibroin-coated metal sulfide nanoparticles includes the following steps:

[0025] Step 1) Provide the above-mentioned silk fibroin nanocage;

[0026] Step 2) In a strongly alkaline environment, metal ions are added to coordinate with silk fibroin;

[0027] Step 3) Add a sulfur source and react within the confined space of the nanocage to generate the metal sulfide nanoparticles.

[0028] A pharmaceutical composition comprising the aforementioned silk fibroin-coated metal sulfide nanoparticles and a pharmaceutically acceptable carrier.

[0029] The above-mentioned silk fibroin nanocages are used as templates in the preparation of metal sulfide nanoparticles.

[0030] The above-mentioned silk fibroin-coated metal sulfide nanoparticles can be used in the preparation of any of the following drugs or formulations:

[0031] (a) Photothermal therapy preparations;

[0032] (b) Medical imaging contrast agents;

[0033] (c) Antibacterial, anti-inflammatory or tissue-repairing drugs or medical dressings.

[0034] The beneficial effects of this invention are as follows:

[0035] (1) This invention innovatively proposes to directly control the size of the "nanocage" formed by the self-assembly of silk fibroin precursors by regulating their molecular weight, thereby achieving precise and continuous control over the particle size (5~50 nm) of different types of metal sulfides (such as Ag2S, CuS, Bi2S3). This cascade control strategy of "molecular weight-cage size-particle size" is something that traditional protein templates such as albumin (fixed molecular weight) and ferritin (fixed cavity) cannot achieve. Experimental data show that different molecular weight fragments can induce the formation of Ag2S nanoparticles of large (approximately 18.7 nm), medium (approximately 8.1 nm), and small (approximately 6.0 nm) sizes, respectively, and this pattern is universally applicable to CuS and Bi2S3 systems, solving the problem of wide and uncontrollable particle size distribution in traditional methods.

[0036] (2) This invention utilizes the abundant natural functional groups (carboxyl groups, hydroxyl groups, etc.) of silk fibroin to efficiently complex various metal ions (Ag) in a normal temperature, normal pressure, and aqueous environment. + Cu 2+ Bi 3+ Cd 2+ This process requires no high temperature or pressure, organic solvents, or additional reducing agents. It is mild, environmentally friendly, and low-cost, providing a unified solution for the green synthesis of various biomedical metal sulfides, overcoming the harsh conditions and lack of versatility of traditional synthesis methods.

[0037] (3) The present invention utilizes the precise confinement effect of silk fibroin templates to prepare nanoparticles with good crystallinity and uniform dispersion, exhibiting excellent functional properties. Experimental data show that the photothermal conversion efficiency of Ag2S nanoparticles prepared from low molecular weight fragments is as high as 51.02%, and efficient heating (ΔT > 35℃) can be achieved within 5 min under 808 nm laser irradiation, which is significantly better than many previously reported similar materials. In addition, Bi2S3 nanoparticles prepared from medium molecular weight fragments exhibit excellent CT imaging performance, with a contrast efficiency approximately 2.6 times that of clinical iodine contrast agents, achieving targeted optimization of diagnostic and therapeutic performance.

[0038] (4) The silk fibroin used in this invention has low immunogenicity and is biodegradable. Its complete outer shell effectively isolates the potential toxicity of the core metal sulfide and prevents ion leakage. Cell experiments show that the cell survival rate of the material exceeds 90% at effective concentrations, and no obvious systemic toxicity was observed in in vivo experiments. At the same time, the core-shell structure endows the nanoparticles with storage stability (4~37℃) for several months and good physiological environment dispersibility. Based on this, the material of this invention can not only be used as a highly efficient photothermal therapy agent and imaging contrast agent, but also exhibits significant antibacterial, anti-inflammatory (ROS scavenging) and tissue repair promotion functions, realizing "one material for multiple uses".

[0039] (5) The silk fibroin nanocages constructed in this invention can not only be used to synthesize single metal sulfides, but their surface active groups and cage-like structure are also easily functionalized. Experimental results show that these silk fibroin nanocages can be conveniently coupled with targeting peptides (such as RGD) to achieve active tumor targeting, or loaded with chemotherapeutic drugs (such as doxorubicin) to construct a "photothermal-chemotherapy" synergistic treatment system. This modular design enables the platform to flexibly integrate multiple functions such as diagnosis, treatment, and targeting, providing a core technology and material basis for the development of a new generation of intelligent nanotherapeutic agents. Attached Figure Description

[0040] Figure 1 Here are the protein electrophoresis images and average molecular weights of different silk fibroin fragments in Example 3;

[0041] Figure 2 Optimization of reaction conditions for silk fibroin silver sulfide nanoparticles in Example 4: A is a graph showing the relationship between reaction time and absorbance; B is a graph showing the relationship between different silk fibroin molecular fragments and absorbance; C is a graph showing the relationship between different silk fibroin molecular fragments and the effect of heating; D is a graph showing the effect of Ag... + :S 2- The relationship between feed ratio and absorbance; E represents Ag. + :S 2- The graph shows the relationship between the feed ratio and the heating effect; F shows the relationship between the reaction concentration and absorbance; G shows the relationship between the reaction concentration and the heating effect; H shows the relationship between the reaction temperature and absorbance; and I shows the relationship between the reaction temperature and the heating effect.

[0042] Figure 3 Morphology and structure of silver sulfide nanoparticles of different molecular fragments of silk fibroin in Example 5: A is a size comparison diagram of each nanoparticle; B is an X-ray diffraction pattern; C is a transmission electron microscope image of each nanoparticle.

[0043] Figure 4 Example 6 shows the silk fibroin template regulating multiple metal sulfide nanoparticles: A is the average particle size diagram of copper sulfide nanoparticles with different silk fibroin molecular fragments; B is the average particle size diagram of bismuth sulfide nanoparticles with different silk fibroin molecular fragments; C is the transmission electron microscope image of two metal sulfides with different valence states.

[0044] Figure 5 The photothermal properties of low molecular weight silk protein silver sulfide nanoparticles in Example 7: A represents different Ag... + A is the graph showing the relationship between concentration and absorbance; B is the graph showing the photothermal conversion efficiency (the inset shows the linear fitting graph during the cooling stage); C is the graph showing the temperature rise stability under photothermal cycling.

[0045] Figure 6The stability evaluation of the low molecular weight silk protein silver sulfide nanoparticles in Example 8 is as follows: A is the particle size stability under different storage environments and at different times; B is the temperature rise stability under different storage environments and at different times.

[0046] Figure 7 To illustrate the in vitro cellular biocompatibility of the low molecular weight silk fibroin silver sulfide nanoparticles in Example 9: A shows the cytotoxic cell morphology image of U87 MG cells incubated with the low molecular weight silk fibroin silver sulfide nanoparticles; B shows the cytotoxicity statistics; C shows the uptake of U87 MG cells by the low molecular weight silk fibroin silver sulfide nanoparticles.

[0047] Figure 8 The in vitro photothermal properties of the low molecular weight silk fibroin fragment in Example 10;

[0048] Figure 9 The following is a comparison of the in vivo animal-level photothermal therapy effect of low molecular weight silk fibroin fragments in Example 11: A is a comparison of the temperature change of the tumor site in mice of different experimental groups under near-infrared laser irradiation over time; B is a comparison of the size of the tumor tissue in mice of different experimental groups; C is a comparison of the weight change of mice of different experimental groups over time; D is a comparison of the tumor volume change of mice of different experimental groups over time.

[0049] Figure 10 For the pathological analysis of tumor tissue after in vivo photothermal therapy of low molecular weight silk fibroin in Example 12: A is Ki67 immunofluorescence image of U87 MG tumors with different treatments after 18 days of treatment; B is a comparison of the proportion of positive cells in different experimental groups; C is H&E staining of U87 MG tumors with different treatments after 18 days of treatment.

[0050] Figure 11 The antibacterial properties of copper sulfide nanoparticles with polymeric fragments in Example 13 are shown in Figure A: images of copper sulfide nanoparticles with polymeric fragments at different concentrations; Figure B: inhibition rate of copper sulfide nanoparticles with polymeric fragments at different concentrations against Escherichia coli; Figure C: inhibition rate of copper sulfide nanoparticles with polymeric fragments at different concentrations against Staphylococcus aureus.

[0051] Figure 12 Evaluation of the in vitro anti-inflammatory and reactive oxygen species scavenging properties of the high molecular weight fragment silk fibroin copper sulfide nanoparticles in Example 14: A is an inverted fluorescence microscope image of intracellular ROS levels in RAW264.7 cells under different treatments; B is a fluorescence intensity statistical graph.

[0052] Figure 13 This is a comparison chart of the CT imaging efficiency (slope) of medium molecular weight silk fibroin bismuth sulfide nanoparticles and iohexol in Example 15.

[0053] Figure 14This is a quantitative graph showing the wound closure rate of mouse skin lesions repaired by the high molecular weight fragment silk fibroin silver sulfide nanoparticles in Example 16.

[0054] Figure 15 This is a statistical graph showing the distribution of fluorescence intensity of the polypeptide-modified silk fibroin inorganic nanoparticle drug in mice in Example 17. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0056] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0057] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] Example 1: Preparation of silk fibroin templates with different molecular weight distributions

[0059] The core of this invention lies in using silk fibroin with different molecular fragments as templates to regulate the size of nanocages, thereby regulating the size of metal sulfide nanoparticles. The length of the silk fibroin molecular chain, i.e. the size of the molecular fragment, directly determines its spatial confinement ability and coordination site density.

[0060] To verify this pattern, this embodiment used silk fibroin with three different molecular weight ranges: low, medium, and high. It should be noted that the method for obtaining silk fibroin with a specific molecular weight distribution is not limited to the thermal degradation method described in this embodiment. Those skilled in the art can also obtain silk fibroin fragments with specific molecular weights that meet the requirements of this invention through enzymatic hydrolysis (e.g., controlled hydrolysis using trypsin or papain), physical degradation (e.g., ultrasonic disruption, high-pressure homogenization), chemical reagent degradation (e.g., acid-base hydrolysis), or membrane separation / gel chromatography fractionation (for fractionating commercially available broadly distributed silk fibroin). As long as the molecular weight of the silk fibroin falls within the scope of protection of this invention, subsequent size control effects can be achieved.

[0061] The specific preparation process (taking thermochemical degradation as an example) is one implementation method selected in this embodiment. The degree of molecular chain breakage is controlled by adjusting the degumming time (thermal degradation time) and the dissolution system. Three different molecular fragments of silk fibroin were prepared by setting three different degumming times and dissolution systems, namely high molecular weight silk fibroin, medium molecular weight silk fibroin, and low molecular weight silk fibroin.

