Melittin nanocomplex targeting tumor and preparation method and application thereof

Abstract

This invention discloses a bee venom peptide nanocomposite targeting tumors, its preparation method, and its applications. The nanocomposite exhibits a core-shell structure, with a bee venom peptide nanoparticle as the core and a polydopamine shell, coated with hyaluronic acid. The average particle size is 211.5 nm, and the bee venom peptide encapsulation rate is 76.3%. This invention utilizes the salting-out effect to regulate the self-assembly of bee venom peptides into bee venom peptide nanoparticles. After polydopamine coating, further surface modification with hyaluronic acid results in composite nanomedicines (MPHA NPs) with active tumor targeting capabilities. It significantly reduces the hemolytic toxicity of bee venom peptides, with a hemolysis rate of only 1.54%, and enhances its accumulation in tumor tissues. Following CD44 receptor-mediated cellular uptake, the polydopamine coating degrades, releasing bee venom peptides that exert anti-tumor effects through mitochondrial-dependent apoptosis, achieving an in vivo tumor growth inhibition rate of 76.8%.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nanomaterials and biomedicine, specifically to a bee venom peptide nanocomposite targeting tumors, its preparation method and application, and particularly to a bee venom peptide nanodelivery system based on salting-out effect-induced self-assembly, polydopamine coating and hyaluronic acid surface modification, and its application in anti-tumor therapy. Background Technology

[0002] Clinical treatment of malignant tumors commonly suffers from problems such as non-specific drug distribution, poor efficacy, and high systemic toxicity. Tumor-targeted drug delivery technology aims to achieve targeted enrichment and controlled release of drugs at tumor lesions through specific structural design, thereby improving therapeutic efficacy and reducing toxicity to normal tissues. While nanodelivery systems have achieved clinical translation, they generally suffer from limitations such as limited drug loading compatibility and insufficient controllability of drug release.

[0003] Melipotassium peptides are the main active components of bee venom. They possess broad-spectrum and highly effective antitumor activity due to their unique membrane cleavage mechanism, making them promising antitumor peptide drug candidates. However, their membrane cleavage action lacks cell specificity, and after entering the circulation, they can non-specifically damage normal cells, easily causing severe hemolysis and systemic toxicity, which greatly restricts the clinical application of melipotassium peptides. Therefore, achieving targeted delivery, controlled release, and toxicity shielding of melipotassium peptides are currently core technical challenges that urgently need to be solved in this field.

[0004] Biocompatible polymers are core carriers for targeted delivery of peptide drugs. Among them, polydopamine possesses excellent biocompatibility, interfacial polymerization ability, and reactive oxygen species scavenging properties, while also exhibiting good near-infrared photothermal conversion characteristics, enabling synergistic therapeutic effects. Hyaluronic acid can specifically bind to CD44 receptors highly expressed on the surface of various tumor cells, providing active targeting capabilities for the delivery system. Both have been widely used in the field of tumor drug delivery and have a mature application foundation.

[0005] In summary, existing applications of melivexin in antitumor therapy suffer from severe systemic toxicity issues, and conventional drug delivery systems cannot achieve precise targeting, controllable assembly, and responsive drug release of melivexin. Summary of the Invention

[0006] Based on the above background, this invention develops a tumor-targeting melive venom peptide nanocomposite (Mel@PDA@HANPs, abbreviated as MPHA NPs). This nanocomposite integrates salting-out effect-induced melive venom peptide self-assembly technology, polydopamine encapsulation to shield hemolytic toxicity, and hyaluronic acid active targeting modification, achieving safe, targeted delivery, photothermal synergy, and controlled release of melive venom peptides. The synergistic effect of each component significantly reduces the hemolytic toxicity of melive venom peptides and improves tumor targeting and anti-tumor efficacy.

