Mellitin-m nanocomposite, preparation method and application thereof

Abstract

The application discloses a mast cell peptide-M nanocomposite and a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. In the application, indocyanine green is used as a morphology mediator, and a dendritic tetra-silicon with an ultrasmall particle size is prepared by reacting the indocyanine green with triethylamine, cetyltrimethylammonium bromide, tetraethyl orthosilicate and bis[3-(triethoxysilyl)propyl]tetrasulfide in water, and then mast cell peptide-M is loaded on the dendritic tetra-silicon to prepare the mast cell peptide-M nanocomposite. In-vitro experiments prove that the nanocomposite can significantly kill 4T1 breast cancer cells, and the nanocomposite also has good targeted enrichment, significant anti-tumor effect and excellent safety in a breast cancer brain metastasis model due to the ultrasmall particle size, and the application provides a novel and efficient nanomedicine delivery system for brain tumor treatment and development of related therapeutic drugs.

Description

Technical Field

[0001] This invention specifically relates to a wasp venom peptide-M nanocomposite and its preparation method, belonging to the field of medicinal chemistry technology. Background Technology

[0002] With the gradual improvement of comprehensive tumor diagnosis and treatment, the clinical intervention effects of tumor-related extracranial lesions have been effectively improved. Cancer brain metastases are increasingly becoming one of the leading causes of death in cancer patients. The core reason for this situation is that the presence of the blood-brain barrier (BBB) ​​severely hinders the effective delivery of most chemotherapy drugs to brain lesions, leading to treatment failure in cancer brain metastases. Therefore, developing a targeted delivery system that can efficiently penetrate the BBB is of significant clinical importance for improving the current treatment status of brain metastases.

[0003] Mastoparan-M (Mast-M) is a naturally derived bee venom peptide. Previous studies have confirmed that this active peptide can cross the blood-brain barrier and effectively intervene in Parkinson's disease. It can also induce apoptosis by disrupting mitochondrial membrane potential and has a potent killing effect on various tumor cells, thus showing great potential in the treatment of brain tumors. However, Mastoparan-M alone suffers from high systemic toxicity, a short circulating half-life, and poor targeted delivery capabilities, which greatly limits its clinical translation and application. Therefore, using nanocarriers to encapsulate and deliver Mastoparan-M has become a highly promising strategy for reducing its biotoxicity and improving its targeting.

[0004] In recent years, responsive nanomedicine delivery systems have attracted widespread attention due to their ability to specifically release drugs within the tumor microenvironment. Glutathione (GSH) is the main reducing agent in cells, and the concentration of GSH in tumor cells is significantly higher than in normal tissues (up to 3-10 times), especially in drug-resistant tumors. Dendritic tetrasulfide containing tetrasulfide bonds is a promising GSH-responsive drug carrier. This carrier can undergo specific cleavage triggered by the high concentration of GSH in the tumor microenvironment: on the one hand, it weakens the antioxidant defense capacity of tumor cells by consuming GSH; on the other hand, it releases hydrogen sulfide (H2S) gas during the cleavage process and promotes the degradation of the carrier material, ultimately achieving controlled drug release at the tumor site. However, as disclosed in Chinese patents CN113413462A, CN118460204A, and CN115227671A, existing dendritic tetrasulfide carriers generally have the problem of large particle size (the ability to cross the blood-brain barrier will be significantly reduced when the particle size is greater than 50 nm). When such materials are used as drug carriers, the biotoxic peptide drug delivery system often has difficulty crossing the blood-brain barrier and cannot achieve effective enrichment in the brain. As a result, the targeting and bioavailability of wasp venom peptide-M in the brain cannot be fundamentally improved.

[0005] In summary, there is currently a lack of a nanosystem for treating brain metastases that is both ultra-small in size and possesses GSH-responsive functionality, and can safely and efficiently deliver wasp venom peptide-M. Summary of the Invention

[0006] To address existing problems, this invention provides a wasp venom peptide-M nanocomposite, its preparation method, and its applications. The wasp venom peptide-M nanocomposite is synthesized by loading wasp venom peptide-M onto dendritic tetrasulfide silica. The dendritic tetrasulfide silica exhibits good GSH responsiveness and ultra-small particle size, providing favorable conditions for the targeted transport of wasp venom peptide-M and the crossing of the blood-brain barrier by the prepared nanocomposite. In a breast cancer brain metastasis model, the wasp venom peptide-M nanocomposite exhibits good targeted enrichment, safety, and significant anti-tumor efficacy, which is of great significance for the treatment of brain tumors and the development of related drugs.

