Yolk-shell mesoporous silica-coated gold nanostars and preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, in particular to a kind of egg yolk shell mesoporous silica coated gold nanostar and its preparation method and application.The egg yolk shell mesoporous silica coated gold nanostar provided by the present application removes the internal structure of traditional mesoporous silica, retains the shell, thereby maximizes the drug loading efficiency, retains the ability of drug loading and sustained release.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a mesoporous silica-coated gold nanostar in egg yolk shell, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease threatening human health. Cervical cancer is the second most common malignant tumor in women after breast cancer, and its incidence and mortality rates continue to rise. Currently, single-treatment methods are insufficient to meet clinical needs, necessitating the intervention of new treatment approaches. Combination therapy has become a new hope for cancer treatment, and multifunctional, biocompatible drug carriers will provide significant support for combination therapy.

[0003] Therefore, multimodal combination therapy is receiving increasing attention, with PD-1 combined with chemotherapy and EGFR combined with chemotherapy becoming first-line treatment options for their respective cancer types. It is evident that multimodal therapy will be the mainstream of cancer treatment in the future, making the development of drug delivery systems suitable for multimodal therapy highly significant. Summary of the Invention

[0004] This invention provides a method for preparing and applying a gold nanostar coated with mesoporous silica from an egg yolk shell.

[0005] A type of egg yolk shell mesoporous silica-coated gold nanostar (GNS@YSMS) comprises:

[0006] The core is a gold nanostar;

[0007] The shell is mesoporous silica.

[0008] This invention provides gold nanostars coated with egg yolk-shell mesoporous silica. The yolk-shell structure of the mesoporous silica removes the internal structure of traditional mesoporous silica while retaining the shell, thereby maximizing drug loading efficiency and preserving both drug loading and sustained release capabilities. Gold nanoparticles also possess the advantage of high biocompatibility, making them promising photothermal agents for clinical tumor photothermal therapy. Compared to spherical gold nanoparticles, the shape changes of non-spherical gold nanoparticles such as gold nanorods and gold nanostars lead to a redshift in surface plasmon resonance wavelength, thereby improving near-infrared absorption and photothermal conversion efficiency. However, non-spherical gold nanoparticles are unstable. This invention constructs gold nanostars coated with mesoporous silica into egg yolk-shell mesoporous silica, preparing a gold nanostar coated with egg yolk-shell mesoporous silica (GNS@YSMS). The introduction of GNS imparts a photothermal effect, and the egg yolk-shell mesoporous silica increases the stability of GNS.

[0009] Specifically, the gold nanostars are coated in the mesoporous silica of the egg yolk shell, and the diameter of the core is 50-100 nm.

[0010] Specifically, the gold nanostars are coated in the mesoporous silica of the egg yolk shell, and the thickness of the shell is 75-150 nm.

[0011] The present invention also provides a method for preparing gold nanostars coated with mesoporous silica from egg yolk shells as described above, comprising:

[0012] 1) Preparation of gold nanostars (GNS);

[0013] 2) Coating the surface of the gold nanostars with dense silica to form GNS@SiO2 (gold nanostars coated with dense silica);

[0014] 3) GNS@SiO2 reacts with tetraethyl orthosilicate (TEOS), dimethyldimethoxysilane (BTSE), and hexadecyltrimethylammonium bromide (CTAB) to prepare GNS@SiO2@mSiO2;

[0015] 4) Etch GNS@SiO2@mSiO2 with sodium carbonate to produce egg yolk shell mesoporous silica-coated gold nanostars (GNS@YSMS).

[0016] Preferably, polyvinylpyrrolidone (PVP) is used for functionalization during the preparation of the gold nanostars. Studies have found that gold nanostars functionalized with PVP can be better coated with silica compared to those functionalized with other materials (such as thiolated PEG, PEG-SH).

[0017] Specifically, the preparation method of the gold nanostars includes: reacting HAuCl4, concentrated hydrochloric acid and gold seeds in solution; then adding AgNO3 and ascorbic acid to react; and finally adding polyvinylpyrrolidone to react, to obtain an aqueous solution of gold nanostars.

[0018] Preferably, 100 mL of 0.25 mM HAuCl4 deionized water solution is prepared, 8.33 μL of 36%-38% concentrated hydrochloric acid is added, and the mixture is stirred magnetically until homogeneous. Then, 1 mL of gold seed aqueous solution is added, and stirring is continued. At the same time, 1 mL of 3 mM AgNO3 deionized water solution and 0.5 mL of 100 mM ascorbic acid deionized water solution are added. After reacting for 1 min, 0.3 mL of 15 mg / mL polyvinylpyrrolidone deionized water solution is added, and the mixture is stirred in the dark for 24 h. After centrifugation at 3000 g for 15 min, the mixture is washed once with deionized water. The resulting precipitate is dispersed in 1 mL of deionized water to obtain gold nanostar aqueous solution.

[0019] The gold seed aqueous solution can be prepared using existing conventional methods. For example, the preparation method of the gold seed aqueous solution includes: preparing 100 mL of 1 mM HAuCl4 deionized water, heating to boiling, adding 15 mL of 10 mg / mL sodium citrate deionized water, and continuing to boil for 15 min to obtain the gold seed aqueous solution.

[0020] This invention reveals that coating the surface of gold nanostars with dense silica to form GNS@SiO2 not only protects the integrity of the gold nanostar structure but also effectively prevents the aggregation of gold nanostars in CTAB solution, thus contributing to the formation of a uniform structure. If gold nanostars react directly with CTAB, BTSE, or TEOS under alkaline conditions, a uniform and stable product cannot be obtained due to the repulsive effect between the polymers (such as PVP and PEG) introduced during the gold nanostar synthesis process and CTAB.