[0062] After equilibration at room temperature, the liquid sample was transferred to a dialysis bag with a molecular weight cutoff of 3500 kDa and immersed in ultrapure water for dialysis for 72 hours, with the pure water replaced every 6 hours to thoroughly remove residual electrolytes through the concentration gradient. The purified silk fibroin solution was then placed in a refrigerated centrifuge (constant 4°C) and centrifuged at 9000 rpm for 20 minutes to effectively separate the precipitate from the target components. The clear liquid layer was finally collected to obtain a high-purity silk fibroin solution containing different molecular fragments.

[0063] Concentration determination of silk fibroin solution: The content of active ingredients in the solution was determined by oven drying and weighing. Three replicate sample groups were required during the experiment to ensure data accuracy. The concentration of the silk fibroin solution (c, w / w) was calculated using the following formula.

[0064]

[0065] In the formula: W0 is the initial mass of the empty dish, W1 is the mass of the silk fibroin solution before drying, and W2 is the mass of the silk fibroin after drying.

[0066] This invention selects three specific ranges: 65–220 kDa (SF1), 34–135 kDa (SF2), and 10–66 kDa (SF3), based on the nonlinear structural evolution and amino acid composition gradient of silk fibroin during degradation. Specifically, the high molecular weight fragment (SF1) retains a relatively complete heavy chain structure and long chain entanglement network, with moderate content of Asp / Glu and other coordinating groups. It mainly stabilizes nanoparticles through high viscosity and strong steric hindrance, preventing excessive aggregation and tending to form medium-sized, well-dispersed particles. The medium molecular weight fragment (SF2) is in an intermediate degradation state, where hydrophilic random coil regions preferentially hydrolyze, resulting in a low total content of metallophilic amino acids (Asp, Glu, Ser, Tyr, Cys) in this fragment. Due to the fewest nucleation sites, crystal growth is strictly limited, making this range a key window for preparing small-sized nanoparticles. Low molecular weight fragments (SF3) have a large number of peptide bonds broken due to deep degradation, exposing a high density of terminal residues (such as thiol groups of Cys and phenolic hydroxyl groups of Tyr). They have a high content of metalophilic amino acids, and the high density of reducing end groups and extremely short molecular chains endow them with extremely fast nucleation kinetics and reduction ability. They tend to induce rapid crystal growth to form large-sized particles, making them the optimal range for preparing highly efficient photothermal agents.

[0067] Table 1. Preparation processes and distribution ranges of silk fibroin molecular weight fragments from different sources.

[0068]

[0069] As shown in Table 1, although silk fibroin from different sources differ in sequence structure and physicochemical properties (e.g., tussah silk has a more compact structure and requires dissolution using a LiSCN system, while recombinant proteins require genetic engineering or enzymatic digestion for control), through targeted process optimization, all can obtain high, medium, and low molecular weight ranges that match those of silkworm fibroin. As long as the silk fibroin fragments from the aforementioned different sources fall within the molecular weight range defined in this invention, they all expose sufficient carboxyl and hydroxyl groups and other metal-loving sites, exhibiting consistent metal ion confinement and mineralization regulation functions, thus strongly supporting the scope of protection of this invention regarding the wide range of silk fibroin sources.

[0070] Example 2

[0071] To verify the core mechanism by which the molecular weight of the silk fibroin fragments determines the confined spatial topology of the nanocages described in this invention, this embodiment employs dynamic light scattering (DLS) technology to non-destructively measure the hydrodynamic particle size (hydrated particle size) and dispersibility of nanocages formed by the self-assembly of different molecular fragments under a non-metallic initial environment (strongly alkaline conditions). Specifically:

[0072] The high-molecular-weight silk fibroin fragments (SF1, average molecular weight approximately 130 kDa), medium-molecular-weight silk fibroin fragments (SF2, average molecular weight approximately 68 kDa), and low-molecular-weight silk fibroin fragments (SF3, average molecular weight approximately 35 kDa) prepared in Example 1 were dissolved in deionized water to prepare solutions with a concentration of 5 mg / mL. The pH of each solution was precisely adjusted to 12 using 1 M sodium hydroxide solution, placing them in the same strongly alkaline cage-forming environment as used in the preparation of metal sulfides. The solutions were stabilized at room temperature for 2 hours to allow the silk fibroin fragments to fully expand and self-assemble into nanocages. A suitable amount of the above solutions was placed in a sample cell, and the hydrated particle size distribution and polydispersity index (PDI) were measured using a dynamic light scattering instrument at 25°C. Each sample was measured in triplicate, and the average value was taken.

[0073] (1) Medium-molecular-weight silk fibroin (SF2) group: DLS results showed that it exhibited an extremely narrow single-peak distribution (PDI < 0.15) and an extremely small average hydrated particle size (approximately 3.5 ± 0.8 nm). This hydrodynamic dimension perfectly matches the theoretically dense radius of a 68 kDa protein monomer after high folding, physically confirming that it did not undergo intermolecular crosslinking or aggregation in solution. This objective data strongly supports its product characteristic of forming a "monomer dense spherical structure with rigid confined cavities". It is this monomolecular-level physical confinement space that enables the nanocage to strictly limit the growth of crystal nuclei.

[0074] (2) Low molecular weight silk fibroin (SF3) group: Although its molecular weight is the smallest among the three, the hydrated particle size measured by DLS is significantly larger (about 10.4±3.5 nm), and the particle size distribution is relatively wide. This anomalous hydrodynamic phenomenon of the smallest molecular weight but a surge in physical size directly proves that low molecular weight fragments cannot exist stably as monomers under strong alkali, but spontaneously assemble into larger "multi-molecular aggregate structures". This loose aggregate composed of multiple short fragments gives the nanocage "elastic deformation ability to adapt to crystal growth", and can be expanded as the crystal grows like a micro network, thereby accommodating the formation of large-sized metal sulfides such as silver sulfide with a particle size of 18.7±2.0 nm.

[0075] (3) The silk fibroin (SF1) polymer fragment group: DLS showed that its hydrated particle size was approximately 6.8 ± 2.1 nm. This size is significantly larger than the theoretical value of a tightly folded single molecule, but smaller than the large-scale aggregation size of SF3. Combined with the hydrodynamic properties of the polymer, it can be seen that the folding of its macromolecular backbone forms a dense cage-like core, while the excess folded long chain segments extend fully into the aqueous phase, greatly increasing its hydrodynamic friction radius. This feature confirms that it forms a "steric hindrance structure with a cage-like core and excess chain segments on the surface". These excess chain segments constitute a strong steric hindrance shielding layer, preventing excessive aggregation of the final synthesized nanoparticles.

[0076] This embodiment directly and non-destructively verifies, through macroscopic hydrodynamic physical parameters, that the confined spatial topology (monomolecule compact, multimolecule aggregate, or steric hindrance) of the silk fibroin nanocage product, which is the core invention, is entirely determined by the molecular weight of the selected fragment.

[0077] Example 3: Molecular weight determination and amino acid composition of different silk fibroin fragments

[0078] 1. Molecular weight detection

[0079] SDS-PAGE analysis of silk fibroin proteins of different molecular weights was performed, as follows:

[0080] (1) The lyophilized silk protein powder obtained in Example 1 was reconstituted with deionized water to prepare a 1% (w / v) solution. It was then mixed with 5×SDS-PAGE loading buffer in proportion. The protein was fully denatured by vortexing and boiling in a water bath for 5 min. The protein was then centrifuged at 4℃ and 12000 rpm for 2 min. The supernatant was taken as the sample to be tested.

[0081] (2) Vertical electrophoresis was performed using a 10% precast gel. 10 μL of sample and 5 μL of protein marker were added to the sample wells, and electrophoresis was performed in Tris-MOPS-SDS buffer at 80 V for 2 h.

[0082] (3) After electrophoresis, stain the gel according to the instructions of Beyotime rapid silver staining kit.

[0083] (4) Each sample was tested three times. The gray values ​​of the bands were analyzed using ImageJ software. The molecular weight distribution of the protein marker was compared with that of the sample. The proportion of each molecular weight range was calculated, and the average molecular weight and main distribution range of the sample were determined. By controlling the degumming time and the dissolution system, silk fibroin with different molecular weight distributions were obtained, as shown in Table 2.

[0084] 2. Verification of amino acid composition

[0085] To verify the amino acid composition characteristics of structurally stable functional fragments, this example performed amino acid analysis on different prepared silk fibroin molecular fragments, as detailed below:

[0086] 100 mg of the lyophilized sample prepared in Example 1 was placed in a hydrolysis tube, and an appropriate amount of 6 mol / L hydrochloric acid solution was added. The tube was vacuum sealed and hydrolyzed at 110°C for 24 h. After cooling, the volume was adjusted, filtered, and evaporated to dryness. Then, 0.02 mol / L hydrochloric acid solution was added and the tube was air-dried. The amino acid content was then determined using an amino acid analyzer, as shown in Table 3. The formula for calculating the amino acid content is as follows:

[0087]

[0088] In the formula: c n c represents the molar concentration of a certain amino acid within silk fibroin. 总 This represents the total molar concentration of all amino acids in silk fibroin.

[0089] 3. Experimental Results and Analysis

[0090] Table 2. Molecular weight test results of silk fibroin of different molecular fragments

[0091]

[0092] Depend on Figure 1As shown in Table 2, the high molecular weight fragments are mainly concentrated in a continuous distribution between 65 kDa and 220 kDa, with an average molecular weight of 130 kDa. A clear band exists at 25 kDa, representing the L-chain or P25 chain of silk fibroin. However, with prolonged degumming time, this molecular chain segment is further decomposed, and it disappears in the bands of low molecular weight silk fibroin fragments. The medium molecular weight silk fibroin fragments are mainly distributed in the range of 34–135 kDa, with a molecular weight between that of the high and low molecular weight fragments, and an average molecular weight of 68 kDa. The low molecular weight silk fibroin fragment bands are mainly distributed between 10–66 kDa, with an average molecular weight of 35 kDa. Analysis and comparison of the average molecular weights of the three silk fibroin fragments show that their average molecular weight changes in a gradient with changes in the silk fibroin preparation process.

[0093] Table 3. Test results (range) of amino acid content (mol) of different molecular fragments of silk fibroin.

[0094]

[0095] Table 3 shows that the amino acid composition of silk fibroin changes significantly with decreasing molecular weight. Analysis of the main metalophilic amino acid content reveals that the contents of Asp, Glu, Ser, Tyr, and Cys generally increase with decreasing molecular weight, but these amino acids are lowest in medium molecular weight silk fibroin. High molecular weight silk fibroin (approximately 130 kDa) has a higher content of metalophilic amino acids, exhibiting a certain coordination ability, resulting in medium-sized nanoparticles. Medium molecular weight silk fibroin (approximately 68 kDa) has the lowest proportion of metalophilic amino acids, resulting in the fewest active sites that can bind to metal ions, leading to a slow nucleation rate, limited crystallization, and the smallest generated particles. Low molecular weight silk fibroin (approximately 35 kDa) has the highest contents of Asp, Glu, Ser, Tyr, and trace amounts of Cys, exhibiting the strongest coordination and reduction abilities, inducing rapid nucleation and easy aggregation growth, ultimately forming the largest nanoparticles.