[0007] The technical solution of the present invention is as follows: This invention provides a method for preparing a wasp venom peptide-M nanocomposite for the treatment of brain tumors. First, using triethylamine (TEA) as a base source, hexadecyltrimethylammonium bromide (CTAB) as a template agent, indocyanine green (ICG) as a morphology mediator, tetraethyl orthosilicate (TEOS) as a silicon source, and bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES) as a tetrasulfinic source, an ultra-small particle size dendritic tetrasulfinic support is prepared. Then, wasp venom peptide-M is loaded onto this support to obtain the wasp venom peptide-M nanocomposite. The method includes the following steps: S1. Preparation of dendritic tetrasulfide silicon: ICG was used as a morphology mediator and dissolved in water (H2O) with TEA and CTAB to form a solution. Then TEOS and BTES were added to the solution, and the mixture was heated and stirred to react fully to synthesize dendritic tetrasulfide silicon with a particle size of less than 50 nm. After the reaction was completed, the dendritic tetrasulfide silicon was centrifuged, washed and stored in ethanol solution for later use. S2, Wasp Venom Peptide-M Composite: The dendritic tetrathionine is centrifuged and washed, then dispersed in ethanol, and wasp venom peptide-M solution is added to it. After ultrasonic treatment or stirring and mixing, the wasp venom peptide-M nanocomposite is obtained.

[0008] Furthermore, in step S1, the mass ratio of TEA, H2O, CTAB, ICG, TEOS, and BTES is 1 : 564.3 : 8.58 : 1.13 : 42.09 : 42.67.

[0009] Furthermore, maintaining the stirring speed during the reaction in step S1 at 1000 r / min to 1200 r / min can control the particle size of the synthesized dendritic tetrasulfide to below 50 nm. This size characteristic helps to ensure the drug delivery capability of the dendritic tetrasulfide across the blood-brain barrier. Preferably, maintaining the stirring speed during the reaction at 1000 r / min or 1200 r / min, when the speed is 1000 r / min, the particle size of the synthesized dendritic tetrasulfide is 38.17 nm ± 1.1%, and when the speed is 1200 r / min, the particle size of the synthesized dendritic tetrasulfide is 27.49 nm ± 3.0%.

[0010] Furthermore, the specific steps for preparing dendritic tetrasulfide silicon in step S1 are as follows: S11. Disperse TEA in ultrapure water according to the formula amount, stir at 600~800 rpm until homogeneous to obtain solution A; S12. Add CTAB to solution A and stir at 600~800 rpm until homogeneous to obtain solution B; S13. Add ICG to solution B and stir until the solution turns into a uniform transparent green color to obtain solution C; S14. Add TEOS and BTES to solution C and stir the reaction at 80℃ and 1000~1200 rpm to obtain crude dendritic tetrasulfide silicon. S15. Centrifuge the crude dendritic tetrasulfide, discard the supernatant, wash with ethanol and repeat the centrifugation several times to obtain dendritic tetrasulfide, and store it in an ethanol solution.

[0011] More preferably, steps S11 to S14 are carried out sequentially under constant temperature stirring conditions at 80°C.

[0012] Furthermore, the concentration of dendritic tetrathionine dispersed in the homogeneous suspension in step S2 is 1 mg / mL.

[0013] Further, the concentration of the wasp venom peptide-M solution in step S2 is 1 mg / mL; the mass ratio of wasp venom peptide-M in the added wasp venom peptide-M solution to the mass of dendritic tetrathionite dispersed in the homogeneous suspension is 1:1.

[0014] The present invention also provides a wasp venom peptide-M nanocomposite prepared according to the above preparation method, wherein the wasp venom peptide-M nanocomposite exhibits a dendritic morphology and is composed of dendritic tetrasulfide silicon and wasp venom peptide-M uniformly loaded on the surface of the dendritic tetrasulfide silicon.

[0015] Furthermore, the average particle size of the wasp venom peptide-M nanocomposite is 30 nm, and the particle size of its dendritic tetrathiosilicon carrier is 27.49 nm ± 3.0%.

[0016] Furthermore, the average particle size of the wasp venom peptide-M nanocomposite is 40 nm, and the particle size of its dendritic tetrathiosilicon carrier is 38.17 nm ± 1.1%.

[0017] Furthermore, the hornet venom peptide-M is derived from the giant hornet in Dehong, Yunnan, and has the amino acid sequence INLKAIAALAKKLL-NH2. It is artificially modified by C-terminal amidation (-CONH2) of the sequence shown in SEQ ID NO:1, in order to enhance the stability and biological activity of the peptide.