[0021] Specifically, step 2) involves coating the surface of the gold nanostars with dense silica, which includes reacting the gold nanostars with tetraethyl orthosilicate (TEOS) in the presence of deionized water, anhydrous ethanol and ammonia to form GNS@SiO2.

[0022] Preferably, in step 2), the step of coating the surface of the gold nanostars with dense silica involves mixing 1 mL of gold nanostar aqueous solution with 3 mL of anhydrous ethanol, adding 72 μL of 28 wt% ammonia, stirring magnetically for 10 min, and then adding 20 μL of tetraethyl orthosilicate (dispersed in 1 mL of anhydrous ethanol, i.e., adding a mixture of 20 μL of tetraethyl orthosilicate and 1 mL of anhydrous ethanol dropwise). The mixture is stirred for 15-16 h, centrifuged at 10000 rpm for 10 min, washed twice with anhydrous ethanol, and once with deionized water. The resulting precipitate is dispersed in 7.5 mL of deionized water to obtain a GNS@SiO2 aqueous solution.

[0023] Specifically, step 3) involves preparing GNS@SiO2@mSiO2 by reacting GNS@SiO2 with tetraethyl orthosilicate (TEOS), dimethyldimethoxysilane (BTSE), and hexadecyltrimethylammonium bromide (CTAB) in the presence of deionized water, anhydrous ethanol, and ammonia to produce GNS@SiO2@mSiO2.

[0024] Preferably, step 3) of preparing GNS@SiO2@mSiO2 includes: mixing 7.5 mL of GNS@SiO2 aqueous solution with 3 mL of anhydrous ethanol, adding 25 mg of cetyltrimethylammonium bromide, dissolving by sonication, adding 100 μL of 28 wt% ammonia, stirring for 1 h in a 35°C water bath, adding a mixture of 20 μL of tetraethyl orthosilicate and 20 μL of dimethyldimethoxysilane, continuing to stir for 4-5 h, centrifuging at 10000 rpm for 10 min, washing twice with anhydrous ethanol, washing once with deionized water, and retaining the precipitate, which is GNS@SiO2@mSiO2, for later use.

[0025] Specifically, in step 4), GNS@SiO2@mSiO2 is contacted with a sodium carbonate solution with a concentration of 200-300 mg / mL (preferably 254.4 mg / mL) to prepare egg yolk shell mesoporous silica-coated gold nanostars (GNS@YSMS).

[0026] Specifically, in step 4), 2.5 mL of a Na2CO3 solution with a concentration of 254.4 mg / mL was added to GNS@SiO2@mSiO2, shaken on a shaker at room temperature for 2 h, centrifuged at 10000 rpm for 10 min, washed twice with deionized water, and the precipitate was collected. The precipitate was added to anhydrous methanol-sodium chloride solution (i.e., 7.9 mg sodium chloride per mL of anhydrous methanol), and magnetically stirred for 12 h. This process was repeated 3 times to remove hexadecyltrimethylammonium bromide. Finally, the precipitate was washed twice with anhydrous ethanol and then freeze-dried to obtain egg yolk shell mesoporous silica-coated gold nanostars for later use.

[0027] Preferably, the preparation method of the gold nanostar coated with mesoporous silica from egg yolk shell further includes:

[0028] 5) The mesoporous silica-coated gold nanostars (GNS@YSMS) in egg yolk shells were amination with 3-aminopropyltrimethoxysilane (APTES).

[0029] Experiments have shown that aminated eggshell mesoporous silica-coated gold nanostars can significantly improve the loading capacity for metal ions, protein drugs, and nucleic acid drugs.

[0030] Specifically, step 5) includes: reacting egg yolk shell mesoporous silica-coated gold nanostars with 3-aminopropyltrimethoxysilane in the presence of anhydrous ethanol and ammonia to obtain aminated egg yolk shell mesoporous silica-coated gold nanostars (aminated GNS@YSMS).

[0031] Preferably, in step 5), 40 mL of anhydrous ethanol is added to 20 mg of egg yolk shell mesoporous silica-coated gold nanostars. After dissolving completely, 0.2 mL of 28 wt% ammonia is added and stirred evenly. Then, 0.15 mL of APTES is added, and the mixture is magnetically stirred for 12 h. After centrifugation at 10,000 rpm for 10 min, the mixture is washed twice with anhydrous ethanol and once with deionized water. The precipitate is collected, which is the aminated egg yolk shell mesoporous silica-coated gold nanostar (aminated GNS@YSMS).

[0032] In this invention, the concentration of ammonia water can be 25-28 wt%.

[0033] The present invention also provides gold nanostars coated with mesoporous silica in egg yolk shells prepared by the above method.

[0034] This invention is the first to prepare GNS@YSMS in a mild manner. Compared with bare GNS, the YSMS coating improves the stability (including freeze-drying and photothermal stability) of GNS without significantly affecting its photothermal properties, thereby improving the accessibility of this drug delivery platform. More importantly, the GNS@YSMS of this invention can load a variety of drugs, including metal ions, proteins, and nucleic acids, demonstrating its potential as a multi-drug delivery platform.

[0035] This invention also provides the application of the aforementioned egg yolk shell mesoporous silica-coated gold nanostars as a drug delivery platform. The aforementioned egg yolk shell mesoporous silica-coated gold nanostars (GNS@YSMS) can serve as a multi-drug delivery platform, effectively loading chemotherapeutic drugs, photosensitizers, metal ions, proteins, and nucleic acid drugs.

[0036] The present invention also provides a composition comprising the above-mentioned mesoporous silica-coated gold nanostars of egg yolk shells and the substrate.

[0037] Specifically, the carrier includes one or more of the following: chemotherapy drugs, photosensitizers, metal ions, protein drugs, and nucleic acid drugs.