[0096] Table 4. Nanocage structures and amino acid composition characteristics of silk fibroin formed by different molecular fragments.

[0097]

[0098] The silk fibroin nanocages of this invention possess a dual trapping structure of "physical confinement-chemical anchoring" for metal ions, with a size ranging from 5 to 50 nm. The chemical anchoring structure refers to the nanocage surface or interior cavity being rich in polydentate coordination sites composed of aspartic acid (Asp), glutamic acid (Glu), serine (Ser), tyrosine (Tyr), and cysteine ​​(Cys) residues, used for complexing metal ions. Furthermore, the physical confinement topology of the nanocage is determined by the molecular weight of its silk fibroin fragments, as shown in Table 4, and takes one of the following forms:

[0099] (1) Adaptive elastic nanocage: When the molecular weight is between 10 and 66 kDa (low molecular fragment), a multi-molecular aggregate structure with elastic deformation capability to adapt to crystal growth is formed.

[0100] (2) Rigid single-molecule nanocage: When the molecular weight is 34~135 kDa (medium molecular fragment), a single-molecule dense spherical structure with rigid confined cavity is formed.

[0101] (3) Steric hindrance type composite nanocage: When the molecular weight is 65~220 kDa (polymer fragment), a three-dimensional steric hindrance structure with a cage-like core and abundant chain segments on the surface is formed.

[0102] The experimental results of this embodiment show that, compared with traditional protein carriers such as albumin, gelatin, and ferritin, the silk fibroin fragments screened in this invention exhibit significant and unique advantages in terms of amino acid composition and molecular weight regulation. In terms of amino acid composition, unlike albumin which mainly relies on a limited number of free thiol groups and contains only one Cys site for single-point coordination, the silk fibroin fragments prepared in this invention are rich in high-density carboxyl groups (Asp / Glu, total content > 2.6%) and hydroxyl groups (Ser / Tyr, total content > 12%), constructing a stronger multidentate coordination network. This not only greatly improves the capture efficiency of metal ions but also effectively avoids the potential immunogenicity risk caused by the complex amino acid sequence of albumin. At the same time, compared with the disordered amino acid arrangement of gelatin, the unique GAGAGS repeating sequence of silk fibroin endows it with the ability to form β-sheet crystalline regions after nucleation, providing structural stability superior to that of gelatin. Regarding molecular weight tunability, as demonstrated in Example 1, silk fibroin overcomes the bottleneck of ferritin's fixed rigid cavity (approximately 8 nm inner diameter), which limits its loading capacity and size adjustment space. It also surpasses the size control limitations imposed by albumin's fixed molecular weight (approximately 66 kDa). This invention utilizes the controllable degradation characteristics of silk fibroin's long chains, employing a simple molecular weight grading strategy (high / medium / low) to achieve continuous, wide-range size control from ultrasmall quantum dots to highly efficient photothermal large particles, exhibiting universality and application flexibility unmatched by other single-molecular-weight protein carriers.

[0103] Based on the above correspondence between amino acid composition and nucleation mechanism, the silk fibroin described in this invention is not limited to silkworm fibroin. For tussah silkworm fibroin or recombinant silk fibroin, although their overall amino acid sequences may differ, such as the alanine repeat sequence, as long as they are degraded to expose sufficient metalophilic amino acid residues, especially carboxyl and hydroxyl sites, and fall within the molecular weight range described in this invention, the same metal ion confinement and mineralization regulation effects can be achieved.

[0104] Example 4: Investigation of the process window and reaction condition tolerance of silk fibroin inorganic crystalline nanoparticles

[0105] To verify the universality and process tolerance of the method for preparing inorganic crystalline nanoparticles using silk fibroin templates described in this invention, and to determine the effective parameter range for obtaining stable core-shell nanocage structures, silver sulfide (Ag2S) was selected as a typical representative (model material) in this embodiment. Using a controlled variable method, the general influence of silk fibroin molecular weight fragments, reactant feed ratios (metal / non-metal source), metal / protein ratio, and reaction temperature on the formation and properties of inorganic crystalline nanoparticles was systematically investigated. Although silver sulfide (Ag2S) was used as the model material, based on the general coordination mechanism of Asp, Glu, Cys groups in silk fibroin, this preparation process is also applicable to the preparation of other metal sulfide nanoparticles such as CuS, Bi2S3, and CdS.

[0106] 1. Preparation of metal sulfide nanoparticles

[0107] The silk fibroin silver sulfide nanoparticles were prepared using a biomineralization method, as detailed below:

[0108] First, silk fibroin solutions of different concentrations (5–80 mg / mL) were prepared. 12.5 mL of each solution was accurately transferred to a temperature-controlled magnetic stirrer. The temperature was gradually increased to the preset temperature (40°C–80°C), and then high-speed stirring was activated. 1 mL of 20 mM silver nitrate solution was slowly added dropwise to the reaction system using a pipette to promote uniform dispersion of the components. Next, the pH was adjusted using 1 M sodium hydroxide solution until the pH of the reaction system reached a strongly alkaline level (approximately 12). Then, 100 mM sodium sulfide solution was precisely added at a silver / sulfur molar ratio of 1:0.5–1:10. A constant-temperature water bath environment was maintained, and high-speed mechanical stirring was continuously performed for 2–12 hours to ensure complete mineralization. After the reaction was terminated, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and purified by continuous circulation in deionized water day and night to thoroughly remove any residual reaction precursors.

[0109] 2. Dynamic tracking of the growth process of silk protein silver sulfide nanoparticles

[0110] During the experiment, for the silver / sulfur reaction system with a molar ratio of 1:2, the silk fibroin solution was maintained at 20 mg / mL, and a constant temperature of 60℃ was set in a water bath. Samples were collected in real time at seven key time points (0, 120, 240, 360, 480, 600, and 720 min). Specifically, the absorption intensity of the mixture at 808 nm wavelength was measured using a UV-Vis spectrophotometer at each time point. A constant temperature environment was maintained during data acquisition to minimize interference from temperature fluctuations. The acquired time-series data were plotted as a growth curve, where the X-axis represents the reaction duration and the Y-axis corresponds to the optical response value at a specific wavelength. A nonlinear fitting algorithm was used to mathematically model the curve, and its first derivative curve was calculated to analyze the nucleation rate and growth stage characteristics of the nanoparticles.

[0111] To demonstrate that the target nanoparticles were "successfully generated" across a wide range of processes, this embodiment sets the following judgment thresholds based on the optical and thermal properties of semiconductor nanocrystals:

[0112] Optical determination line: The ultraviolet-visible absorbance of the sample at 808 nm must be ≥0.1 (after deducting background) to prove that an inorganic band structure with near-infrared absorption characteristics has been formed in the solution.

[0113] Functional determination line: Sample under 808 nm laser (1.5 W / cm²) 2 After irradiation for 5 minutes, the solution temperature rise needs to be ≥5.0℃, which proves that the material has effective photothermal conversion capability.

[0114] Colloid determination line: No macroscopic layering or precipitation was observed after the reaction solution stood for 24 hours, proving that a stable nanoscale dispersion system was formed.

[0115] 3. Investigation of the process range of silk protein silver sulfide nanoparticles

[0116] The photothermal properties of silver sulfide nanoparticles prepared from silk fibroin were improved by optimizing different silk fibroin fragments, reactant ratios, silk fibroin concentrations, and reaction temperatures. Parallel control experiments were conducted using three different silk fibroin fragments (SF1, SF2, and SF3); multiple Ag... + :S 2- Molar ratios (1:0.5, 1:1, 1:2, 1:3, 1:5, 1:7, 1:10); multiple silk fibroin concentrations (5, 10, 20, 40, 60, 80 mg / mL, corresponding to a molar ratio of metal salt to silk fibroin of approximately 1:5 to 1:20) and different reaction temperatures (40, 60, 80℃) were used to prepare silk fibroin silver sulfide nanoparticles. The preparation method was the same as above, and their properties were tested.

[0117] 4. Experimental Results

[0118] (1) Reaction time (0~720 min)

[0119] like Figure 2 As shown in Figure A, the absorbance gradually increases with increasing reaction time. The curves indicate that the reaction tends to be complete and stable around 120–240 min, suggesting that the silk fibroin template can effectively control crystal nucleation and growth termination.

[0120] (2) Silk fibroin molecular fragments (SF1, SF2, SF3)

[0121] like Figure 2 As shown in Figures B and C, nanoparticles prepared from the low molecular weight silk fibroin fragment (SF3) exhibit the highest absorbance and optimal heating effect (reaching approximately 53.7°C after 5 minutes of illumination); the high molecular weight fragment (SF1) can also induce nucleation, but the resulting crystals have smaller particle sizes. This embodiment lists three fragments, but is not limited to them; any fragment within this molecular weight range can serve as a mineralization template for inorganic crystals.

[0122] (3) Ag + :S 2- Feed ratio (1:0.5 to 1:10)

[0123] like Figure 2 As shown in D and E, with the non-metallic source (such as S) 2- As the ratio increases, the number of nuclei increases. After the ratio reaches 1:2, the performance improvement tends to plateau or saturate. This indicates that this ratio range is applicable to the synthesis of various metal sulfides or oxides.

[0124] (4) Reaction concentration (5~80 mg / mL, corresponding to a molar ratio of metal salt to silk fibroin of approximately 1:5~1:20)

[0125] like Figure 2 As shown in F and G, crystal growth can be effectively controlled within the range of 5–80 mg / mL. The optical / photothermal properties of the sample reach their peak at 20 mg / mL. Although the photothermal properties differ at concentrations that are too high (1:20) or too low (1:5), they each have advantages in colloidal stability or the preparation of large-size particles, respectively, and are both effective process points.

[0126] (5) Reaction temperature (40, 60, 80℃)

[0127] like Figure 2 As shown in Figures H and I, the sample prepared at 60℃ exhibits the best balance between crystallinity and dispersibility. The reaction kinetics are slower at 40℃, but rapid at 80℃. This indicates that the biomineralization process can proceed in the temperature range from room temperature to high temperatures.

[0128] Based on the experimental results of the above factors, and using silver sulfide as a verification model, the method described in this invention has a wide range of process adaptability. This embodiment lists the specific feed ratios, temperatures, and concentrations mentioned above, but is not limited to these. Within a metal salt to silk fibroin molar ratio of 1:5 to 1:20 and a temperature range of 40 to 80°C, silk fibroin can successfully regulate the nucleation and growth of inorganic crystals, preparing nanoparticles with a core-shell structure. Although the specific performance indicators (such as the photothermal heating amplitude) of the products differ under different process conditions (such as concentration and temperature), they all conform to the structural characteristics of silk fibroin encapsulating inorganic crystals. Among them, nanoparticles prepared using low-molecular-weight silk fibroin fragment (SF3) as a template, controlling the silk fibroin concentration at 20 mg / mL (corresponding to a metal / protein ratio of approximately 1:10), the precursor feed molar ratio at 1:2, and reacting under a constant temperature water bath at 60°C for 240 min, exhibit excellent photothermal conversion performance.