[0007] This invention aims to construct a novel drug delivery system that combines the potent antitumor activity of meliofemort peptides with the safety and targeting properties of nanodelivery systems. Through a simple and low-cost preparation process, this nanocomposite can be efficiently applied in antitumor drug delivery and tumor therapy.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned technical objectives is as follows: a bee venom peptide nanocomposite targeting tumors, wherein the nanocomposite has a core-shell structure, wherein the core is a bee venom peptide nanoparticle, the shell is a polydopamine coating layer, and the surface is modified with hyaluronic acid; the bee venom peptide nanoparticle is formed by the self-assembly of bee venom peptide under the salting-out effect; the polydopamine shell is coated on the surface of the bee venom peptide nanoparticle by the oxidative self-polymerization of dopamine under alkaline conditions; and the hyaluronic acid is connected to the surface of the polydopamine shell through amide bonds.

[0009] Preferably, the bee venom peptide nanoparticles are spherical and formed by self-assembly induced by dipotassium hydrogen phosphate; the particle size can be controlled by the salt concentration, and as the concentration of dipotassium hydrogen phosphate increases, Melz self-assembly is enhanced, and the average particle size continues to increase.

[0010] Preferably, the MPHA NPs nanocomposite has a particle size of 211.5 nm and is spherical.

[0011] Preferably, the encapsulation rate of meliosteum in the MPHA nanocomposite NPs is 76.3%, and the drug loading of meliosteum is 0.26 mg / mg.

[0012] The preparation method of the aforementioned tumor-targeting melivitin nanocomposite MPHA NPs comprises the following steps:

[0013] Step 1: Dissolve bee venom peptide in water, add dipotassium hydrogen phosphate (K2HPO4), and stir to allow the bee venom peptide to self-assemble into bee venom peptide nanoparticles under the induction of salting-out effect.

[0014] Step 2: Add dopamine hydrochloride to the solution obtained in Step 1, and carry out an oxidative self-polymerization reaction under alkaline conditions to coat the surface of the bee venom peptide nanoparticles with polydopamine. After centrifugation, the polydopamine-coated bee venom peptide nanoparticles Mel@PDA NPs, abbreviated as MP NPs, are obtained.

[0015] Step 3: After activating hyaluronic acid, react it with the polydopamine-coated bee venom peptide nanoparticles obtained in Step 2 in a buffer solution, so that hyaluronic acid is modified on the surface of the polydopamine shell through amide bonds. After purification, the bee venom peptide nanocomplex Mel@PDA@HA NPs, abbreviated as MPHA NPs, is obtained.

[0016] Preferably, in step 1, 5 mg of melitin is dissolved in 30 mL of deionized water and stirred until completely dissolved. 20 mg of K₂HPO₄ and 18 mg of Tris are added, and the mixture is stirred for 5 minutes to allow the melitin to self-assemble into melitin nanoparticles under the induction of the salting-out effect.

[0017] Preferably, 40 mg of dopamine hydrochloride is added to the solution obtained in step (1), and the mixture is stirred at room temperature for 8 hours. After the reaction is completed, the resulting suspension is centrifuged at 11,000 rpm for 5 minutes, and the precipitate is washed twice with deionized water to obtain polydopamine-coated bee venom peptide nanoparticles (MP NPs).

[0018] Preferably, the activation method of hyaluronic acid in step (3) is to dissolve 30 mg EDCI in 50 mL of pure water, add 85 mg hyaluronic acid, activate for 0.5 hours, then add 35 mg NHS, and continue stirring for 12 hours to obtain activated hyaluronic acid.

[0019] Preferably, the modification method of hyaluronic acid in step (3) is to mix 1 mL of activated hyaluronic acid solution with 10 mg of MP NPs in 5 mL of PBS buffer and react for 8 hours. The resulting product is washed three times with deionized water and collected by centrifugation at 11000 rpm for 5 min.

[0020] Application of the tumor-targeting meliotide nanocomposite in the preparation of drug delivery and tumor treatment drugs.

[0021] Preferably, by using polydopamine to coat melitoxin nanoparticles, the hemolytic toxicity of melitoxin is effectively shielded. The resulting nanocomposite has a hemolysis rate of only 1.54%, which significantly reduces the systemic toxicity of melitoxin. This achieves safe, targeted delivery, photothermal synergy, and controllable release of melitoxin, thereby improving tumor targeting and anti-tumor efficacy.