[0018] The wasp venom peptide-M nanocomposite provided by this invention has a small particle size, which provides a strong guarantee for its crossing of the blood-brain barrier. This wasp venom peptide-M nanocomposite has broad application prospects in the treatment of brain tumors, including breast cancer brain metastases and gliomas, and in the development of related therapeutic drugs.

[0019] Unlike existing technologies, this invention has the following advantages: 1. This invention provides a novel method for preparing dendritic tetrasulfide silicon loaded with wasp venom peptide-M, using ICG as a morphology mediator for the first time. This morphology mediator has a relatively large molecular spatial conformation and is negatively charged (sulfonic acid group) and has an amphiphilic structure. In a TEA / CTAB micelle environment, ICG can participate in the early nucleation stage, thereby forming uniformly dispersed micronuclei that can be embedded in CTAB micelles or interfaces, exerting a spatial / interface confinement effect and effectively inhibiting micronucleus aggregation. By reacting it with TEA, CTAB, TEOS, and BTES, and combining it with precise control of stirring speed, the preparation of ultra-small dendritic tetrasulfide silicon was achieved, and the size of the final wasp venom peptide-M nanocomposite was limited to the range that can be penetrated by the blood-brain barrier.

[0020] 2. The hornet venom peptide-M nanocomposite provided by this invention retains the GSH-responsive drug release capability of dendritic tetrasulfide, while also possessing good structural stability and small particle size. It can fully leverage the synergistic effect of dendritic tetrasulfide-hornet venom peptide-M in "drug delivery-antitumor" and exhibits the characteristic of significant enrichment of hornet venom peptide-M in the brain in a breast cancer brain metastasis model. This nanocomposite effectively solves the key technical problems of high systemic toxicity, short half-life, and difficulty in targeted delivery of single hornet venom peptide-M, as well as the large particle size of traditional drug carriers and their inability to cross the blood-brain barrier. The raw materials are widely available and common, and the preparation method has high economic efficiency and operability, which is of great significance for the drug development and treatment of brain metastases. Attached Figure Description

[0021] Figure 1 The image shows a transmission electron microscope (TEM) image of dendritic tetrasulfide silicon (DMS) prepared in Example 1 using ICG as a morphology mediator.

[0022] Figure 2 The image shows the TEM image of the wasp venom peptide-M nanocomposite (DMS@Mast-M) prepared in Example 1.

[0023] Figure 3 The image shows the TEM image of DMS prepared in Example 2 with ICG as the morphology mediator.

[0024] Figure 4 The image shows the TEM image of DMS@Mast-M prepared in Example 2.

[0025] Figure 5 TEM image of tetrasulfide silicon (NGS) prepared for Comparative Example 1.

[0026] Figure 6 DMS (with sodium salicylate (NaSaI) as the morphology mediator, was prepared as a comparative example 2. Figure 6 a, Figure 6 b) and its DMS@Mast-M loaded with wasp venom peptide-M ( Figure 6 c) TEM image and DMS ( Figure 6 d) and DMS@Mast-M ( Figure 6 e) Corresponding particle size distribution.

[0027] Figure 7 The image shows the Fourier transform infrared (FTIR) effect of the DMS@Mast-M fabricated in Example 1.

[0028] Figure 8 The image shows the XRD pattern of DMS@Mast-M prepared in Example 1.

[0029] Figure 9 The graph shows the effect of DMS@Mast-M on GSH consumption detected in vitro using ultraviolet light.

[0030] Figure 10 A diagram showing the effect of using lead acetate test paper to verify the in vitro generation of hydrogen sulfide gas for DMS@Mast-M.

[0031] Figure 11 This is a schematic diagram of DMS@Mast-M uptake in 4T1 cells.

[0032] Figure 12 This is a schematic diagram illustrating the killing effect of DMS@Mast-M on 4T1 cells.

[0033] Figure 13 A schematic diagram of the IC50 values ​​of DMS@Mast-M (30nm / 40nm) on 4T1 cells.

[0034] Figure 14 This is a schematic diagram illustrating the tumor targeting of DMS@Mast-M in breast cancer-bearing mice.

[0035] Figure 15 Fluorescent images of 4Tlluc tumor-bearing mice treated with DMS@Mast-M on different days.

[0036] Figure 16 Survival curves for tumor-bearing mice treated with DMS@Mast-M.