[0129] Example 5 Morphology and structural characterization of silver sulfide nanoparticles with different molecular fragments of silk fibroin

[0130] Morphological characterization was performed on silk fibroin silver sulfide nanoparticles prepared with different molecular fragments. The macroscopic morphology of the protein nanoparticles was scanned using a transmission electron microscope (HT7700 TEM), and the crystal planes of the samples were measured using a high-resolution transmission electron microscope (Talos F200X G2 Talos field emission transmission electron microscope, HR-TEM). The samples were ultrasonically dispersed and then dropped onto a copper mesh for high-resolution imaging to obtain the morphological characteristics and interplanar spacing of the nanoparticles. The size of the silk fibroin silver sulfide nanoparticles prepared with different molecular fragments was statistically analyzed, with a statistical measure of 100. To determine the elemental composition and crystal characteristics of the silk fibroin silver sulfide nanoparticles, X-ray diffraction (XRD) was used to perform crystal phase analysis on the silk fibroin silver sulfide nanoparticles with different molecular fragments. The crystal structure was confirmed by matching characteristic diffraction peaks with JCPDS standard cards and the measured interplanar spacings, as shown in Table 5.

[0131] Table 5. Size and crystal form of silver sulfide nanoparticles of different molecular fragments of silk fibroin

[0132]

[0133] From Table 5 and Figure 3As shown in Figure A, the nanoparticle sizes of 6.0 nm, 8.1 nm, and 18.7 nm represent typical sizes under different process conditions. Combined with the control rules in Example 4, it can be seen that this invention can achieve continuous tunability of particle size within the range of 5 nm to 50 nm. The sizes of silk fibroin silver sulfide nanoparticles prepared from different molecular fragments of silk fibroin are different and show significant differences, with the nanoparticles prepared from low molecular fragments of silk fibroin having the largest size. Combined with the photothermal heating of silk fibroin silver sulfide nanoparticles with different molecular fragments and the test of the photothermal capacity of silver sulfide crystals at 808 nm UV absorbance in Example 4, the experimental results are consistent with the literature reports, that is, within the range of silver sulfide crystals smaller than 100 nm, its photothermal capacity is dependent on its size. It also demonstrates that the size of silk fibroin molecular fragments can regulate the content of specific amino acids, thereby further controlling the nucleation and growth of silver sulfide crystals. (See Table 5 and...) Figure 3 As shown in Figure B, X-ray diffraction (XRD) was used to analyze silk fibroin silver sulfide nanoparticles. The XRD patterns showed that the peaks were consistent with those of the standard monoclinic silver sulfide, indicating that the silver sulfide crystals within the nanoparticles were all monoclinic. Further high-resolution transmission electron microscopy (HRTEM) was used to analyze silk fibroin silver sulfide nanoparticles with different molecular fragments, and the interplanar spacing of each nanoparticle was calculated. The results showed that the crystal planes observed under high-resolution XRD matched the XRD results. Figure 3 As shown in Figure C, transmission electron microscopy of the selected three molecular fragments of silk fibroin silver sulfide nanoparticles after protein restaining reveals that the clearly visible silk fibroin shell (thickness 1~5nm) is the key feature that distinguishes this invention from existing bare nanoparticles. All three molecular fragments of silk fibroin silver sulfide nanoparticles have a protein-crystal core-shell structure.

[0134] In summary, the molecular weight of silk fibroin fragments and the particle size of metal sulfide nanoparticles exhibit a specific regulatory relationship. For the same metal sulfide system, the particle size (D) follows the trend of D (low molecular weight) > D (high molecular weight) > D (medium molecular weight), with the specific numerical range covering:

[0135] (1) Nanocages with a molecular weight distribution in the range of 10~66 kDa (low molecular weight) are suitable for guiding the formation of relatively large nanoparticles, with an average particle size usually in the range of 6~50 nm.

[0136] (2) Nanocages with a molecular weight distribution in the range of 34~135 kDa (medium molecular weight) are suitable for guiding the formation of ultra-small nanoparticles or quantum dots, with an average particle size usually in the range of 2~10 nm.

[0137] (3) Nanocages with a molecular weight distribution in the range of 65~220 kDa (high molecular weight) are suitable for guiding the formation of medium-sized nanoparticles with an average particle size in the range of 4~15 nm.

[0138] Example 6 Evaluation of the universality and size controllability of silk fibroin template-regulated multi-metal sulfide nanoparticles

[0139] To verify that the "silk fibroin molecular weight hierarchical regulation strategy" described in this invention is not only applicable to silver sulfide, but also a universal preparation platform applicable to various metal sulfides, this embodiment selects copper sulfide (CuS) and bismuth sulfide (Bi2S3), which have important applications in photothermal therapy and photoacoustic / CT imaging, as extended models to investigate the regulatory effects of silk fibroin proteins of different molecular weights (SF1, SF2, SF3) on the nucleation and growth of these different metal sulfide crystals.

[0140] To verify the universality of the "silk fibroin molecular weight hierarchical regulation strategy" described in this invention in the synthesis of inorganic crystals with different valence states and types, the same general biomineralization preparation process as described in Example 4 was used. Silk fibroin solutions with three different molecular weight distributions (high (SF1), medium (SF2), and low (SF3)) prepared in Example 1 (with a uniform concentration of 20 mg / mL) were used as mineralization templates to conduct parallel synthesis experiments of various metal sulfides. Details are as follows:

[0141] For the preparation of copper sulfide (CuS) nanoparticles, a copper acetate solution (20 mM) was used as the copper source precursor. This solution was added to the aforementioned silk fibroin solution, and the pH of the system was adjusted to 12 using 1 M sodium hydroxide solution under high-speed stirring to allow the protein chains to fully extend and expose binding sites. Subsequently, the CuS nanoparticles were prepared according to the Cu... 2+ :S 2- A 100 mM sodium sulfide solution was slowly added dropwise at a molar ratio of 1:1, and the reaction was carried out in a constant temperature water bath at 60°C for 4 h.

[0142] For the preparation of bismuth sulfide (Bi2S3) nanoparticles, bismuth nitrate solution (20 mM) was selected as the bismuth source. After adjusting the pH to 12, the bismuth was then... 3+ :S 2- Sodium sulfide solution was added at a molar ratio of 2:3, and the mixture was reacted in a constant temperature water bath at 60°C for 4 hours.

[0143] After the reaction was completed, the resulting dark colloidal solutions were placed into dialysis bags with a molecular weight cutoff of 3500 Da and dialyzed in flowing deionized water for 48 h to remove unreacted ions and small molecule byproducts, and finally a pure dispersion of CuS and Bi2S3 nanoparticles encapsulated in silk fibroin was obtained.

[0144] like Figure 4 As shown, TEM characterization and particle size statistical analysis of the two metal sulfides with different valence states revealed that the growth of inorganic crystals regulated by silk fibroin of different molecular weights exhibited a highly consistent and universal rule.

[0145] like Figure 4 As shown in Figure A, for the divalent metal model (SF@CuS NP), the high molecular weight group (SF1) formed dispersed particles with a particle size of approximately 5.0 ± 1.3 nm; the medium molecular weight group (SF2) exhibited a significant size confinement effect, forming ultrasmall quantum dots with an average particle size of only 2.4 ± 0.7 nm; while the low molecular weight group (SF3) induced the formation of larger particles with an average particle size of approximately 6.9 ± 1.5 nm. This trend confirms that the rule that "medium molecular weight tends to confine crystal nucleus growth, while low molecular weight tends to induce the formation of large particles" still holds true in the copper ion system.

[0146] like Figure 4 As shown in Figure B, for the trivalent metal model (SF@Bi2S3 NP), TEM characterization shows that its particle size regulation is consistent with that of the copper system: the high molecular weight fragment silk fibroin bismuth sulfide nanoparticle group forms uniformly distributed particles with an average particle size of about 4.5±1.2 nm; the medium molecular weight fragment silk fibroin bismuth sulfide nanoparticle group again exhibits the strongest size confinement effect, with extremely small particles and an average particle size of about 2.8±0.9 nm; while the low molecular weight fragment silk fibroin bismuth sulfide nanoparticle group induces the formation of highly crystalline large particles with an average particle size of about 8.2±1.8 nm.

[0147] This embodiment lists two metal sulfides, CuS and Bi2S3, but is not limited to them.

[0148] The experimental results of this embodiment strongly demonstrate the strong versatility and universal applicability of the silk fibroin template method described in this invention. Based on the abundant universal coordination sites such as Asp, Glu, Cys, and Ser in the silk fibroin chain, this method not only successfully regulates Ag + Cu 2+ Bi 3+ Beyond mineralization, the inventors have further utilized this universal system to successfully prepare CdS, ZnS, PbS, MoS2, and other transition metal or main group metal sulfide nanoparticles. Experimental results confirm that these extended systems can all achieve well-dispersed core-shell structures and strictly adhere to the aforementioned regulatory principle that "the molecular weight of silk fibroin determines the particle size."

[0149] In summary, this embodiment strongly supports the broad definition of "metal sulfide nanoparticles" in this invention with conclusive experimental facts, proving that the method is not limited to specific metals, but has broad applicability to various metal sulfide nanoparticles and their complexes, all of which fall within the protection scope of this invention.

[0150] Example 7: General Evaluation of the Photothermal Properties of Silk Fibroin Inorganic Crystal Nanoparticles

[0151] To evaluate the efficiency of the silk fibroin inorganic crystalline nanoparticles prepared in this invention in converting light energy into heat energy and their potential as a photothermal therapy agent, this embodiment selects silver sulfide nanoparticles prepared from low-molecular-weight silk fibroin fragments as a typical representative, and systematically evaluates their light absorption characteristics, photothermal heating capacity, and photothermal stability. Details are as follows:

[0152] The same Ag was analyzed using a UV-Vis spectrophotometer. + The absorption intensity of silver sulfide nanoparticle solutions prepared from low-molecular-weight silk fibroin fragments at concentrations was measured at 808 nm. To evaluate its photothermal heating capability, a series of solutions with different Ag concentrations were prepared. + Silver sulfide nanoparticle solutions prepared from low molecular weight silk fibroin fragments at concentration gradients (0.02, 0.05, 0.10, 0.20, 0.50, 1.00 mM) were aliquoted into centrifuge tubes at 300 μL each. The samples were then irradiated laterally using an 808 nm near-infrared laser with a power density of 1.5 W / cm². 2 The irradiation time was 10 minutes, and the solution temperature change was recorded in real time using an infrared thermal imager. Furthermore, to test the photothermal stability of the material, a 0.50 mM sample solution was selected and subjected to a cyclic test of "laser irradiation for 5 minutes followed by natural cooling to room temperature," repeated for 5 cycles, with the temperature change curve recorded. Finally, based on the heating-cooling data, the photothermal conversion efficiency was calculated using the heat balance formula.