[0022] This invention utilizes the salting-out effect to regulate the self-assembly of meliosteum peptides. The salting-out effect in this invention is not the traditional method of precipitating and discarding the protein, but rather utilizes salt ions to regulate the hydration layer and surface charge of meliosteum peptide Mel, controllably and reversibly inducing Mel to self-assemble into nanoparticles. Mel is an amphiphilic α-helical polypeptide containing multiple basic lysine and arginine residues, carrying a strong positive charge in neutral aqueous solutions. It is originally dispersed in water by its hydration layer and intermolecular electrostatic repulsion. Adding a certain concentration of salt causes competition for water molecules, disrupting the hydration layer. Salt ions dissociate in water and tightly bind to water molecules, reducing free water and destroying the hydration shell on the Mel surface, exposing hydrophobic domains. The high ionic strength shields the positive charge on the Mel surface, weakening intermolecular electrostatic repulsion. When hydration and electrostatic repulsion are weakened, hydrophobic interactions dominate, causing Mel molecules to approach and aggregate, forming nanoscale self-assembled particles, i.e., Mel NPs. Under the induction of the salting-out effect, meliosteum peptides self-assemble into nanoparticles. Subsequently, dopamine undergoes oxidative self-polymerization on the surface of the meliothrix nanoparticles under weakly alkaline conditions (around pH 8.5), forming a polydopamine shell. This polydopamine shell shields the meliothrix from hemolytic toxicity and endows the nanocomposite with photothermal conversion properties and reactive oxygen species scavenging capabilities. Hyaluronic acid is modified onto the polydopamine shell surface via amide bonds, giving the nanocomposite the ability to actively target tumor cells with high CD44 receptor expression. When the nanocomposite enters tumor cells via CD44 receptor-mediated endocytosis, the high concentration of hydrogen peroxide in the tumor microenvironment triggers the degradation of the polydopamine shell, releasing the active meliothrix monomer. The released meliothrix activates caspase-9 and caspase-3 sequentially by upregulating the Bax / Bcl-2 ratio, increasing mitochondrial outer membrane permeability, and releasing cytochrome C, thus initiating the mitochondrial-dependent apoptosis pathway. In addition, under near-infrared laser irradiation, polydopamine produces a photothermal effect, which directly kills tumor cells on the one hand, and accelerates the degradation of polydopamine to promote the release of melilotinib, thus achieving chemotherapy-photothermal synergistic therapy.

[0023] Beneficial effects:

[0024] 1. This invention is the first to apply the salting-out effect to the controllable self-assembly of melitoxic peptides, inducing the formation of uniformly sized nanoparticles from melitoxic peptides using dipotassium hydrogen phosphate. This method is simple to operate, operates under mild conditions, and does not damage the bioactivity of melitoxic peptides, overcoming the technical bottlenecks of traditional melitoxic peptide formulations. The innovation of the MPHA NPs system lies in system integration, rather than a breakthrough in a single technology. It is the first to organically integrate several technical modules, including salting-out self-assembly, PDA encapsulation, HA targeting, H2O2-responsive release, and photothermal synergy, constructing a multifunctional, programmable, intelligent delivery platform for melitoxic peptides. This solves the three core challenges in the clinical application of melitoxic peptides: hemolytic toxicity, poor stability, and lack of targeting.

[0025] 2. Existing melivitin antitumor preparations mostly employ chemical synthesis and covalent cross-linking methods, which suffer from drawbacks such as irreversible assembly, uneven particle size, high biotoxicity, and lack of targeting, easily causing damage to normal tissues and low drug utilization. This technology abandons traditional preparation processes, utilizing the salting-out effect to regulate the hydrophobic and electrostatic interactions between melivitin molecules, precisely driving the autonomous assembly of melivitin into uniform and controllable micro / nano particles. This assembly method possesses excellent reversibility, enabling controlled drug release based on changes in the in vivo microenvironment, effectively solving the problems of poor in vivo stability, premature degradation, and severe drug efficacy loss of melivitin, and significantly improving the in vivo circulation stability of the drug.

[0026] 3. The polydopamine and hyaluronic acid in the nanocomposite prepared by this invention have been shown to have good biocompatibility and can be safely used in in vivo experiments.