[0037] Figure 17 For the DMS@Mast-M hemolysis rate experiment. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0039] This invention provides a method for preparing wasp venom peptide-M nanocomposite. Using ICG as a morphology mediator, it reacts with TEA, CTAB, TEOS, and BTES in water to prepare an ultra-small particle size dendritic tetrasulfide silicon, and then loads wasp venom peptide-M onto it to obtain the wasp venom peptide-M nanocomposite.

[0040] The wasp venom peptide-M nanocomposite prepared by this invention has a small particle size and can efficiently cross the blood-brain barrier. It can be used to prepare drugs for the treatment of brain tumors and has broad application prospects in the treatment of brain tumors, including brain metastases of breast cancer and gliomas.

[0041] The following specific embodiments further illustrate the wasp venom peptide-M nanocomposite and its preparation method of the present invention.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0044] In some preferred embodiments, the wasp venom peptide-M is derived from the giant wasp *Vespa mandshurica* of Dehong, Yunnan Province, and has the amino acid sequence INLKAIAALAKKLL-NH2. This amino acid sequence is obtained by C-terminal amidation modification of the sequence shown in SEQ ID NO:1, aiming to enhance the stability and bioactivity of the peptide. To meet the tracer requirements in the experiment, commercially available Mastoparan-M-FITC was selected as the raw material for synthesizing the wasp venom peptide-M nanocomposite in the following examples. Based on the wasp venom peptide-M peptide chain sequence, its N-terminus is also labeled with a FITC (fluorescein isothiocyanate) fluorescent group.

[0045] Example 1 This embodiment provides a method for preparing wasp venom peptide-M nanocomposite, comprising the following steps: S1. Preparation of dendritic tetrasulfide: In this embodiment, ICG was used as a morphology mediator to synthesize dendritic tetrasulfide. During the synthesis process, the amount of ICG used was 100 mg, and the components such as TEA, H2O, CTAB, TEOS, and BTES were added in the mass ratio of TEA : H2O : CTAB : ICG : TEOS : BTES = 1 : 564.3 : 8.58 : 1.13 : 42.09 : 42.67. According to the formulation, the amount of TEA was first added to an appropriate amount of ultrapure water and placed in a constant temperature environment of 80°C. The mixture was stirred at 600 rpm for 15 minutes to ensure that the triethylamine was fully dispersed. Then, CTAB was added, and the mixture was stirred at 80°C and 600 rpm for 2 hours to ensure that the CTAB was completely dissolved and evenly distributed. Next, an appropriate amount of ICG was added to the above solution, and the mixture was stirred at 80°C for 15 minutes (600 rpm) until a green, uniform, transparent, and clear solution was obtained. Finally, TEOS and BTES were added, and the mixture was stirred at 80°C and 1200 rpm for 12 hours to obtain crude dendritic tetrasulfide. The crude dendritic tetrasulfide was then repeatedly centrifuged and washed 8-10 times until the supernatant showed no obvious fluorescence. The supernatant was discarded, and the dendritic tetrasulfide was obtained and stored in an ethanol solution for later use. S2. Loading of wasp venom peptide-M: The dendritic tetrasulfide obtained in step S1 was collected by centrifugation to remove residual ethanol. 10 mg of dendritic tetrasulfide was added to 10 mL of ultrapure water and dispersed by ultrasonication to obtain a homogeneous suspension of dendritic tetrasulfide. Subsequently, 10 mg of commercially available Mastoparan-M-FITC was dissolved in 10 mL of ultrapure water to prepare an aqueous solution of wasp venom peptide-M. Under continuous stirring (800 rpm), the aqueous solution of wasp venom peptide-M was slowly added to the homogeneous suspension of dendritic tetrasulfide and stirred at room temperature for 1 h to achieve loading of wasp venom peptide-M on the surface of branched silicon nanoparticles.

[0046] After loading was completed, the nanoparticles were collected by centrifugation and washed with ultrapure water to remove unloaded Matoparan-M-FITC. The loading amount of FITC in the supernatant after centrifugation could be quantitatively analyzed by detecting the fluorescence intensity of FITC.

[0047] The wasp venom peptide-M nanocomposite prepared in this embodiment consists of dendritic tetrasulfide silicon and wasp venom peptide-M uniformly loaded on the surface of dendritic tetrasulfide silicon.