[0038] Specifically, the metal ions include iron ions (Fe). 2+ ).

[0039] Specifically, the protein drug includes one or more of glucose oxidase (GOX) and horseradish peroxidase (HRP).

[0040] In some embodiments, the carrier is iron ions (Fe). 2+ ) and the protein drug glucose oxidase (GOX).

[0041] This invention selects Fe 2+ GOX was used as a model drug and loaded into GNS@YSMS. 2+GNS@YSMS synergistically inhibited the growth of cervical SiHa cells when used in conjunction with GOX-mediated chemokinetic therapy and laser-induced photothermal therapy, while no significant toxicity was observed when GNS@YSMS was used alone. The GNS@YSMS-based multi-drug delivery platform has proven to be highly effective and safe.

[0042] This invention utilizes metal ions, specifically iron ions (Fe). 2+ Both Fe and the protein drug glucose oxidase (GOX) were introduced into the drug delivery platform (GNS@YSMS) to prepare Fe-loaded drugs. 2+ And GOX's GNS@YSMS (GNS@YSMS-Fe 2+ &GOX). The glucose content in the tumor microenvironment is higher than in normal tissue. GOX catalyzes the in situ generation of hydrogen peroxide from glucose within the tumor, followed by Fe... 2+ The catalysis of H2O2 into highly toxic hydroxyl radicals (•OH) efficiently induces tumor cell death. GNS@YSMS-Fe 2+ &GOX combines Fe 2+ Both H2O2-mediated chemokinetic therapy and GNS-mediated photothermal therapy demonstrated significant synergistic killing effects on SiHa cervical cancer tumor cells in vitro and in vivo, with good safety profiles. The experiments demonstrate the potential of GNS@YSMS as a multi-drug delivery platform and provide a novel and effective option for the local treatment of cervical cancer. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the synthesis scheme of GNS@YSMS in a preferred embodiment of the present invention.

[0044] Figure 2 The images are TEM images of GNS (a), GNS@SiO2 (b), GNS@MS (c), and GNS@YSMS (d) in a preferred embodiment of the present invention.

[0045] Figure 3 The UV-Vis spectra and photographs of GNS (before and after freeze-drying) and GNS@YSMS (before and after freeze-drying) are shown in the preferred embodiments of the present invention.

[0046] Figure 4 Temperature curves of GNS and GNS@YSMS after 6 laser irradiation cycles (40 min per cycle).

[0047] Figure 5 The temperature rise curves of GNS@YSMS at different concentrations under 808nm laser irradiation are shown.

[0048] Figure 6 This is an electron microscope image of the silica-coated gold nanostars prepared in Comparative Example 1 of this invention.

[0049] Figure 7 This is an electron microscope image of the silica-coated gold nanostars prepared in Comparative Example 2 of this invention.

[0050] Figure 8 This is an electron microscope image of the silica-coated gold nanostars prepared in Comparative Example 3 of this invention.

[0051] Figure 9 The nitrogen adsorption and desorption curves (a) and pore size distribution (b) of GNS@YSMS in a preferred embodiment of the present invention are shown.

[0052] Figure 10 The diagram below shows GNS@YSMS as a platform for delivering metal ions, proteins, and nucleic acids in a preferred embodiment of the present invention.

[0053] Figure 11 The loading efficiency (a) and zeta potential (b) of GNS@YSMS for glucose oxidase, horseradish peroxidase and iron ions in a preferred embodiment of the present invention are shown.

[0054] Figure 12 In a preferred embodiment of the present invention, the quantitative (a) and qualitative results (b) of DCFH-DA staining of SiHa tumor cells after treatment with GNS@YSMS are shown.

[0055] Figure 13 The quantitative (a) and qualitative (b) detection results of SiHa tumor cells after different concentrations of GNS@YSMS were applied to SiHa tumor cells in a preferred embodiment of the present invention.

[0056] Figure 14 This is a schematic diagram of an in vivo tumor suppression experiment protocol.

[0057] Figure 15 In a preferred embodiment of the present invention, GNS@YSMS-Fe 2+ &GOX in vivo temperature rise curve (a) and thermal imaging (b) under 808nm laser irradiation.

[0058] Figure 16 In a preferred embodiment of the present invention, GNS@YSMS-Fe 2+ The inhibitory effect of &GOX on mouse tumors: a, b, c, d, e, and f are mouse tumor photographs at different time points, tumor volume, in vitro tumor photographs at the endpoint, tumor mass, and HE staining of tumor tissue, respectively.

[0059] Figure 17 The effect of GNS@YSMS on the activity of two cell lines (SiHa and Tu212) in a preferred embodiment of the present invention.

[0060] Figure 18 In a preferred embodiment of the present invention, GNS@YSMS-Fe 2+ Effects of GOX injection on blood physiological and biochemical indicators in mice. Detailed Implementation

[0061] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0062] Preparation method of gold seed aqueous solution: Prepare 100 mL of 1 mM HAuCl4 deionized water solution and place it in a round-bottom flask. Heat to boiling with an electric heating mantle, then add 15 mL of 10 mg / mL sodium citrate deionized water solution. Continue boiling for 15 min, keeping the system stable, to obtain the gold seed aqueous solution. Store at 4℃ for later use.

[0063] Example 1

[0064] 1) Preparation of gold nanostars (GNS)

[0065] Prepare 100 mL of 0.25 mM HAuCl4 deionized water solution, add 8.33 μL of 37% concentrated hydrochloric acid, stir magnetically to mix well, then add 1 mL of gold seed aqueous solution, continue stirring, and simultaneously add 1 mL of 3 mM AgNO3 deionized water solution and 0.5 mL of 100 mM ascorbic acid deionized water solution. The solution changes from red to dark blue. After reacting for about 1 min, add 0.3 mL of 15 mg / mL PVP deionized water solution, stir in the dark for 24 h, centrifuge at 3000 g for 15 min, wash once with deionized water, and disperse the resulting precipitate in 1 mL of deionized water to obtain gold nanostar aqueous solution.