[0027] 4. This invention utilizes polydopamine to coat melitoxic peptide nanoparticles, effectively shielding the hemolytic toxicity of melitoxic peptides. Experimental results show that the hemolysis rate of the obtained nanocomposite is only 1.54%, significantly reducing the systemic toxicity of melitoxic peptides and making safe systemic administration possible.

[0028] 5. This invention achieves active tumor-targeted delivery of nanocomposites by modifying the surface of a polydopamine shell with hyaluronic acid and utilizing the specific binding of hyaluronic acid to the CD44 receptor highly expressed on the surface of tumor cells.

[0029] 6. The complex prepared by this invention exhibits good stability and photothermal conversion performance. The complex's excellent photothermal conversion efficiency (34.3%) allows it to generate localized high temperatures under near-infrared laser irradiation, directly killing tumor cells on one hand and accelerating the degradation of polydopamine to promote the release of melilothin on the other, thus achieving synergistic chemotherapy-photothermal therapy. The preparation method of this invention is simple, easy to implement, and low in cost, making it suitable for large-scale production and utilization. Figure 14 The A-14C results showed that the tumor growth inhibition rate of the MPHA NPs+808 nm group reached 76.8%, which was significantly better than that of other groups. Attached Figure Description

[0030] The invention will now be further described with reference to the accompanying drawings.

[0031] Figure 1 This is a schematic diagram illustrating the preparation process of the MPHA NPs, a bee venom peptide nanocomposite targeting tumors according to the present invention.

[0032] Figure 2 Tyndall effect image of the K2HPO4-mediated self-assembly process of melitoxin.

[0033] Figure 3The graph shows the particle size change curves of three systems: (A) melitoxin, (B) bacitracin, and (C) oyster peptide during the dilution process.

[0034] Figure 4 The particle size distribution of bacitracin nanoparticles (A, K2HPO4; B, Na2SO4; C, CH3COONa) and oyster peptide nanoparticles (D, K2HPO4; E, Na2HPO4; F, K2CO3) in different salt solutions is shown in the figure.

[0035] Figure 5 The particle size distribution of melittin in different salt solutions (C, K2HPO4; D, (NH4)2SO4; E, Na2HPO4; F, K2CO3) was investigated.

[0036] Figure 6 The pH curves as a function of salt concentration are shown for different salt solutions (A, (NH4)2SO4; B, Na2SO4; C, CH3COONa; D, Na2CO3; E, K2CO3; F, Na2HPO4; G, K2HPO4).

[0037] Figure 7 Transmission electron micrographs of (A) Mel NPs and (B) MPHA NPs, and DLS particle size distribution of (C) MPHA NPs. (D) Particle size stability of MPHA NPs in three different solutions: ultrapure water, PBS, and DMEM.

[0038] Figure 8 This is a graph showing the encapsulation efficiency of melioflavones at different reaction times.

[0039] Figure 9 The graph shows (A) a comparison of the hemolysis rates of different drugs and (B) the corresponding absorbance curves.

[0040] Figure 10 To study the photothermal conversion performance of MPHA NPs, (A) the thermal rise of MPHA NPs at different concentrations; (B) the fitting of the natural cooling curve and the logarithmic cooling linear curve of the MPHA NPs aqueous solution after the light source is removed; (C) the thermal cycle experiment of five heating-cooling-cooling cycles.

[0041] Figure 11 The results show the in vitro antitumor activity and apoptosis detection of MPHA NPs.

[0042] Figure 12 (A) In vivo targeted fluorescence imaging of MPHA NPs; (B) Fluorescence distribution in ex vivo organs.

[0043] Figure 13(D) Photothermal images of different drug treatment groups; (E) Corresponding temperature change curves.

[0044] Figure 14 To evaluate the in vivo antitumor efficacy of MPHA NPs, a subcutaneous tumor-bearing mouse model of breast cancer was used to verify the therapeutic effect of MPHA NPs prepared in the specific implementation method on tumors: (A) The results of the in vivo antitumor efficacy evaluation of MPHA NPs in mice during treatment.

[0045] (A) Tumor volume change curve; (B) Differential analysis of tumor volume; (C) Representative images of tumors in mice after treatment.