[0048] Example 2 This embodiment provides a method for preparing wasp venom peptide-M nanocomposite, which is basically the same as that in Example 1, except that: In step S1, TEOS and BTES are added to the green, uniform, transparent, and clear solution, and stirred at 80°C and 1000 rpm for 12 h to obtain crude dendritic tetrasulfide. The crude dendritic tetrasulfide is then repeatedly centrifuged and washed 8-10 times until the supernatant shows no obvious fluorescence. The supernatant is then discarded to obtain the dendritic tetrasulfide, which is stored in an ethanol solution for later use.

[0049] The remaining steps and conditions not mentioned are the same as in Example 1, and will not be repeated here.

[0050] Similar to Example 1, the wasp venom peptide-M nanocomposite prepared in this example is also composed of dendritic tetrasulfide silicon and wasp venom peptide-M uniformly loaded on the surface of dendritic tetrasulfide silicon.

[0051] Comparative Example 1 This comparative example provides a method for preparing tetrathionyl silicon (NGS) nanomedicine carriers, which is basically the same as the method for preparing dendritic tetrathionyl silicon described in Example 2. Except for the absence of the morphology mediator ICG during the NGS synthesis process, the other components in the synthesis process are also added in the same mass ratio as TEA: H2O: CTAB: TEOS: BTES = 1: 564.3: 8.58: 42.09: 42.67. The amount and order of addition are the same as those in the dendritic tetrathionyl silicon in Example 2.

[0052] The specific steps of the NGS preparation method are as follows: According to the formulation dosage, the prescribed amount of TEA was first added to an appropriate amount of ultrapure water and placed in a constant temperature environment of 80℃. The mixture was stirred at 600 rpm for 15 min to ensure that the triethylamine was fully dispersed. Then, CTAB was added, and the stirring conditions of 80℃ and 600 rpm were maintained for 2 h to ensure that the CTAB was completely dissolved and evenly distributed. Then, TEOS and BTES were added, and the mixture was stirred at 80℃ and 1000 rpm for 12 h to obtain crude tetrasulfide silicon. Finally, the crude NGS was centrifuged and washed 8-10 times, the supernatant was discarded, and then dispersed and stored in an ethanol solution.

[0053] Comparative Example 2 This comparative example provides a wasp venom peptide-M nanocomposite, composed of dendritic tetrasulfide silica and wasp venom peptide-M uniformly loaded on the surface of the dendritic tetrasulfide silica. The preparation method of the wasp venom peptide-M nanocomposite is basically the same as that of Example 2, except that: In step S1, sodium salicylate (NaSaI) is used as a morphology mediator instead of ICG.

[0054] The remaining steps and conditions are the same as in Example 2, and will not be repeated here.

[0055] Structural characterization and performance testing (1) Structural characterization The TEM morphology of the dendritic tetrathionine drug carrier and wasp venom peptide-M nanocomposite in Example 1 is as follows: Figures 1-2 As shown, before loading wasp venom peptide-M, the dendritic tetrasulfide exhibits a distinct dendritic morphology. Figure 1 a), with a particle size of 27.49 nm ± 3.0% ( Figure 1 b). Morphology after loading wasp venom peptide-M ( Figure 2 a) Significant changes were observed in the dendritic tetrasulfide compared to the empty carrier, indicating that wasp venom peptide-M was successfully loaded onto the dendritic tetrasulfide. Furthermore, TEM images showed that the particle size of the wasp venom peptide-M nanocomposite was 30.20 nm ± 2.8%. Figure 2(b) The average particle size is 30 nm, which indicates that the particle size of the wasp venom peptide-M nanocomposite did not change significantly compared to the empty carrier, and still maintained a small particle size base, which provides a strong guarantee for its ability to penetrate across the blood-brain barrier.

[0056] The TEM morphology of the dendritic tetrathionine drug carrier and wasp venom peptide-M nanocomposite in Example 2 is as follows: Figures 3-4 As shown, similar to Example 1, the dendritic tetrasulfide also exhibits a distinct dendritic morphology. Figure 3 a), but its particle size showed a relative increase, at 38.17 nm ± 1.1% ( Figure 3 b). Meanwhile, similar to Example 1, the particle size of the prepared wasp venom peptide-M nanocomposite did not show a significant change compared to the empty carrier dendritic tetrasulfide, remaining at 39.58 nm ± 2.4%. Figure 4 (b) The average particle size is 40 nm.

[0057] By comparing Examples 1 and 2, it can be found that in the preparation method of wasp venom peptide-M nanocomposite described in the above examples, the particle size of the obtained dendritic tetrasulfide drug carrier can be controlled by changing the stirring speed of the reaction in step S1. The present invention controls the particle size of the synthesized dendritic tetrasulfide to below 50 nm by using the above-mentioned preferred stirring speed (1200 rpm, 1000 rpm), thereby effectively enhancing its ability to cross the blood-brain barrier.