[0066] 2) Preparation of GNS@SiO2

[0067] Mix 1 mL of gold nanostar aqueous solution with 3 mL of anhydrous ethanol, add 72 μL of ammonia (28 wt%), stir magnetically for 10 min, then add 20 μL of TEOS (dispersed in 1 mL of anhydrous ethanol) dropwise, continue stirring for 15-16 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol, wash once with deionized water, disperse the precipitate in 7.5 mL of deionized water, which is the GNS@SiO2 aqueous solution, for later use.

[0068] 3) Preparation of GNS@SiO2@mSiO2

[0069] Mix 7.5 mL of GNS@SiO2 aqueous solution with 3 mL of anhydrous ethanol in step 2), add 25 mg of CTAB, sonicate to dissolve, then add 100 μL of ammonia (28 wt%), stir in a 35 °C water bath for 1 h, add a mixture of 20 μL of TEOS and 20 μL of BTSE, continue stirring for 4-5 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol and once with deionized water, and collect the precipitate, which is GNS@SiO2@mSiO2, for later use.

[0070] 4) Preparation of GNS@YSMS (Na2CO3 etching method)

[0071] Take GNS@SiO2@mSiO2 from step 3), add 2.5 mL of Na2CO3 solution with a concentration of 254.4 mg / mL, shake on a shaker at room temperature for 2 h, centrifuge at 10000 rpm for 10 min, wash twice with deionized water, keep the precipitate, add anhydrous methanol-sodium chloride solution (7.9 mg / mL) and stir magnetically for 12 h, repeat 3 times to remove CTAB; wash twice with anhydrous ethanol, then freeze-dry the precipitate to obtain egg yolk shell mesoporous silica-coated gold nanostars (GNS@YSMS), for later use.

[0072] 5) APTES amination

[0073] Weigh 20 mg of the lyophilized material from step 4), add 40 mL of anhydrous ethanol, dissolve thoroughly, then add 0.2 mL of ammonia, stir evenly, then add 0.15 mL of APTES, stir magnetically for 12 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol, wash once with deionized water, and retain the precipitate, which is the aminated egg yolk shell mesoporous silica-coated gold nanostar (aminated GNS@YSMS).

[0074] Figure 2 Image d is a transmission electron microscope (TEM) image of the gold nanostars (GNS@YSMS) with mesoporous silica shells prepared in this embodiment. TEM shows a spherical structure with GNS yolk and MS shell.

[0075] Figure 2 Image b is a transmission electron microscope (TEM) image of GNS@SiO2 prepared in step 1) of Example 1. A 5 nm dense silicon dioxide layer is pre-coated on the GNS. At this point, GNS@SiO2 can be well coated by MS (mesoporous silica), and GNS@MS shows a clear spherical structure under TEM with an average diameter of 200 nm. Figure 2 c).

[0076] Figure 2 Image a is a transmission electron microscope image of the gold nanostar (GNS) prepared in step 1), which shows a clear star-shaped structure.

[0077] The fabrication process of GNS@YSMS is as follows: Figure 1 .

[0078] Comparative Example 1: Gold Nanostars without PVP Functionalization

[0079] The only difference from Example 1 is step 1). The method for preparing gold nanostars in this comparative example is as follows:

[0080] Prepare 100 mL of 0.25 mM HAuCl4 deionized water solution, add 8 μL of 37% concentrated hydrochloric acid, mix well, then add 5 mL of the above gold seed, stir gently (700 rpm), then add 1 mL of 3 mM AgNO3 and 0.5 mL of 100 mM ascorbic acid. The solution changes from red to blue-green. Then add 100 μL of 5000 mM PEG-SH (thiolized polyethylene glycol), stir for 15 min, centrifuge at 4000 rpm for 25 min, collect the precipitate, disperse it in 3 mL of deionized water, and store at 4℃ for later use.

[0081] Using the gold nanostars prepared in this comparative example, silica-coated gold nanostars were prepared in the same manner as step 2) of Example 1.

[0082] See electron microscope image Figure 6 The results showed that most of the silica was not coated on the surface of the gold nanostars.

[0083] Comparative Example 2: No SiO2 coating (i.e., one-step method)

[0084] The only difference from Example 1 is that step 2 is omitted, as follows:

[0085] Mix 7.5 mL of gold nanostar aqueous solution with 3 mL of anhydrous ethanol, add 25 mg of CTAB, sonicate to dissolve, then add 100 μL of ammonia (28 wt%), stir in a 35 °C water bath for 1 h, add a mixture of 20 μL of TEOS and 20 μL of BTSE, continue stirring for 4-5 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol and once with deionized water, and keep the precipitate for later use.

[0086] Take the precipitate, add 2.5 mL of Na2CO3 solution with a concentration of 254.4 mg / mL, shake on a shaker at room temperature for 2 h, centrifuge at 10000 rpm for 10 min, wash twice with deionized water, keep the precipitate, add anhydrous methanol-sodium chloride solution (7.9 mg / mL) and stir magnetically for 12 h, repeat 3 times to remove CTAB; wash twice with anhydrous ethanol, then freeze-dry the precipitate for later use.

[0087] Weigh 20 mg of the lyophilized material, add 40 mL of anhydrous ethanol, dissolve it completely, then add 0.2 mL of ammonia water, stir evenly, then add 0.15 mL of APTES, stir magnetically for 12 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol, wash once with deionized water, and retain the precipitate to obtain egg yolk shell mesoporous silica-coated gold nanostars.