[0046] Figure 15 Images of H&E staining and TUNEL apoptosis staining of tumor tissues from each treatment group. Detailed Implementation

[0047] To more clearly illustrate the present invention, the following detailed description is provided in conjunction with embodiments. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, all reagents and equipment are commercially available. The reagents and pharmaceuticals used in the preparation of nanoparticles in this invention are as follows: dopamine hydrochloride and dipotassium hydrogen phosphate were purchased from Aladdin; bee venom peptide (CAS: 20449-79-0) and hyaluronic acid were purchased from Maclean's; Tris was purchased from Sigma.

[0048] Mel is short for free bee venom peptide;

[0049] PHA NPs are PDA@HA nanoparticles, which are polydopamine-hyaluronic acid nanocarriers that do not contain melitoxin (Mel).

[0050] PHA NPs+808 nm refers to PDA@HA nanoparticles illuminated with 808 nm light.

[0051] MP NPs are Mel@PDA core-shell nanoparticles without modified hyaluronic acid (HA);

[0052] MP NPs+808 nm refers to Mel@PDA core-shell nanoparticles illuminated with 808 nm light.

[0053] MPHA consists of Mel@PDA@HA nanoparticles, which are Mel self-assembled cores + PDA shells + (HA) surface modifications.

[0054] MPHA+808 nm refers to Mel@PDA@HA nanoparticles illuminated with 808 nm light.

[0055] Example 1

[0056] This example illustrates the induction effect of different inorganic salts on the self-assembly of melitoxin and the universality of the salting-out effect.

[0057] Step 1: Self-assembly of melivitin and salt selection: Dissolve 5 mg of melivitin in 30 mL of deionized water and stir until completely dissolved. Add 20 mg of K₂HPO₄ (mass concentration: 0.67 mg / mL, molar concentration: 3.85 mmol / L) and stir for 5 minutes. Observe the Tyndall effect and determine the particle size distribution using a dynamic light scattering instrument.

[0058] Figure 2 The results showed that neither pure meliosteum peptide solution nor pure dipotassium hydrogen phosphate solution exhibited the Tyndall effect, while the meliosteum peptide solution with added K₂HPO₄ showed a significant Tyndall effect, indicating the formation of nanoscale aggregates. With increasing amounts of added K₂HPO₄, the average particle size of the meliosteum peptide nanoparticles gradually increased. Figure 5 (C) in the middle. From Figures 3-6 It is known that the higher the amount of K2HPO4 added, the stronger the self-assembly and the larger the particle size. However, excessively large particle size is not conducive to the subsequent in vivo circulation of nanomedicines. It is generally believed that 10–200 nm is a more ideal particle size range for tumor nanomedicines because this range can take into account long circulation, RES escape, and receptor-mediated endocytosis. Particles smaller than 10 nm are easily cleared by the kidneys, while those larger are easily retained by the liver and spleen. In this invention, the average hydrated particle size of MPHA NPs in DLS is 211.5 nm, slightly higher than this traditional upper limit. However, this value includes the hydrated radius of the PDA and HA hydrophilic layers, and the TEM core particle size may be even smaller. More importantly, HA modification endows it with CD44 active targeting capability, the PDA shell can reduce non-specific protein adsorption and RES recognition, and the photothermal effect can also enhance tumor vascular permeability and cellular uptake. These factors together compensate for the shortcomings of the slightly larger particle size, ultimately achieving an in vivo tumor inhibition rate of 76.8%, indicating that reasonable surface functionalization and active targeting can effectively expand the classical particle size window.

[0059] A K₂HPO₄ concentration of approximately 3-4 mM is required to stabilize the solution pH at 8.5, which is the optimal pH for dopamine (DA) oxidative self-polymerization. Testing at this K₂HPO₄ concentration revealed that the final average particle size of the MPHA NPs complex formed was 211.5 nm. In addition to K₂HPO₄, ammonium sulfate, sodium acetate, and potassium carbonate were also tested. All showed salting-out effects, but ammonium sulfate (acidic), sodium acetate (near-neutral), and potassium carbonate (too alkaline) failed to initiate dopamine oxidative polymerization. K₂HPO₄ combined the strongest salting-out induction ability with the most suitable pH environment for subsequent PDA coating. The optimal induction effect of K₂HPO₄ is attributed to its HPO₄²⁻ content. -Anions possess high charge density and strong hydration capacity, effectively disrupting the hydration layer of meliosteum and exposing its hydrophobic domains, thereby inducing molecular aggregation. Microscopic observation reveals that the meliosteum nanoparticles exhibit a spherical morphology.