[0058] Unlike Examples 1 and 2, Comparative Example 1 provides a method for preparing an NGS nanomedicine carrier, and the TEM image of the prepared tetrathionylsilane is shown below. Figure 5 As shown, the average particle size of the prepared NGS is 50 nm, and the morphology is a simple sphere without any branching structure. This result directly confirms that ICG, as a morphology mediator, is the key to inducing the formation of dendritic structures in silicon-based materials.

[0059] It should be noted that the spherical NGS, lacking the hierarchical pores and ultra-large specific surface area characteristic of dendritic structures, can only achieve drug adsorption through a small number of surface sites, thus significantly limiting its drug loading efficiency. In contrast, dendritic tetrasulfide, with its unique branching morphology, not only possesses abundant pore structures and an ultra-high specific surface area, enabling efficient and high-capacity drug loading, but also achieves slow drug release through its structural characteristics, greatly improving drug loading stability and targeted delivery. In comparison, the spherical NGS in this comparative example is far inferior to the dendritic tetrasulfide in Examples 1 and 2 in terms of drug loading capacity, antitumor activity, and practical application potential.

[0060] Unlike Examples 1 and 2, Comparative Example 2 used NaSaI as a morphology mediator to prepare a wasp venom peptide-M nanocomposite. Compared with the examples, it prepared dendritic tetrasulfide silicon (… Figure 6 a, Figure 6 b) and its wasp venom peptide-M nanocomposite ( Figure 6 c) The particle size of all particles increased significantly, reaching 167.2 nm ± 1.8%. Figure 6 d) and 171.8 nm ± 1.7% (6e), at this size it is difficult to achieve effective crossing of the blood-brain barrier, and the transmembrane delivery capability is significantly limited.

[0061] By comparing Examples 1-2 with Comparative Examples 1-2, it can be seen that the present invention achieves the synthesis of wasp venom peptide-M nanocomposites with ultra-small particle size and dendritic morphology through the use of ICG morphology mediator and precise control of reaction conditions.

[0062] Since the preparation methods of the dendritic tetrasulfide silicon carrier and the wasp venom peptide-M nanocomposite in Examples 1 and 2 are similar, this invention takes the dendritic tetrasulfide silicon carrier and the wasp venom peptide-M nanocomposite in Example 2 as an example to further test their chemical structure and crystal structure.

[0063] The Fourier transform infrared spectrum of the wasp venom peptide-M nanocomposite (labeled DMS@Mast-M in the figure) in Example 2 is shown below. Figure 7 As shown, it was compared with a physical mixture obtained by grinding and mixing dendritic tetrathiosilicon empty carrier (DMS), wasp venom peptide-M (Mast-M), and DMS and Mast-M powders at a mass ratio of 1:1. It can be found that chemical complexation occurred between Mast-M and DMS in DMS@Mast-M, which is significantly different from the infrared spectrum of ordinary physical mixtures.

[0064] The XRD pattern of the wasp venom peptide-M nanocomposite in Example 2 is shown below. Figure 8 As shown, no sharp characteristic peaks were observed in the XRD diffraction pattern, only at 2... θ The sample exhibits a broadened diffuse peak in the 20°~30° range, indicating that the drug sample is an amorphous substance.

[0065] (2) In vitro GSH response test Since the dendritic tetrathionine carrier and wasp venom peptide-M nanocomposite prepared in Examples 1 and 2 are similar in preparation methods and have similar chemical properties and crystal structures, this invention takes the sample of Example 2 as an example to conduct GSH response testing.

[0066] The DMS@Mast-M prepared in Example 2 was formulated into a dispersion with a concentration of 100 μg / mL. After incubating it with 10 mM GSH solution at 37°C, 1.5 mg / mL 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution was added for reaction. The absorbance was then measured using a UV-Vis spectrophotometer. The decrease in absorbance was used to quantify the residual GSH, which indirectly reflected the degree of drug release triggered by GSH, and was compared with that of pure DMS empty carrier.

[0067] like Figure 9 As shown, both DMS and DMS@Mast-M can consume GSH.

[0068] The 100 μg / mL DMS@Mast-M dispersion from Example 2 was reacted with 10 mM GSH solution for 0–30 min, and the reaction solution was dropped onto lead acetate test paper at different time points, with color changes recorded. Figure 10 As shown, the lead acetate test paper darkens in color over time, confirming that hydrogen sulfide gas was produced during the reaction.