[0088] See electron microscope image Figure 7 The results showed that some egg yolk shells had no gold nanostars in the center of the mesoporous silica, while some egg yolk shells had multiple gold nanostars in the center of the mesoporous silica, and the protrusions on the surface of the gold nanostars were shorter.

[0089] The comparative example of GNS@YSMS was synthesized using a one-step method, where GNS directly reacted with CTAB, BTSE, and TEOS under alkaline conditions. However, due to the repulsive effects of polymers (such as PVP and PEG) introduced during the GNS synthesis and CTAB, a homogeneous product could not be obtained.

[0090] Comparative Example 3: Etching without sodium carbonate

[0091] The only difference from Example 1 is step 4), as follows:

[0092] Take GNS@SiO2@mSiO2 from step 3 of Example 1, wash once with anhydrous ethanol, wash once with deionized water, disperse the collected precipitate in 10 mL of deionized water, incubate at 60°C for 8 h, centrifuge at 10000 rpm for 10 min, wash three times with anhydrous ethanol, and collect the precipitate. Take 60 mL of anhydrous ethanol, add 120 μL of concentrated HCl (37%), and mix well. Add the collected precipitate to this mixture, stir at 60°C for 3 h to remove CTAB, and obtain egg yolk shell mesoporous silica-coated gold nanostars.

[0093] See electron microscope image Figure 8 The results showed that the star-shaped structure of gold nanostars was destroyed and transformed into a spherical structure, thus losing the advantage of high absorption in the near-infrared region.

[0094] Experiment Example 1: Stability Assessment

[0095] Compared to GNS, GNS@YSMS exhibits superior stability under various conditions.

[0096] Stability is crucial for clinical applications. This study evaluated the freeze-thaw and photothermal stability of GNS and GNS@YSMS.

[0097] Prepare aqueous solutions of GNS and GNS@YSMS nanoparticles (prepared in Example 1) with the same gold content. Take 2.5 mL of each solution, centrifuge, freeze-dry the precipitate, and then reconstitute it in 2.5 mL of deionized water. Take 2 mL of each of the four solutions before and after freeze-drying and place them in quartz cuvettes. Use a UV-1800 ultraviolet-visible spectrophotometer to measure the absorption spectra of the four samples in the range of 200-1100 nm.

[0098] 200 μL of each of the GNS and GNS@YSMS aqueous solutions were added to small, self-made, transparent glass tubes. The tubes had a wall thickness of approximately 0.5 mm and a height of approximately 2 cm. The tube openings were sealed with sealing film to prevent evaporation of water and heat loss during the experiment. The sealed glass tubes were then placed in a fitted foam bath, with the initial temperature controlled at approximately 25°C. An 808 nm laser was used to irradiate the glass tubes vertically downwards from approximately 10 cm directly above them. Both solutions underwent six laser irradiations for both heating and cooling. An infrared thermal imager was used to track and measure the liquid temperature inside the tubes.

[0099] Take 200 μL of deionized water and GNS@YSMS aqueous solution (0.05 mg / mL and 0.1 mg / mL respectively), place them in glass tubes, seal them, and irradiate them with an 808 nm laser. Record the liquid temperature at 0, 0.5, 1, 2, 3, 5, 10, 15 and 20 min during the laser irradiation process using an infrared thermal imager. Repeat the test three times for each liquid and take the average value.

[0100] GNS@YSMS exhibits a similar physical appearance, with no significant aggregation after freeze-drying, while GNS shows significant aggregation after freeze-drying, consistent with the UV-Vis absorption spectrum. Figure 3 Furthermore, the photothermal properties remained almost unchanged after six ON / OFF laser cycles, demonstrating the high photothermal stability of GNS@YSMS. Figure 4 Meanwhile, different concentrations of GNS@YSMS exhibited rapid temperature rise under 808nm laser irradiation. Figure 5 These results all demonstrate that GNS@YSMS possesses stable performance and excellent photothermal properties.

[0101] Experiment Example 2: Investigation of Drug Loading Capacity

[0102] GNS@YSMS has the ability to load multiple drugs.

[0103] Considering the positively charged mesoporous structure of the yolk shell of GNS@YSMS, this experiment explores its potential as a platform for delivering various drugs.

[0104] Prepare a 0.2 mol / mL FeSO4 aqueous solution, an 8 mg / mL GNS@YSMS aqueous solution, a 10 mg / mL GOX aqueous solution, and an HRP aqueous solution.

[0105] Carrying Fe 2+ Take 0.5 mL of the above GNS@YSMS aqueous solution, 0.25 mL of FeSO4 aqueous solution, and add 0.25 mL of deionized water. Mix well and place in a shaker at room temperature in the dark. Shake at 200 rpm for 2 hours. After centrifugation and washing with water, obtain GNS@YSMS-Fe 2+ .

[0106] GOX loading: Take 0.25 mL of the above GNS@YSMS aqueous solution, 0.04 mL of GOX aqueous solution, add 0.75 mL of deionized water, mix well, and place in a shaker at room temperature in the dark. Shake at 200 rpm for 1 h. After centrifugation and washing with water, GNS@YSMS-GOX is obtained.

[0107] HRP loading: Take 0.25 mL of the above GNS@YSMS aqueous solution, 0.04 mL of HRP aqueous solution, add 0.75 mL of deionized water, mix well, and place in a shaker at room temperature in the dark. Shake at 200 rpm for 1 h. After centrifugation and washing with water, GNS@YSMS-HRP is obtained.