[0153] Table 6 Different Ag + Temperature rise of silk fibroin silver sulfide nanoparticles at different concentrations

[0154]

[0155] As shown in Table 6 and Figure 5 As shown in Figure A, silver sulfide nanoparticles (SF3@Ag2S NP) prepared from low-molecular-weight silk fibroin fragments exhibit a significant absorption peak in the near-infrared region. Photothermal testing reveals that these nanoparticles exhibit a significant concentration-dependent photothermal effect: when Ag... + At a concentration of 1.00 mM, the temperature rapidly increased by approximately 50°C after irradiation for 5 minutes; when Ag... +At a concentration of 0.50 mM, the temperature rises by approximately 35°C, which is sufficient to meet the requirements of photothermal therapy for tumors (typically requiring 42-46°C). Calculations show that the photothermal conversion efficiency of the low-molecular-weight silk fibroin silver sulfide nanoparticles prepared in this invention is as high as 51.02%, significantly superior to silver sulfide nanodots synthesized from conventional bovine serum albumin (approximately 35.0%). In five photothermal cycle tests (… Figure 5 In the B and C sections, the maximum heating temperature of the nanoparticles remained stable throughout, without any obvious photobleaching or performance degradation, proving that the material has excellent photothermal stability and is suitable for long-term or repeated photothermal therapy.

[0156] Example 8: Stability evaluation of silk fibroin inorganic crystalline nanoparticles

[0157] To investigate the universal stability of the silk fibroin inorganic crystal nanoparticles prepared in this invention under different application scenarios, such as long-term shelf storage and in vivo circulation, this example selects Ag... + A solution of silver sulfide nanoparticles prepared from a 0.50 mM low-molecular-weight silk fibroin fragment was used as a typical example and stored in a light-protected, sealed environment under three representative temperature conditions: room temperature, 4°C, and 37°C. This example illustrates these three typical temperature conditions but is not limited to them. The experiment aims to demonstrate that the nanoparticles prepared by this invention possess excellent structural integrity and functional stability over a wide range (4°C~37°C) from conventional storage temperatures to biological physiological temperatures. Details are as follows:

[0158] Samples were taken at weeks 0, 1, 2, and 4 of storage. Dynamic light scattering (DLS) was used to detect changes in hydrated particle size, while an 808 nm near-infrared laser (1.5 W / cm²) was used for analysis. 2 Irradiate for 3 minutes and test the maintenance of its photothermal heating capacity. Three parallel samples are set up for each group to ensure data reliability.

[0159] like Figure 6 As shown in Figure A, the stability test results indicate that after storage at 4°C for 4 weeks, the hydrated particle size of the nanoparticles fluctuated only within a very small range from 31.29±1.83 nm to 35.78±0.84 nm, demonstrating excellent dimensional stability. At room temperature and a higher temperature of 37°C, although the particle size values ​​fluctuated slightly, no significant aggregation or precipitation was observed, indicating that the silk fibroin shell effectively prevented particle aggregation. This result strongly supports the protective effect of the silk fibroin shell on inorganic crystals, proving that the silk fibroin layer provides sufficient steric hindrance and electrostatic repulsion, effectively preventing the oxidation and aggregation of metal sulfides.

[0160] like Figure 6As shown in Figure B, in terms of photothermal performance, the temperature rise of samples stored at different temperatures and times under laser irradiation remained stable within the range of 32~35℃, with no significant difference from freshly prepared samples.

[0161] Although this embodiment uses silver sulfide as an example, given that all nanoparticles described in this invention (including CuS, Bi2S3, etc.) have the same "silk fibroin shell" structure, this stability conclusion applies to all silk fibroin inorganic nanoparticles covered by this invention. Therefore, it is demonstrated that the silk fibroin inorganic crystalline nanoparticles prepared by this invention possess excellent colloidal and chemical stability, which is beneficial for long-term preservation and clinical application. Furthermore, the excellent colloidal stability exhibited under a simulated physiological environment at 37°C proves that it can maintain a monodisperse state for a long time after entering the body, meeting the clinical requirements for use as a long-acting drug delivery carrier, blood pool contrast agent, or photothermal therapy agent, thus supporting the protection of this invention regarding the use of "drug delivery or tissue repair complexes" and "imaging enhancement agents."

[0162] Example 9: Evaluation of the biosafety and cellular uptake of silk fibroin inorganic crystalline nanoparticles

[0163] To evaluate the biocompatibility of the silk fibroin inorganic crystalline nanoparticles prepared in this invention at the cellular level and their ability to be targeted and taken up by tumor cells, and to determine their safe therapeutic concentration window, silver sulfide nanoparticles prepared from low-molecular-weight silk fibroin fragments were selected as a typical representative (validation model) in this embodiment. Human glioma cells (U87 MG) were used as model cells to conduct a comprehensive evaluation of cytotoxicity and endocytic behavior, as detailed below:

[0164] U87 MG human astroblastoma cells in the logarithmic growth phase were selected, digested with trypsin, and then injected with 3.0 × 10⁻⁶ cells. 3 Inoculate 10 cells / well into a 96-well plate and incubate for 24 h. Discard the old culture medium and add culture medium containing different Ag. + Fresh culture medium containing silk fibroin silver sulfide nanoparticles at concentrations (0.06, 0.13, 0.25, 0.50, 0.75 mM) was used for further incubation for 24 h. The experiment included a light-treated group and a control group without light. After incubation, the light-treated group received fresh culture medium and was exposed to an 808 nm laser (1.5 W / cm²). 2 Irradiate vertically for 3 min. After culturing for another 4 h, cell viability is assessed using the Alamar Blue assay kit.

[0165] U87 MG cells were also taken at 3.0 × 10⁻⁶. 4Cells were seeded per well in 24-well plates with pre-placed slides. After adhesion, culture medium containing 0.50 mM rhodamine B-labeled nanoparticles was added and incubated for 24 h. After culture was terminated, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and the cytoskeleton was stained with SF488-labeled phalloidin, with the nuclei counterstained with DAPI. Finally, the slides were placed under a laser confocal microscope for image acquisition.

[0166] like Figure 7 As shown in Figures A and B, the cytotoxicity test results indicate that, under conditions without near-infrared light irradiation, when Ag... + At concentrations of 0.50 mM and below, the survival rate of U87 MG cells remained consistently above 90%. This excellent biocompatibility is primarily attributed to the silk fibroin shell on the surface of the nanoparticles. This bioprotein layer not only effectively shields against the potential toxicity of the core inorganic crystals (such as metal ions) but also improves the material's hydrophilicity and cell affinity. Based on this "core-shell shielding" mechanism, other metal sulfides described in this invention (such as CuS, Bi2S3, CdS, etc.) also possess equivalent biocompatibility when encapsulated by silk fibroin.

[0167] Highly efficient cellular uptake: such as Figure 7 As shown in Figure C, laser confocal imaging reveals that red fluorescent signals (representing nanoparticles) are widely distributed within the cytoplasm and surround the cell nucleus, indicating that the nanoparticles can be efficiently internalized by tumor cells. This provides a cellular basis for the material's potential as a universal drug delivery carrier.

[0168] Example 10: Evaluation of the in vitro photothermal therapeutic efficacy of silk fibroin inorganic crystal nanoparticles

[0169] To visually verify the photothermal therapeutic effect (i.e., "light-controlled switching" killing ability) of the silk fibroin inorganic crystal nanoparticles prepared in this invention within cells, this embodiment selects copper sulfide nanoparticles (SF3@CuS NP) prepared from low molecular weight silk fibroin fragments as a typical representative. Using a live-dead cell double staining technique, the specific killing effect of this type of material on tumor cells under near-infrared light triggering was visualized. U87 MG cells were seeded in 96-well plates and cultured for 24 h before being divided into four groups: a blank control group (Control), a light-only group (NIR), a material-only group (SF@CuS NP), and a photothermal therapy group (SF@CuS NP+NIR). The material group and the therapy group were incubated with 0.50 mM silk fibroin copper sulfide nanoparticles for 24 h. Subsequently, the culture medium was aspirated and the cells were washed with PBS, and fresh culture medium was added. The light-only group and the photothermal therapy group received an 808 nm laser (1.5 W / cm²). 2Irradiate for 3 min, ensuring the light spot covers the central area of ​​the well plate. After culturing for another 4 h, discard the supernatant and add 100 μL of PBS working solution containing Calcein-AM (live cells stain green) and PI (dead cells stain red) to each well. Incubate at 37°C in the dark for 30 min. Finally, observe and photograph using a fluorescence inverted microscope.

[0170] like Figure 8 As shown in the fluorescence microscope images, the control group and the material-only group exhibited almost entirely high-brightness green fluorescence, indicating intact cell membrane structure and good growth, further confirming the conclusion in Example 9 that the material had no dark toxicity. In stark contrast, the photothermal therapy group showed a large area of ​​red fluorescence in the central region covered by the laser spot, with almost no green fluorescence, indicating that the cell membrane in this area was damaged, PI dye entered the cell nucleus, and the cells underwent necrosis and death. This further confirms that the cell survival rate in the photothermal therapy group was significantly reduced. This result confirms the high efficiency of the synergistic therapeutic mechanism of "silk fibroin-assisted endocytosis + inorganic crystal photothermal conversion". Since the silk fibroin shell does not hinder heat transfer and can carry various inorganic crystals with photothermal effects (such as CuS, Bi2S3, CdS, etc.) into the cell, any inorganic core with photothermal conversion properties is expected to achieve a similar photothermal killing effect after being encapsulated by the silk fibroin described in this invention, while providing conclusive in vitro efficacy evidence for subsequent in vivo anti-tumor experiments.

[0171] Example 11 Evaluation of the in vivo photothermal therapeutic efficacy of silk fibroin inorganic crystal nanoparticles

[0172] To evaluate the therapeutic potential of the silk fibroin inorganic crystalline nanoparticles prepared in this invention at the in vivo level, this embodiment selected silver sulfide nanoparticles prepared from low-molecular-weight silk fibroin fragments as a typical representative. A tumor-bearing mouse model was constructed, and the temperature-increasing behavior and tumor-suppressing effect of this type of material with a "core-shell structure" under intratumoral injection and light irradiation conditions were systematically investigated. Details are as follows:

[0173] U87 MG cells were subcutaneously instilled into the back of female BALB / c nude mice; tumors were allowed to grow to 60-100 mm. 3 When the difference in mouse weight does not exceed 10%, individuals that meet the criteria are randomly assigned to four experimental groups (n=4 in each group).

[0174] The saline group (PBS) was injected at specific points in the tumor area with 50 μL of sterile saline.

[0175] Saline combined with light irradiation group (PBS+NIR): After injection of an equal volume of saline, the patient was irradiated with 808 nm near-infrared light (power density 1.5 W / cm²). 2 (lasts 3 minutes)

[0176] Silk fibroin silver sulfide nanoparticle assembly (SF@Ag2S NP): Ag injection + Working solution for silver sulfide nanoparticles prepared from low molecular weight silk fibroin fragments at a concentration of 0.50 mM;

[0177] Silk fibroin silver sulfide nanoparticles combined with phototherapy (SF@Ag2S NP+NIR): Photothermal therapy was performed simultaneously on the basis of the SF@Ag2S NP group.