[0069] (3) Test on the uptake capacity of tumor cells of wasp venom peptide-M nanocomposite Taking breast cancer cell line 4T1 as an example, breast cancer cell line 4T1 was seeded into 12-well plates and cultured in an incubator for 12 hours. Then, the medium was replaced with DMS@Mast-M-FITC and cultured in an incubator for 2 hours, 4 hours and 6 hours before DAPI staining. After staining, the uptake of the drug by the cells was observed using a laser confocal microscope.

[0070] The results are as follows Figure 11 As shown, with the extension of time, the uptake of DMS@Mast-M by breast cancer cells gradually increased, and it mainly accumulated in the cytoplasm, which indicates that DMS@Mast-M can be effectively taken up by breast cancer cells.

[0071] (4) Cytotoxicity of wasp venom peptide-M nanocomplex This invention investigates the in vitro inhibitory and cytotoxic effects of the wasp venom peptide-M nanocomposite on tumor cell proliferation, using breast cancer cell line 4T1 as an example. The steps are as follows: 4T1 breast cancer cell line was seeded into 96-well plates and cultured for 12 h. DMS@Mast-M drug group: The culture medium was changed to contain different concentrations of the composite nanoparticles (20, 40, 80, 160 μg / mL), and cultured for 24 h. Cell viability was then detected using CCK-8 assay.

[0072] like Figure 12As shown, DMS@Mast-M (Example 2) can effectively inhibit the growth and reproduction of tumor cells. The IC50 of cancer cells in the drug treatment group was significantly lower than that in the Mast-M treatment group. Compared with the DMS group alone, the DMS@Mast-M drug group has the effect of inhibiting the growth and reproduction of tumor cells.

[0073] like Figure 13 As shown, the hornet venom peptide-M complexes loaded with Mast-M in DMS of different particle sizes (Example 1 and Example 2) all showed good killing effects on 4T1 cells, but the hornet venom peptide-M complex in Example 1 may have benefited from its smaller particle size, and its IC50 was lower than that of the treatment group in Example 2.

[0074] (5) Tumor targeting ability test of wasp venom peptide-M nanocomplex The DMS@Mast-M samples obtained in Examples 1 and 2 were fluorescently labeled with ICG-NHS fluorescent dye. Then, the two complexes (ICG-NHS-DMS@Mast-M), an equal amount of ICG-NHS fluorescent dye, and an equal amount of Mast-M complex (ICG-NHS-Mast-M) labeled with the same ICG-NHS fluorescent dye were injected into breast cancer brain metastases mice via the tail vein. At six time points after administration, namely 3 h, 12 h, 24 h, 48 h, 72 h, and 96 h, the in vivo fluorescence signals of each group of mice were dynamically evaluated using a small animal imaging system.

[0075] The results are as follows Figure 14 As shown, both groups of ICG-NHS-labeled DMS@Mast-M-ICG exhibited good enrichment effects in brain tumor sites at different time points, and their tumor enrichment ability was significantly improved compared with the ICG-NHS group and the ICG-NHS-Mast-M group.

[0076] (7) Experiment on a model of breast cancer brain metastasis Six- to eight-week-old female BALB / c mice were used. Before the experiment, the mice were acclimatized under SPF conditions for one week. 4T1luc breast cancer cells were dispersed in PBS (Phosphate Buffered Saline) solution to prepare a cell suspension. After thorough mixing, 10,000 cells were sampled and placed 2 mm to the right posterolateral side of the coronal suture of the mice to construct a tumor model using a stereotaxic instrument.

[0077] The mice were randomly divided into 4 groups and treatment was initiated.

[0078] 1) 4T1 tumor-bearing mice were randomly divided into 4 groups and then intravenously injected with PBS, DMS, Mast-M, and DMS@Mast-M (Example 2). The drugs were administered intravenously once every two days, for a total of three administrations.

[0079] 3) Detect mouse weight and record mouse biofluorescence signals during drug administration.

[0080] like Figure 15 As shown, analysis of tumor fluorescence signals in mice revealed that DMS@Mast-M exhibited better tumor inhibition effects compared to PBS, Mast-M, and DMS.

[0081] like Figure 16 As shown, DMS@Mast-M can effectively improve the survival of mice with brain metastases from breast cancer.