[0108] The N2 absorption-desorption isotherm and the corresponding pore size distribution further confirm the explicit mesoporous structure. Figure 9 (a) and (b), the surface area, pore volume, and average pore diameter of GNS@YSMS are 944.00 m², respectively. 2 / g, 1.13cc / g, and 3.94nm. Next, metal ions, proteins, and nucleic acid drugs were loaded into GNS@YSMS ( Figure 10 The results showed that protein drugs, represented by glucose oxidase and horseradish peroxidase, and Fe... 2+ Metal ion drugs, represented by these, can be efficiently loaded into GNS@YSMS, with loading rates of 4.07%, 4.37%, and 1.96%, respectively. Figure 11 a). After loading protein drugs, the surface potential of GNS@YSMS decreased; after loading metal ions, the surface potential of GNS@YSMS increased. Figure 11 (b) These results demonstrate that GNS@YSMS can serve as a universal drug delivery platform, playing a crucial role in multimodal cancer therapy.

[0109] Experimental Example 3 GNS@YSMS-Fe 2+ &GOX-mediated photothermal therapy and chemokinetic therapy synergistically inhibit tumor cell growth

[0110] To confirm the synergistic inhibitory effect of GNS@YSMS on tumor growth, GOX and Fe were used. 2+ Simultaneously loaded into GNS@YSMS, resulting in GNS@YSMS-Fe 2+ &GOX.

[0111] GNS@YSMS-Fe 2+ The preparation method of GNS@YSMS is as follows: Take 0.5 mL of 8 mg / mL GNS@YSMS aqueous solution, 0.25 mL of 0.2 mol / mL FeSO4 aqueous solution, and add 0.25 mL of deionized water. Mix well and place in a shaker at room temperature in the dark. Shake at 200 rpm for 2 hours. Then add 0.08 mL of 10 mg / mL GNS@YSMS aqueous solution and continue shaking for 1 hour. After centrifugation and washing with water, GNS@YSMS-FeSO4 is obtained. 2+ &GOX.

[0112] Preparation method of GNS@YSMS-GOX: Take 0.25 mL of 8 mg / mL GNS@YSMS aqueous solution, 0.04 mL of 10 mg / mL GOX aqueous solution, add 0.75 mL of deionized water, mix well, and place in a shaker at room temperature in the dark. Shake at 200 rpm for 1 h. After centrifugation and washing with water, GNS@YSMS-GOX is obtained. GNS@YSMS-Fe 2+ Preparation method: Take 0.5 mL of 8 mg / mL GNS@YSMS aqueous solution, 0.25 mL of 0.2 mol / mL FeSO4 aqueous solution, add 0.25 mL of deionized water, mix well, and place in a shaker at room temperature in the dark. Shake at 200 rpm for 2 hours. After centrifugation and washing with water, obtain GNS@YSMS-Fe 2+ .

[0113] In vitro cell experiments showed that only when GOX and Fe... 2+ Simultaneous loading is necessary for a significant increase in intracellular reactive oxygen species (ROS) levels. Figure 12 (a and b) indicate that only when GOX catalyzes glucose as H2O2 and Fe 2+ Intracellular reactive oxygen species levels can only increase when H₂O₂ is catalyzed to •OH. H₂O₂ and Fe 2+ Neither GOX nor Fe significantly increased the ROS level of cells, thus preliminarily verifying the presence of GOX and Fe. 2+ The effectiveness of GNS-mediated chemokinetic therapy was assessed. Furthermore, GNS-mediated photothermal therapy was introduced. At the cellular level, we observed synergistic killing effects of the two treatment modalities. Specifically, 150 μg / mL GNS@YSMS-Fe 2+ The viability of SiHa cells treated with GNS@YSMS+L was 44.9%, while that treated with GNS@YSMS+L was 60.1%. However, the viability of SiHa cells treated with GNS@YSMS-Fe...2+ The cell viability after treatment with &GOX+L was only 12.3% ( Figure 13 (a and b) indicates that the combination of the two treatment modalities can significantly kill SiHa cells in vitro.

[0114] Experimental Example 4 GNS@YSMS-Fe 2+ &GOX-mediated photothermal therapy and chemodynamic therapy inhibit solid tumor growth

[0115] To further investigate GNS@YSMS-Fe 2+ To investigate the therapeutic effect of &GOX on solid tumors, this experiment constructed a SiHa cell xenograft model, and waited for the tumor volume to grow to 50 mm. 3 Different therapeutic agents were administered via intratumoral injection, followed by laser irradiation at 6 hours and 48 hours post-injection. Figure 14 ).

[0116] After cervical cancer cells were cultured to the logarithmic growth phase, the cells were digested and collected in 1.5 mL centrifuge tubes, at a concentration of approximately 10⁻⁶. 7 Cells / mL. 50 μL of cell suspension was drawn up with a syringe and subcutaneously injected into the axilla of the right forelimb of nude mice. Tumors were allowed to grow to approximately 50 mm. 3 The experiment can begin at the left or right.

[0117] Twenty nude mice bearing cervical cancer tumors were randomly divided into four groups. The tumors were injected with 50 μL of PBS solution, 50 μL of GNS@YSMS-Fe, and other solutions, respectively. 2+ &GOX (1mg / mL), 50μL GNS@YSMS (1mg / mL), 50μL GNS@YSMS-Fe 2+ &GOX (1 mg / mL), i.e., the "Tumor + PBS" group, "Tumor + GNS@YSMS-Fe 2+ The "&GOX" group, the "GNS@YSMS + L" group, and the "GNS@YSMS-Fe" group 2+ The &GOX + L group. Six hours after injection into the tumor, the mice underwent their first laser irradiation (10 min), during which the temperature of the tumor site was recorded using an infrared thermal imager (0, 0.5, 1, 2, 3, 4, 5, and 10 min). Forty-eight hours after injection, a second laser irradiation (10 min) was performed. Starting from the day of injection, the size of the mouse tumor was measured and photographed every two days. On day 16, the mice were sacrificed, and the tumors were dissected, photographed, and weighed. Finally, the tumor tissue was fixed for subsequent histological analysis.