[0178] The treatment regimen lasted for 15 days, with intratumoral injections every 72 hours (for a total of 5 times). Four hours after each injection, the combined light irradiation group received 808 nm near-infrared light irradiation (power density 1.5 W / cm²). 2 (3 min). During the light exposure, infrared thermal imaging cameras were used to record infrared thermal images to monitor temperature changes at the tumor site; simultaneously, typical mouse photographs of different groups receiving different treatments at different times were recorded; over the next 18 days, mice from each group were removed every 2 days to measure their weight; and the tumor volume was estimated using the following formula:

[0179]

[0180] In the formula: L represents the length of the tumor; W represents the width of the tumor.

[0181] The relative volume of the tumor is calculated as: V / V0, where: V0 is the original tumor volume before photothermal therapy.

[0182] The tumor tissue inhibition rate is calculated using the following formula:

[0183]

[0184] The results are as follows Figure 9 As shown in Figure A, in the control group (PBS+NIR), the tumor temperature remained between 38°C and 39°C under near-infrared laser irradiation, showing no significant difference from the basal body temperature, confirming that near-infrared light irradiation could not produce an effective photothermal therapeutic effect. In contrast, the experimental group (SF@Ag2S NP+NIR) showed a significant temperature rise (41.6°C) within 1 minute of near-infrared light irradiation, reaching 45.1°C after 3 minutes of continuous irradiation. This temperature gradient met the threshold for irreversible tumor cell damage (42-46°C), and the temperature of adjacent normal tissue remained below 41°C. This indicates that silk fibroin nanoparticles are highly efficient at accumulating in the tumor site and possess excellent photothermal conversion capabilities, enabling tumor thermal ablation while effectively avoiding the risk of thermal damage to normal tissues.

[0185] In addition, different groups of tumor-bearing mice were treated with drugs, and their body weight and tumor volume were measured over the following 18 days. Figure 9 As shown in Figure D, under both near-infrared laser irradiation and non-near-infrared conditions, the tumor volume in the control groups (PBS and PBS+NIR) gradually increased, increasing approximately 12-fold compared to pre-treatment levels, further demonstrating that near-infrared laser irradiation itself does not have a therapeutic effect on tumors. Under non-illuminated conditions, compared to the PBS group, the tumors in the SF@Ag2S NP group showed consistent growth rates for the first 4 days, followed by a slowdown. After 18 days, the tumor volume was approximately 6 times that before treatment, indicating that silk fibroin silver sulfide nanoparticles have a certain inhibitory effect on tumor growth, but the tumor volume still showed an increasing trend. Under 808nm near-infrared light (1.5W / cm²), the tumor volume was... 2 Under near-infrared irradiation, the tumor growth curve of the SF@Ag2S NP+NIR group was basically consistent with that of the SF@Ag2S NP group for the first 12 days, with the same growth rate. However, after the 12th day, the tumor volume decreased significantly (vs. control group, P≤0.01). This indicates that during the first 12 days, the rate of tumor cell death in the silk fibroin silver sulfide nanoparticle group after near-infrared irradiation was lower than its growth rate. After the 12th day, the cell death rate exceeded the tumor growth threshold, resulting in tumor ablation. Clearly, hyperthermia with increased temperature leads to destructive tumor cell death. After 18 days, the tumor volume was only 1.2 times that before treatment, and the tumor inhibition rate reached 89.85±5.50% (vs. control group, P≤0.01), demonstrating that the silver sulfide nanoparticles prepared from low-molecular-weight silk fibroin fragments have good photothermal tumor ablation capabilities.

[0186] like Figure 9 As shown in Figure C, during the 18-day observation period, the body weight of mice in each group fluctuated slightly, but there was no significant difference between the groups, indicating that the silk fibroin silver sulfide nanoparticles had low cytotoxicity and did not significantly affect the physiological functions of mice during treatment. After treatment, tumor tissues from different experimental groups were excised and collected, as shown in Figure C. Figure 9 As shown in Figure B, the tumor volume in the SF@Ag2S NP+NIR group was significantly reduced, showing a significant difference compared to the control groups (PBS and PBS+NIR) that only received saline injection. The tumor size in each group also conformed to the differences observed in the tumor growth curve.

[0187] The experimental results of this embodiment confirm that the nanoparticles of the present invention, represented by silk fibroin silver sulfide nanoparticles, can achieve "highly efficient and low-toxicity" tumor thermal ablation at conventional dosages. The data from this embodiment strongly support the present invention's protection regarding the use of nanoparticles in "preparation of anti-tumor drugs," "tumor photothermal therapy formulations," and "tissue repair / anti-inflammatory materials." Based on the universal mechanism of photothermal ablation, this protection is not limited to gliomas but also covers the treatment of other solid tumors such as breast cancer and liver cancer.

[0188] Example 12 Pathological analysis of tumor tissue after in vivo photothermal therapy with silk fibroin inorganic crystal nanoparticles

[0189] To reveal the specific mechanism by which the inorganic crystalline nanoparticles of silk fibroin prepared in this invention inhibit tumor growth from a microscopic histological perspective (i.e., to verify the dual effect of "thermal ablation" and "proliferation inhibition"), this embodiment selected silver sulfide nanoparticles (SF3@Ag2S NP) prepared from low molecular weight silk fibroin fragments as a typical representative (verification model). Detailed H&E staining and Ki67 immunofluorescence analysis were performed on the tumor tissue after treatment in Example 11. After the in vivo treatment experiment (day 18), mice were sacrificed and tumor tissue was dissected. The tumor tissue was fixed, embedded, and sectioned, and subjected to H&E staining and Ki67 immunofluorescence staining, respectively. H&E staining was used to observe tissue morphology and necrosis, while Ki67 staining was used to assess the proliferative activity of tumor cells (the higher the proportion of Ki67-positive cells, the more active the proliferation). Microscopic observation of the sections was performed, and image acquisition and data analysis were conducted. Details are as follows:

[0190] H&E staining of tumor tissue: After fixation, the samples were dehydrated in five gradients of 75% to 100% ethanol (30 min per grade), cleared with xylene (30 min), and infiltrated with melted paraffin (≥5 h). The samples were then shaped with a mold, frozen and solidified to obtain complete paraffin blocks, and then precisely sectioned (6 μm), spread in a warm water bath, transferred to anti-detachment glass slides, and baked in a constant temperature oven at 60℃ for 2 h to complete the slide preparation. For staining, the slides were first dewaxed with a clearing solvent (2 times × 15 min), rehydrated with a gradient of 100% (2 times) to 80% ethanol (5 min per grade), and rinsed with running water for 15 min. Then, the nuclei were stained with hematoxylin for 5 min (running water to remove excess stain), and the cytoplasm was quickly stained with eosin for 5 s. After rapid dehydration with a gradient of 85%, 90%, and 100% ethanol (5 s per grade), dried at room temperature, and sealed with a mounting agent, the morphological characteristics of the tissue were finally observed using a conventional microscopic imaging system.

[0191] Ki67 immunofluorescence staining of tumor tissue: Sample slides were dewaxed with a biodegradable dewaxing agent (3 times × 10 min), dehydrated with anhydrous ethanol (3 times × 5 min), and thoroughly rinsed with distilled water. They were then placed in a special container containing EDTA retrieval solution at pH 8.0, and antigen retrieval was performed using microwave heating. After natural cooling to room temperature, the slides were rinsed with pH 7.4 phosphate buffer (3 times × 5 min). A hydrophobic barrier was constructed along the outer edge of the tissue using an anti-permeability pen. Bovine serum albumin blocking solution was added and the slides were allowed to stand for 30 min. After removing the blocking solution, a proportionally diluted primary antibody working solution was added, and the slides were incubated at 4°C for ≥12 h in a constant humidity environment. The next day, after rinsing with phosphate buffer, the corresponding species secondary antibody complex was added and reacted at room temperature for 50 min. The slides were then treated with DAPI nuclear labeling dye in the dark for 10 min, followed by treatment with a fluorescence background inhibitor for 5 min, and rinsed with running deionized water for 10 minutes. After thorough cleaning (3 times), gently shake off any residual liquid, cover with an anti-fluorescence attenuation sealing medium to complete the slide preparation, and finally collect fluorescence signals through the multispectral channels of an inverted fluorescence microscope to analyze cell proliferation activity.

[0192] H&E staining results are as follows Figure 10 As shown in Figure C, tumor cells in the control group and the light-only group exhibited intact morphology and vigorous growth. In contrast, the silk fibroin silver sulfide nanoparticle group and the silk fibroin silver sulfide nanoparticle group irradiated with near-infrared light showed varying degrees of nuclear atrophy and necrosis. The silk fibroin silver sulfide nanoparticle group irradiated with near-infrared light showed a significant reduction in the number of cell nuclei, irregular widening of interstitial spaces, and a significant increase in the number of necrotic cells, demonstrating greater tumor tissue damage. This indicates that under near-infrared laser irradiation, these silk fibroin inorganic crystalline nanoparticles have a significant photothermal damage therapeutic effect on tumor cells. This pathological change confirms that "irreversible physical thermal damage caused by photothermal effect" is its main anti-tumor mechanism. Since this is based on physical heating-induced destruction, theoretically, this mechanism is applicable to all types of solid tumor tissues and to all photothermal nanomaterials described in this invention.

[0193] Ki67 immunofluorescence results are as follows: Figure 10As shown in Figures A and B, both the control group (PBS(-)) and the light-only group (PBS(+)) exhibited significant enrichment of red fluorescence signals, with positive cell proportions of 21.14±1.46% and 20.87±3.1%, respectively, significantly higher than the other two treatment groups (P≤0.001). The addition of silk fibroin silver sulfide nanoparticles (SF@Ag2S NP(-)) resulted in a decrease in red fluorescence intensity, with the positive cell proportion dropping to 13.97±0.24%, indicating that the silk fibroin inorganic crystalline nanoparticles themselves possess a basal level of tumor cell proliferation and inhibition effect. However, near-infrared light-excited silk fibroin silver sulfide nanoparticles (SF@Ag2S NP(+)) exhibited a stronger photothermal therapeutic effect, with the positive cell proportion significantly decreasing to 8.2±0.41% (P≤0.001). This gradient change clearly reveals the decisive role of photothermal conversion efficiency. Under 808nm laser irradiation, silk fibroin silver sulfide nanoparticles significantly inhibited the proliferation of U87 MG malignant glioma cells through the localized thermal effect generated by efficient photothermal conversion. This phenomenon is in high agreement with the in vitro tumor inhibition experiment curve, further verifying the feasibility of the photothermal therapy strategy. This significant difference indicates that the nanoparticles prepared in this invention can not only directly "burn" tumor cells under light irradiation, but also significantly inhibit the proliferative activity of residual tumor cells by disrupting the tumor microenvironment, thereby effectively preventing tumor recurrence.