[0082] (9) Biocompatibility test of DMS@Mast-M Mix PBS, H2O, and DMS@Mast-M at different concentrations (1, 10, 50, 100 ug / mL) with red blood cell suspension at a 1:1 ratio, incubate at 37℃ for 1~1.5 h, centrifuge at 3000 rpm for 5 min, and then observe the hemolysis.

[0083] The results are as follows Figure 17 As shown, DMS@Mast-M exhibits a low hemolysis rate, indicating good biocompatibility.

[0084] In summary, the wasp venom peptide-M nanocomposite of the present invention showed good brain enrichment effect in in vitro tests and breast cancer brain metastasis models, demonstrating good targeting enrichment, safety and significant anti-tumor efficacy. The wasp venom peptide-M nanocomposite of the present invention has an average particle size as small as 30 nm, and thanks to its small size and penetrability, it has the best effect.

[0085] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a wasp venom peptide-M nanocomposite, characterized in that, Includes the following steps: S1. Using indocyanine green as a morphology mediator, it was fully dissolved in water with triethylamine and hexadecyltrimethylammonium bromide to obtain a solution. Then, tetraethyl orthosilicate and bis[3-(triethoxysilyl)propyl]tetrasulfide were added to the solution, and the mixture was stirred and allowed to react fully to synthesize dendritic tetrasulfin with a particle size of less than 50 nm. After the reaction, the dendritic tetrasulfin was centrifuged, washed, and stored for later use. S2. The dendritic tetrathionine obtained in step S1 is redispersed in water to obtain a homogeneous suspension; hornet venom peptide-M solution is added to the homogeneous suspension, and after ultrasonic treatment or stirring, the hornet venom peptide-M nanocomposite is obtained.

2. The method for preparing the wasp venom peptide-M nanocomposite according to claim 1, characterized in that, In step S1, the mass ratio of triethylamine, water, hexadecyltrimethylammonium bromide, indocyanine green, tetraethyl orthosilicate, and bis[3-(triethoxysilyl)propyl]tetrasulfide is 1 : 564.3 : 8.58 : 1.13 : 42.09 : 42.

67.

3. The method for preparing the wasp venom peptide-M nanocomposite according to claim 1, characterized in that, In step S1, the stirring speed during the reaction is maintained at 1000 r / min to 1200 r / min.

4. The method for preparing the wasp venom peptide-M nanocomposite according to claim 2, characterized in that, The specific steps for preparing dendritic tetrasulfide in step S1 are as follows: S11. Disperse triethylamine in ultrapure water and stir at 600-800 rpm until homogeneous to obtain solution A; S12. Add hexadecyltrimethylammonium bromide to solution A and stir at 600-800 rpm until homogeneous to obtain solution B; S13. Add indocyanine green to solution B and stir until the solution is a uniform transparent green to obtain solution C; S14. Tetraethyl orthosilicate and bis[3-(triethoxysilyl)propyl]tetrasulfide were added to solution C and stirred at 80°C and 1000~1200 rpm to obtain crude dendritic tetrasulfide silicon. S15. Centrifuge the crude dendritic tetrasulfide, discard the supernatant, wash with ethanol and repeat the centrifugation several times to obtain dendritic tetrasulfide, and store it in an ethanol solution.

5. The method for preparing the wasp venom peptide-M nanocomposite according to claim 1, characterized in that, The concentration of dendritic tetrathionine dispersed in the homogeneous suspension in step S2 is 1 mg / mL.

6. The method for preparing the wasp venom peptide-M nanocomposite according to claim 1, characterized in that, The concentration of the wasp venom peptide-M solution in step S2 is 1 mg / mL; the mass ratio of wasp venom peptide-M in the added wasp venom peptide-M solution to the mass of dendritic tetrathionine dispersed in the homogeneous suspension is 1:

1.

7. A wasp venom peptide-M nanocomposite prepared by any one of the preparation methods according to claims 1 to 6.

8. The wasp venom peptide-M nanocomposite according to claim 7, characterized in that, The wasp venom peptide-M nanocomposite exhibits a dendritic morphology, with wasp venom peptide-M uniformly loaded on the surface of dendritic tetrasulfide silicon; the amino acid sequence of wasp venom peptide-M is INLKAIAALAKKLL-NH2.

9. The wasp venom peptide-M nanocomposite according to claim 7, characterized in that, The dendritic tetrasulfide has a particle size of 27.49 nm ± 3.0% or 38.17 nm ± 1.1%.

10. The use of the wasp venom peptide-M nanocomposite according to claim 7 in the preparation of a medicament for treating brain metastases of breast cancer.

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