[0118] During the first laser irradiation treatment, the temperature of the solid tumor site in mice was monitored. The results showed that the temperature of the mouse tumors treated with GNS@YSMS rapidly reached about 49°C after laser irradiation, while PBS and laser irradiation alone could not significantly increase the temperature of the mouse tumors, only raising it to about 40°C. Figure 15 (a and b). A local tumor temperature of 43°C inhibits tumor cell growth; therefore, GNS@YSMS combined with laser irradiation can effectively inhibit tumor growth. Subsequent mouse tumor photographs ( Figure 16 a) and mouse tumor growth curve ( Figure 16 (b) and (c) confirmed that GNS@YSMS combined with laser irradiation significantly inhibited the growth of solid tumors in mice, with a tumor growth inhibition rate (TGI) of 66.1% after 16 days of treatment. GNS@YSMS-Fe 2+ The TGI after GOX processing was 53.2%, GNS@YSMS-Fe 2+ &GOX combined with laser irradiation, i.e., photothermal therapy of GNS combined with GOX and Fe 2+ Following mediated chemokinetic treatment, the TGI was 92.1%, demonstrating that the combination of the two treatment modalities significantly inhibited the growth of SiHa solid tumors. Ex vivo mouse tumor photographs and weights further corroborated these results. Figure 16 ,d and e). Results of HE staining of mouse tumor tissue showed ( Figure 16 f) In the PBS group, the tumor tissue was dense, with intact tumor cell nuclei and cytoplasm, and a clear structure, indicating vigorous tumor growth at this time; GNS@YSMS-Fe 2+ &GOX, GNS@YSMS + L and GNS@YSMS-Fe 2+ The loss of cytoplasm and nucleus, and increased intercellular spaces in the &GOX + L group indicate that the tumor cells are in an apoptotic and necrotic state. Among these, GNS@YSMS-Fe... 2 + The necrosis was particularly pronounced in the &GOX + L group. These results indicate that the necrosis in GNS@YSMS-Fe 2+ &GOX can effectively inhibit the growth of SiHa solid tumors under laser excitation.

[0119] Experimental Example 5: Both GNS@YSMS and GNS@YSMS-Fe2+&GOX showed good biocompatibility.

[0120] In confirming GNS@YSMS-Fe 2+ While acknowledging the therapeutic efficacy of &GOX, safety is a key consideration. Only by ensuring a certain level of safety can the treatment be considered more valuable and meaningful.

[0121] SiHa and Tu212 cells were cultured to the logarithmic growth phase, digested with trypsin, and passaged into the wells of 96-well cell culture plates. The cell suspension concentration was adjusted to approximately 6000 cells per well, with two plates containing 25 wells each. The plates were incubated overnight at 37°C with 5% CO2 to allow cell adhesion. The culture medium was removed from the wells, and 200 μL of serum-free GNS@YSMS medium solutions with concentrations of 0.1, 0.2, 0.3, and 0.4 mg / mL were added, respectively. A serum-free medium solution without nanomaterials was used as a control. Five wells were used as replicates for each group. The 96-well plates were then placed in a cell culture incubator and cultured at 37°C with 5% CO2 for 24 h. Cell viability was then assessed for each group.

[0122] Nine healthy female mice were randomly divided into three groups: one group was untreated, with blood samples taken directly to detect various physiological and biochemical indicators in the blood as a control; the other two groups were injected subcutaneously with GNS@YSMS-Fe 2+ Blood samples of 2-GOX (1 mg / mL, 50 μL) were collected on day 1 and day 7 after injection to detect various physiological and biochemical indicators in the blood.

[0123] Based on this, this experiment first analyzed the effects of different concentrations of GNS@YSMS on the activity of two cell lines, SiHa and Tu212, in vitro. Figure 17 The results showed that even 400 μg / mL GNS@YSMS had no significant effect on the growth of either cell line, confirming the good biocompatibility of the GNS@YSMS drug delivery platform. We further analyzed the effects of subcutaneous injection of GNS@YSMS-Fe into healthy mice. 2+ Blood physiological and biochemical indicators of &GOX on day 1 and day 7 ( Figure 18 Compared with healthy mice, there were no significant differences in any of the indicators, proving that GNS@YSMS-Fe 2+ Treatment with &GOX had minimal impact on the overall physiological state of mice and showed good biocompatibility.

[0124] In summary, this invention constructs a drug delivery platform with excellent photothermal properties, which can be combined with various treatment modalities. In vitro and in vivo therapeutic experiments, after in combination with chemokinetics, have confirmed its highly effective tumor suppression effect, while also demonstrating good biocompatibility. This therapeutic system has potential clinical application value for the combined treatment of cervical cancer.

[0125] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A gold nanostar coated with mesoporous silica from an egg yolk shell, characterized in that, include: The core is a gold nanostar; The shell is mesoporous silica; The preparation method of the gold nanostars coated with mesoporous silica from egg yolk shells includes: 1) Preparation of gold nanostars: In solution, HAuCl4, concentrated hydrochloric acid and gold seeds are reacted first; then AgNO3 and ascorbic acid are added and reacted; finally, polyvinylpyrrolidone is added and reacted to obtain gold nanostars; 2) Dense silica is coated onto the surface of the gold nanostars to form GNS@SiO2; 3) GNS@SiO2 reacts with tetraethyl orthosilicate, dimethyldimethoxysilane and hexadecyltrimethylammonium bromide to prepare GNS@SiO2@mSiO2; 4) Etch GNS@SiO2@mSiO2 with sodium carbonate to produce egg yolk shell mesoporous silica-coated gold nanostars.

2. The egg yolk shell mesoporous silica-coated gold nanostar according to claim 1, characterized in that, The gold nanostars are encapsulated in mesoporous silica, with the core having a diameter of 50-100 nm and the shell having a thickness of 75-150 nm.