[0060] The second step, verification of the reversibility of salting out, involved diluting the solution of meliofemol nanoparticles formed under high-salt conditions with distilled water and monitoring particle size changes using dynamic light scattering. The results showed that as the peptide concentration decreased, the meliofemol nanoparticles gradually dissociated into monomers, and the particle size continuously decreased. Figure 3 (A) indicates that the salting-out induced self-assembly process is reversible, which is crucial for the restoration of the bioactivity of melittin.

[0061] Step 3: Verification of the universality of the salting-out effect in peptide self-assembly: To verify the universality of the salting-out effect in inducing peptide self-assembly, the same salting-out scheme was applied to bacitracin and oyster peptide, respectively. The results showed that K₂HPO₄ could also effectively induce the self-assembly of bacitracin, and sodium sulfate and sodium acetate also showed some inducing ability. Figure 4 China A- Figure 4 (C); For oyster peptides, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and potassium carbonate can all effectively induce their self-assembly into nanoparticles (C). Figure 4 D- Figure 4 (F). As the salt concentration decreases, both bacitracin and oyster peptide nanoparticles dissociate (F). Figure 3 B, Figure 3 (C). The above results confirm that the salting-out induced self-assembly strategy has good peptide versatility.

[0062] Step 4: pH determination of different salt solutions: The pH changes of aqueous solutions of various inorganic salts, including (NH4)2SO4, Na2SO4, CH3COONa, Na2CO3, K2CO3, Na2HPO4, and K2HPO4, at different concentrations were investigated. Figure 6 A-6G). The results showed that aqueous solutions of (NH4)2SO4 and Na2SO4 were acidic (pH<7), CH3COONa solution was nearly neutral (pH≈7), while solutions of K2HPO4, Na2CO3, K2CO3 and Na2HPO4 were alkaline (pH>7).

[0063] In solutions containing alkaline substances such as K2HPO4, Na2CO3, K2CO3, and Na2HPO4, the alkalinity of Na2CO3 and K2CO3 is too strong, which may destroy the melitin or cause uneven coating. Figure 6At very low concentrations (0–4 mM), Na₂HPO₄ causes the pH to rise from 6.5 to approximately 8.5, exhibiting a narrow pH plateau. K₂HPO₄, on the other hand, maintains a stable pH of approximately 8.5 over a wide concentration range (0–300 mM), with an even wider plateau. If a higher salt concentration is required to achieve sufficient salting-out, Na₂HPO₄ may cause the pH to rise further, exceeding the optimal polymerization window for PDA and potentially even disrupting the Mel conformation. Therefore, since the pH of K₂HPO₄ solution increases gently with concentration and stabilizes at approximately 8.5, precisely corresponding to the optimal pH conditions for dopamine oxidative self-polymerization, dipotassium hydrogen phosphate was determined to be the optimal salting-out agent.

[0064] Example 2

[0065] Preparation of tumor-targeting melione peptide nanocomposites (MPHA NPs)

[0066] The first step involved the preparation of melive venom peptide nanoparticles (Mel NPs) and the pretreatment for the synthesis of polydopamine-coated melive venom peptide nanoparticles: 5 mg of melive venom peptide was dissolved in 30 mL of deionized water and stirred until completely dissolved. 20 mg of dipotassium hydrogen phosphate and 18 mg of Tris base were added, and the mixture was stirred for 5 minutes to allow the melive venom peptide to self-assemble into melive venom peptide nanoparticles under the induction of the salting-out effect.

[0067] The second step involved the synthesis of polydopamine-coated bee venom peptide nanoparticles (MP NPs): 40 mg of dopamine hydrochloride was added to the solution obtained in the first step, and the mixture was stirred at room temperature for 8 hours. After the reaction was completed, the resulting suspension was centrifuged at 11,000 rpm for 5 minutes, and the precipitate was washed twice with deionized water to obtain polydopamine-coated bee venom peptide nanoparticles (MP NPs).