[0194] Based on this universal physical therapy mechanism, the anti-tumor applications protected by this invention are not limited to specific gliomas, but can also be extended to the treatment of other solid tumors such as breast cancer, liver cancer, and melanoma.

[0195] Example 13 Evaluation of the antibacterial properties of silk fibroin inorganic crystalline nanoparticles

[0196] To comprehensively evaluate the application potential and optimal effective concentration of the silk fibroin inorganic crystal nanoparticles prepared in this invention in the field of infection control, this embodiment selects copper sulfide nanoparticles (SF1@CuSNP) prepared from high molecular weight silk fibroin fragments as a typical representative. Using clinically common Staphylococcus aureus and Escherichia coli as model strains, a gradient concentration plate coating counting method was used to evaluate their antibacterial performance. To assess the dose-response relationship of their antibacterial ability, a series of different Cu nanoparticles were prepared following the test method for the photothermal performance of silver ions in Example 7. 2+ SF1@CuS NP solutions with concentration gradients (0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM) were respectively compared with bacterial suspensions in logarithmic growth (1×10⁻⁶ mM). 6The bacterial count (CFU / mL) was incubated for 4 hours at 37°C in a shaker. Subsequently, an appropriate amount of the diluted solution was spread onto LB solid agar plates and incubated for 18 hours, followed by counting. After incubation, the number of colonies (CFU) on each plate was counted, with three replicates per group, and the average value was taken. Antibacterial activity was quantitatively assessed using the inhibition rate (Antibacterial Rate), with five repeated measurements and the average value taken as the final result. The calculation formula is as follows:

[0197]

[0198] In the formula: C0 represents the colonies in the control group, and C represents the colonies in the experimental group.

[0199] The results are as follows Figure 11 As shown, the nanoparticles exhibit a significant concentration-dependent antibacterial behavior: the Control group (0 μg / mL) plates were covered with dense bacterial colonies; when Cu... 2+ At a concentration of 0.125 mM, the colony density decreased significantly; when the concentration was increased to 0.25 mM, the number of colonies on the plate showed a visibly large reduction; when Cu 2+ When the concentrations reach 0.5 mM and 1 mM, the surface of the culture medium is extremely clean, with only sporadic or no colony growth observed, and the inhibition rate is as high as 99% or more.

[0200] The experimental results of this embodiment confirm that the synergistic mechanism of "silk fibroin anti-agglomeration / adhesion + inorganic core" ion release has clear dose-regulation characteristics, that is, the bactericidal efficacy can be precisely controlled by adjusting the dosage concentration. This embodiment lists polymer fragments, copper sulfide, and the above-mentioned specific concentration gradients (0~100 μg / mL), but is not limited to these. The experimental results demonstrate that the nanoparticles with a "core-shell structure" prepared by the method described in this invention can effectively inhibit bacterial growth within a certain concentration range. Based on the broad-spectrum bactericidal mechanism of metal ions such as copper (Cu), silver (Ag), and zinc (Zn), the data of this embodiment strongly support the protection of the invention regarding the use of nanoparticles for "preparation of antibacterial coatings," "anti-infective drugs," and "medical dressings"; this antibacterial use is not limited to Gram-positive bacteria, but also covers Gram-negative bacteria such as Escherichia coli.

[0201] Example 14 Evaluation of the in vitro anti-inflammatory and reactive oxygen species scavenging properties of silk fibroin copper sulfide nanoparticles

[0202] To evaluate the application potential of the silk fibroin inorganic crystalline nanoparticles prepared in this invention in the field of inflammation regulation, and considering that scavenging ROS is a key pathway to suppressing cytokine storms, this embodiment selects copper sulfide nanoparticles (SF1@CuS NP) prepared from high molecular weight silk fibroin fragments as a typical representative. Using an LPS-induced RAW264.7 macrophage inflammation model, the intracellular reactive oxygen species (ROS) level was detected using the DCFH-DA fluorescent probe to evaluate the antioxidant and anti-inflammatory properties of the material. Details are as follows:

[0203] Log-phase RAW264.7 macrophages were seeded at a density of 3000 cells / well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. After complete cell adhesion, the experiment was divided into four groups for treatment:

[0204] PBS group (blank control): Cells were not specially treated, only an equal volume of sterile PBS was added;

[0205] LPS group (inflammation model): 100 μL of lipopolysaccharide (LPS, 1 μg / mL) was added to each well and polarized for 24 h to induce inflammatory response and high levels of ROS in cells;

[0206] SF@CuS NP group (material treatment): After LPS induction for 24 h, the culture medium was discarded, and 100 μL of medium containing SF1@CuS NP was added to each well (Cu 2+ (Adjust the concentration to 50 μg / mL), and incubate for 24 h;

[0207] SF@CuS NP+NIR group (photothermal combined therapy): The sample addition and incubation steps were the same as the SF@CuS NP group, followed by replacement with fresh culture medium, and the group was irradiated with near-infrared laser (808 nm, 1.5 W / cm²). 2 After treatment, discard the culture medium in the wells and wash 2-3 times with sterile PBS. Add 100 μL of the reactive oxygen species fluorescent probe DCFH-DA (10 μM) to each well under light-protected conditions and incubate at 37°C for 30 min. Discard the probe solution, wash 2-3 times with PBS to remove any residual extracellular probe, and then observe cell morphology and acquire fluorescence images under an inverted fluorescence microscope.

[0208] like Figure 12As shown, the intensity of green fluorescence directly reflects the level of intracellular ROS accumulation. Only very weak background fluorescence was observed in the PBS group, indicating that normal cells were under low oxidative stress. The LPS group exhibited a strong burst of green fluorescence, indicating that LPS successfully induced macrophages to polarize towards the M1 (pro-inflammatory) type, leading to a surge in intracellular reactive oxygen species and creating a severe oxidative stress environment. Compared to the LPS group, the fluorescence intensity of the SF@CuS NP group showed a significant decrease visible to the naked eye. This indicates that the silk fibroin copper sulfide nanoparticles themselves possess excellent ROS scavenging ability and can effectively neutralize excess free radicals generated by inflammation. The green fluorescence of the SF@CuS NP+NIR group further weakened, approaching the level of the PBS group. This suggests that under near-infrared light excitation, the photothermal or photochemical effects of the nanoparticles may further promote the activation of anti-inflammatory pathways, achieving the optimal anti-inflammatory effect.

[0209] This example illustrates RAW264.7 cells and DCFH-DA detection methods, but is not limited to these.

[0210] The experimental results of this embodiment strongly demonstrate that the silk fibroin inorganic nanoparticles prepared by this invention can not only serve as a physical photothermal agent, but also possess significant biochemical anti-inflammatory activity. Based on the biocompatibility of silk fibroin itself and the potential multi-enzyme mimicry activity of copper sulfide and other transition metal sulfides, this embodiment strongly supports the protection of this invention regarding the use of nanoparticles in the preparation of anti-inflammatory drugs, antioxidant agents, and tissue repair materials.

[0211] Example 15: In vitro and in vivo CT imaging enhancement properties of silk fibroin bismuth sulfide nanoparticles

[0212] To evaluate the application potential of the silk fibroin inorganic crystal nanoparticles prepared in this invention in the field of medical imaging diagnosis, this embodiment selects bismuth sulfide nanoparticles (SF@Bi2S3 NP) prepared from medium molecular weight silk fibroin as a typical representative. Since bismuth (Bi, Z=83) has a higher atomic number and X-ray absorption coefficient than iodine (I, Z=53), it theoretically possesses superior imaging density compared to traditional iodine preparations. Specifically:

[0213] The prepared bismuth sulfide nanoparticle solution made from medium molecular weight silk fibroin was concentrated using an ultrafiltration tube to prepare a series of gradient solutions with different Bi molar concentrations (0, 2, 4, 6, 8, 10, 12 mM), which were then placed in custom-made agar plate molds. Iohexol, a commonly used iodine-based contrast agent, was used as a positive control, and deionized water as a negative control. Scanning was performed using a micro-CT imaging system at 80 kV tube voltage and 450 μA tube current to reconstruct images and measure CT values. A whole-body CT pre-scan of the background signal was performed on U87 tumor-bearing mice from Example 11. Subsequently, the bismuth sulfide nanoparticle solution made from medium molecular weight silk fibroin was injected intratumorally into the tumor site at a dose of 20 mg / kg (it). Fifteen minutes after injection, CT scans were performed on the mice under the same parameters to observe signal changes at the tumor site.

[0214] Table 7. In vitro CT value determination results of different concentrations of SF@Bi2S3 NP and iohexol

[0215]

[0216] As shown in Table 7 and Figure 13 As shown, the CT value of bismuth sulfide nanoparticles prepared from medium molecular weight silk fibroin exhibits an excellent linear increase with concentration. Fitting calculations show that its CT imaging efficiency (slope) is as high as 11.25 ± 0.23 HU / mM. In comparison, the slope of commercial iohexol under the same conditions is only 4.32 ± 0.09 HU / mM. Calculations indicate that the CT contrast enhancement capability of the bismuth-based nanoparticles prepared in this invention is approximately 2.6 times that of traditional iodine preparations. This means that the molar dose required for injection of the material of this invention is significantly reduced to achieve the same imaging clarity, which is beneficial to improving biocompatibility. According to in vivo CT scan images, before injection, the tumor tissue and surrounding muscle tissue have similar densities and blurred boundaries. After intratumoral injection of SF@Bi2S3 NP for 15 min, the tumor area exhibits a significantly bright white signal, with a significantly increased HU value, forming a sharp contrast with the surrounding dark tissue. This proves that the silk fibroin-encapsulated nanoparticles can successfully reside within the tumor tissue and provide clear contour imaging.

[0217] This example illustrates Bi2S3 and the above concentration range and dosage, but is not limited thereto.

[0218] The experimental results of this embodiment strongly demonstrate that the silk fibroin inorganic nanoparticles prepared by this invention possess excellent X-ray attenuation capabilities, significantly superior to iohexol. Based on the physical properties of high atomic number elements such as bismuth (Bi), gold (Au), and tantalum (Ta), this embodiment strongly supports the invention's protection regarding the use of nanoparticles in "preparation of CT imaging enhancers" and "multimodal diagnostic and therapeutic agents."