3. The method for preparing the egg yolk shell mesoporous silica-coated gold nanostars according to claim 1 or 2, characterized in that, include: 1) Preparation of gold nanostars: In solution, HAuCl4, concentrated hydrochloric acid and gold seeds are reacted first; then AgNO3 and ascorbic acid are added and reacted; finally, polyvinylpyrrolidone is added and reacted to obtain gold nanostars; 2) Dense silica is coated onto the surface of the gold nanostars to form GNS@SiO2; 3) GNS@SiO2 reacts with tetraethyl orthosilicate, dimethyldimethoxysilane and hexadecyltrimethylammonium bromide to prepare GNS@SiO2@mSiO2; 4) Etch GNS@SiO2@mSiO2 with sodium carbonate to produce egg yolk shell mesoporous silica-coated gold nanostars.

4. The method according to claim 3, characterized in that, include: (1) In solution, HAuCl4, concentrated hydrochloric acid and gold seeds are reacted first; then AgNO3 and ascorbic acid are added to react; finally, polyvinylpyrrolidone is added to react to obtain gold nanostars; (2) In the presence of deionized water, anhydrous ethanol and ammonia, the gold nanostars were reacted with tetraethyl orthosilicate to prepare GNS@SiO2. (3) In the presence of deionized water, anhydrous ethanol and ammonia, GNS@SiO2 reacts with tetraethyl orthosilicate, dimethyldimethoxysilane and hexadecyltrimethylammonium bromide to prepare GNS@SiO2@mSiO2. (4) GNS@SiO2@mSiO2 was contacted with a sodium carbonate solution with a concentration of 200-300 mg / mL to prepare egg yolk shell mesoporous silica-coated gold nanostars.

5. The method according to claim 4, characterized in that, include: S1. Prepare 100 mL of 0.25 mM HAuCl4 deionized water solution, add 8.33 μL of 36%-38% concentrated hydrochloric acid, stir magnetically to mix well, then add 1 mL of gold seed aqueous solution, continue stirring, and simultaneously add 1 mL of 3 mM AgNO3 deionized water solution and 0.5 mL of 100 mM ascorbic acid deionized water solution. After reacting for 1 min, add 0.3 mL of 15 mg / mL polyvinylpyrrolidone deionized water solution, stir in the dark for 24 h, centrifuge at 3000 g for 15 min, wash once with deionized water, and disperse the resulting precipitate in 1 mL of deionized water to obtain gold nanostar aqueous solution; The preparation method of the gold seed aqueous solution includes: preparing 100 mL of 1 mM HAuCl4 deionized water solution, heating to boiling, adding 15 mL of 10 mg / mL sodium citrate deionized water solution, and continuing to boil for 15 min to obtain the gold seed aqueous solution. S2. Mix 1 mL of gold nanostar aqueous solution with 3 mL of anhydrous ethanol, add 72 μL of 28 wt% ammonia, stir magnetically for 10 min, then add 20 μL of tetraethyl orthosilicate dropwise, continue stirring for 15-16 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol and once with deionized water, disperse the resulting precipitate in 7.5 mL of deionized water to obtain GNS@SiO2 aqueous solution; S3. Mix 7.5 mL of GNS@SiO2 aqueous solution with 3 mL of anhydrous ethanol, add 25 mg of cetyltrimethylammonium bromide, sonicate to dissolve, add 100 μL of 28 wt% ammonia, stir for 1 h in a 35 °C water bath, add a mixture of 20 μL of tetraethyl orthosilicate and 20 μL of dimethyldimethoxysilane, continue stirring for 4-5 h, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol and once with deionized water, and retain the precipitate, which is GNS@SiO2@mSiO2. S4. Add 2.5 mL of Na2CO3 solution with a concentration of 254.4 mg / mL to GNS@SiO2@mSiO2, shake on a shaker at room temperature for 2 h, centrifuge at 10000 rpm for 10 min, wash twice with deionized water, collect the precipitate, add it to anhydrous methanol-sodium chloride solution with a concentration of 7.9 mg / mL, stir magnetically for 12 h, repeat 3 times to remove hexadecyltrimethylammonium bromide; finally, wash twice with anhydrous ethanol, collect the precipitate and freeze-dry to obtain egg yolk shell mesoporous silica-coated gold nanostars; The 7.9 mg / mL anhydrous methanol-sodium chloride solution means that each milliliter of anhydrous methanol contains 7.9 mg of sodium chloride.

6. The method according to claim 3, characterized in that, Also includes: 5) The mesoporous silica coating of egg yolk shells with gold nanostars was aminated with 3-aminopropyltrimethoxysilane; Specifically, in the presence of anhydrous ethanol and ammonia, mesoporous silica-coated gold nanostars from egg yolk shells were reacted with 3-aminopropyltrimethoxysilane to prepare aminated mesoporous silica-coated gold nanostars from egg yolk shells.

7. A gold nanostar coated with mesoporous silica from an egg yolk shell, characterized in that, It is prepared by the method described in any one of claims 3-6.

8. The application of the egg yolk shell mesoporous silica-coated gold nanostars as described in any one of claims 1-2 and 7 in the preparation of a drug delivery platform; The carrier includes one or more of the following: chemotherapy drugs, photosensitizers, metal ions, proteins, and nucleic acid drugs.

9. A composition, characterized in that, Includes the egg yolk shell mesoporous silica-coated gold nanostars and the substrate as described in any one of claims 1-2 and 7; The carrier includes one or more of the following: chemotherapy drugs, photosensitizers, metal ions, proteins, and nucleic acid drugs.

10. The composition according to claim 9, characterized in that, The loaded material is selected from iron ions, protein drugs, and glucose oxidase.

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