[0219] Example 16 Evaluation of the tissue repair and wound healing properties of silk fibroin inorganic nanoparticles

[0220] To evaluate the application potential of the silk fibroin inorganic crystalline nanoparticles prepared in this invention in the field of wound repair, this embodiment specifically selects silver sulfide nanoparticles (SF1@Ag2S NP) prepared from high-molecular-weight silk fibroin fragments, which possess excellent film-forming properties, adhesion, and bioscaffold functions, as a typical representative. Utilizing the physical sealing effect of high-molecular-weight silk fibroin, similar to "bio-glue," and the photothermal antibacterial properties of silver sulfide, a synergistic repair mechanism of "physical sealing - photothermal clearance of infection - protein scaffold-induced regeneration" is constructed. Details are as follows:

[0221] Establishing an ICR mouse model of full-thickness skin defect on the back accompanied by Staphylococcus aureus infection: Hair was removed from the back of mice, and a circular full-thickness skin wound with a diameter of approximately 6 mm was created. Staphylococcus aureus suspension was then inoculated to establish an infection environment. The experiment was randomly divided into 3 groups (n=5):

[0222] Blank control group (PBS): 2 mL of PBS was dripped into the wound;

[0223] High molecular weight silk fibroin group (SF1): An equal volume of high molecular weight silk fibroin solution is dripped into the wound to form a protective film by utilizing the high adhesiveness of high molecular weight silk fibroin;

[0224] Simple material group (SF1@Ag2S NP): SF1@Ag2S NP solution (Ag) was dripped into the wound. + (Concentration 0.5 mM), utilizing the high adhesiveness of high molecular weight silk fibroin to form a protective film;

[0225] Photothermal therapy group (SF1@Ag2S NP+NIR): After material was added, an 808 nm laser (1.0 W / cm²) was applied. 2 Irradiate for 5 minutes to utilize the thermal effect (approximately 45°C) to sterilize and promote blood circulation.

[0226] Wound photographs were taken and wound healing rates were calculated during treatment (days 0, 3, 7, and 14). Mice were sacrificed on days 7 and 14, and regenerated skin tissue was collected for H&E staining to observe epithelial remodeling and Masson's trichrome staining to observe collagen deposition.

[0227] like Figure 14As shown, the experimental results indicated that SF1@Ag2S NP significantly accelerated the healing process of infected wounds: On day 3, the photothermal therapy group showed dry wound surfaces, rapid scab formation, and significant reduction in inflammation and swelling, demonstrating the excellent film-forming and sealing effect of high-molecular-weight silk fibroin; the control group still had significant purulent discharge. On day 7, the photothermal therapy group showed a significant reduction in wound area, with a healing rate of approximately 65%, significantly higher than the control group (~30%). On day 14, the photothermal therapy group showed almost complete wound closure (healing rate >95%), with new skin growing hair and no obvious scarring; while the control group still had a large unhealed area.

[0228] Histological H&E staining results showed that, on day 14, the photothermal therapy group exhibited complete reepithelialization, with an epidermal layer thickness approaching that of normal skin and minimal inflammatory cell infiltration in the dermis. This was attributed to the "bio-breathing membrane" formed by the silk fibroin shell on the wound surface, which both isolated external bacteria and maintained a moist environment. Masson staining results showed that the photothermal therapy group exhibited dense and neatly arranged blue collagen fiber bundles, with significantly higher collagen deposition than the control group, demonstrating that silk fibroin, as an extracellular matrix (ECM) mimic, effectively induced fibroblast migration and collagen synthesis.

[0229] Example 17 Evaluation of a peptide-modified silk fibroin inorganic nanoparticle drug delivery system and its photothermal-chemotherapy synergistic effect.

[0230] This embodiment uses low-molecular-weight silk fibroin silver sulfide (SF3@Ag2S NP) as an example to verify the function of the nanoparticles described in this invention as a "smart drug carrier" and "introducing peptides to achieve active targeting in vivo," strongly supporting the invention's protection regarding the use of "drug delivery complexes" and "targeted diagnosis and treatment." To verify the application potential of the silk fibroin inorganic crystal nanoparticles prepared in this invention in the field of precision medicine, this embodiment constructs a nanoplatform integrating active targeting and drug delivery. Low-molecular-weight silk fibroin (RGD-SF3) modified with RGD peptide (specifically targeting integrin receptors on the surface of glioma cells) was selected as a template. While inducing the mineralization of silver sulfide (Ag2S), the antitumor drug doxorubicin (DOX) was in situ encapsulated, thus preparing the RGD-SF3@Ag2S / DOX nanocomposite. Details are as follows:

[0231] First, peptide modification and drug loading were performed. RGD peptides were coupled to the surface of SF3 via conventional amidation. Then, DOX and a metal precursor were added to the RGD-SF3 solution in a one-pot process, and the pH was adjusted to 12 to initiate mineralization. After dialysis, the targeted nanocomposite was obtained. Subsequently, in vivo targeting ability was evaluated by establishing a U87 tumor-bearing mouse model (subcutaneous xenograft tumor). The tumor was allowed to grow to approximately 100 mm. 3Mice were randomly divided into three groups and administered equal amounts of the drug (DOX concentration was uniformly 5 mg / kg) via tail vein injection:

[0232] Free DOX group: Injection of free doxorubicin solution;

[0233] Targeted experimental group (RGD-SF3@Ag2S / DOX): Injection of drug-loaded nanoparticles modified with RGD peptides;

[0234] Non-targeted control group (SF3@Ag2S / DOX): Injected with unmodified drug-loaded nanoparticles.

[0235] Fluorescence images were acquired using a mouse in vivo imaging system (IVIS) at 1, 4, 8, 12, and 24 hours after injection to observe the distribution and metabolism of the drug in the whole body and at the tumor site (ROI). After 24 hours, the mice were sacrificed, and the tumor and major organs (heart, liver, spleen, lung, and kidney) were dissected for in vitro fluorescence imaging and quantitative analysis of signal intensity.

[0236] The experimental results showed significant differences in distribution among the groups. In the free drug group, the fluorescent signal rapidly spread throughout the body after injection, but was quickly eliminated through renal metabolism over time. After 4 hours, the signal at the tumor site was very weak, indicating that the free drug had a short half-life and lacked tumor specificity. In the non-targeted group, thanks to the EPR effect of the nanoparticles, the drug passively accumulated at the tumor site to some extent, with a higher signal intensity than the free drug group, but this intensity began to decay significantly after 12 hours. In contrast, the targeted experimental group showed extremely high-brightness red fluorescence at the tumor site 8-12 hours after injection, and this high-intensity signal remained clearly visible until 24 hours. This indicates that the RGD peptide specifically bound to tumor angiogenesis and integrins on the surface of U87 cells, significantly prolonging the drug's residence time in tumor tissue. Figure 15 As shown, in vitro organ analysis further confirmed that the fluorescence signal intensity of the targeted drug group in tumor tissue was significantly higher than that of the free drug group and the non-targeted drug group, and the accumulation in non-target organs such as the heart was lower, reducing the risk of systemic toxicity.

[0237] This example illustrates RGD peptides, the U87 model, and doxorubicin (DOX), but is not limited to these.

[0238] The experimental results of this embodiment strongly demonstrate that the silk fibroin inorganic nanoparticles prepared by this invention are a universal, multifunctional loading platform. Based on the abundant functional groups of silk fibroin, this platform is also suitable for loading other hydrophobic drugs such as paclitaxel (PTX) and curcumin, or introducing other functional peptides / antibodies such as TAT ​​and PD-L1. Combined with the inherent photoacoustic / CT imaging capabilities of the inorganic core (such as Ag2S and Bi2S3), this embodiment strongly supports the invention's protection regarding the use of "imaging-guided targeted photothermal-chemotherapy integrated formulations".

[0239] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A silk fibroin nanocage, characterized in that, It is formed by the self-assembly of silk fibroin fragments with molecular weight distributions selected from any of the following groups, and its size is regulated by the molecular weight of the selected silk fibroin fragments: (a) 10~66 kDa, 34~135 kDa or 65~220 kDa; (b) 10~60 kDa, 60~115 kDa or 120~250 kDa; (c) 10~45 kDa, 50~110 kDa or 100~300 kDa; The nanocage is formed and maintained in its unfolded conformation by the self-assembly of silk fibroin fragments in a strongly alkaline environment with a pH value of not less than 11.

2. The silk fibroin nanocage according to claim 1, characterized in that, The surface or inner cavity of the nanocage has a main coordination network composed of aspartic acid, glutamic acid, serine, and tyrosine, as well as auxiliary reduction sites provided by trace amounts of cysteine ​​residues.

3. The silk fibroin nanocage according to claim 1, characterized in that, The molecular weight of the silk fibroin fragment determines the confined space topology of the nanocage, specifically: (1) When the molecular weight distribution is in the range of 10~66 kDa, the nanocage forms a multi-molecular aggregate structure with elastic deformation capability to adapt to crystal growth; (2) When the molecular weight is distributed in the range of 34~135 kDa, the nanocage forms a single-molecule dense spherical structure with a rigid confined cavity; (3) When the molecular weight distribution is in the range of 65~220 kDa, the nanocage forms a three-dimensional steric structure with a cage-like core and abundant chain segments on the surface.

4. The silk fibroin nanocage according to claim 3, characterized in that, The confined space size of the nanocage is significantly correlated with the amino acid and molecular weight of the silk fibroin fragment, such that: (1) Nanocages with molecular weight distribution in the range of 10~66 kDa are suitable for guiding the formation of metal sulfide nanoparticles with an average particle size of 6~50 nm. (2) Nanocages with a molecular weight distribution in the range of 34~135 kDa are suitable for guiding the formation of metal sulfide nanoparticles with an average particle size of 2~10 nm. (3) Nanocages with a molecular weight distribution in the range of 65~220 kDa are suitable for guiding the formation of metal sulfide nanoparticles with an average particle size in the range of 4~15 nm.

5. A type of silk fibroin-coated metal sulfide nanoparticle, characterized in that, The nanoparticles are prepared from silk fibroin nanocages as described in any one of claims 1-4; the nanoparticles have a core-shell structure, with the core being a metal sulfide and the shell being silk fibroin; the particle size of the nanoparticles is 5-50 nm.

6. The silk fibroin-coated metal sulfide nanoparticles according to claim 5, characterized in that, The metal sulfide is Ag2S, CuS, Bi2S3, CdS, ZnS, PbS, or MoS2.

7. A method for preparing silk fibroin-coated metal sulfide nanoparticles as described in claim 5 or 6, characterized in that, Includes the following steps: Step 1) Provide silk fibroin nanocages as described in any one of claims 1-4; Step 2) In a strongly alkaline environment, metal ions are added to coordinate with silk fibroin; Step 3) Add a sulfur source and react within the confined space of the nanocage to generate the metal sulfide nanoparticles.

8. A pharmaceutical composition, characterized in that, It comprises metal sulfide nanoparticles coated with silk fibroin as described in claim 5 or 6, and a pharmaceutically acceptable carrier.

9. The use of silk fibroin nanocages as templates in the preparation of metal sulfide nanoparticles as described in any one of claims 1-4.

10. The use of the silk fibroin-coated metal sulfide nanoparticles as described in claim 5 or 6 in the preparation of any of the following pharmaceuticals or formulations: (a) Photothermal therapy preparations; (b) Medical imaging contrast agents; (c) Antibacterial, anti-inflammatory or tissue-repairing drugs or medical dressings.

Images