[0068] The third step is the activation of hyaluronic acid: Dissolve 30 mg of EDCI in 50 mL of pure water, add 85 mg of hyaluronic acid, activate for 0.5 hours, then add 35 mg of NHS, and continue stirring for 12 hours to obtain activated hyaluronic acid.

[0069] Step 4: Hyaluronic acid surface modification (synthesis of MPHA NPs): 1 mL of activated hyaluronic acid solution was mixed with 10 mg of MP NPs in 5 mL of PBS buffer (pH 7.4) and incubated for 8 hours. The reaction product was washed three times with deionized water and collected by centrifugation at 11,000 rpm for 5 minutes to obtain the tumor-targeting melilotide nanocomposite (Mel@PDA@HA NPs, abbreviated as MPHA NPs).

[0070] Example 3

[0071] Physicochemical characterization of MPHA NPs: The morphology of Mel NPs and MPHA NPs was observed using transmission electron microscopy, and the particle size distribution of MPHA NPs was determined using dynamic light scattering. The results showed that Mel NPs and MPHA NPs exhibited a spherical morphology. Figure 7 The average particle size of MPHA NPs (B) is 211.5 nm. Figure 7 (C). The zeta potential is -32.2 mV.

[0072] Example 4

[0073] Encapsulation efficiency and drug loading: The encapsulation efficiency of melittin was determined by the BCA protein quantification method. The concentration of residual melittin in the supernatant was measured at 2, 4, 6, 8 and 10 hours of reaction. Figure 8 The results showed that the encapsulation rate gradually increased with the extension of reaction time and tended to stabilize after 8 hours. The encapsulation rate of melittin was 76.3% and the drug loading was 0.26 mg / mg.

[0074] Example 5

[0075] Add 100 μL of 2% erythrocyte suspension to each well of an 8-tube tubing, resulting in a final volume of 150 μL per well. Add PBS (negative control), Triton X-100 (positive control), free meliofemort peptide (20.8 μg), hyaluronic acid (8.0 μg), PDA nanoparticles (51.2 μg), and MPHA NPs (80.0 μg), respectively, and incubate at 37°C for different times (0, 10, 20, 30, 60, 90, 120 minutes). After incubation, centrifuge, observe the color of the supernatant, and measure the absorbance at 540 nm.

[0076] Figure 9 The results showed that the free melitoxin group exhibited significant hemolysis, while the hemolysis rate of the MPHA NPs group was only 1.54%, which was not significantly different from the negative control group, indicating that the polydopamine shell can effectively shield the hemolytic toxicity of melitoxin.

[0077] Example 6

[0078] Photothermal performance evaluation of MPHA NPs: An aqueous solution of MPHA NPs with a concentration of 200 μg / mL was prepared and irradiated with an 808 nm laser (1.0 W / cm²) for 6 minutes, with temperature changes and thermal images recorded every 20 seconds. The effects of different concentrations (0, 25, 50, 100, 150, 200 μg / mL) on photothermal performance were investigated. The photothermal conversion efficiency was calculated using cooling curves, and the photothermal stability was evaluated through a five-cycle irradiation experiment.

[0079] Figure 10The results from the study show that the photothermal effect of MPHA NPs is both concentration-dependent and laser power density-dependent. Figure 10 The photothermal conversion efficiency of medium B is 34.3%, and Figure 10 The C-value shows good repeatability of temperature rise during five cycles of irradiation, indicating that MPHA NPs have excellent photothermal stability and photothermal conversion performance.

[0080] Example 7

[0081] Evaluation of the in vitro antitumor activity of MPHA NPs:

[0082] 4T1 mouse breast cancer cells were seeded into 96-well plates (5 × 10³ cells / well) and cultured for 24 hours. Then, cells were treated with different concentrations (5, 10, 15, 20, 25, 30 μg / mL) of MPHA NPs and different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 μg / mL) of Mel or Mel pre-heated at 60 °C for 30 minutes. Cell viability was measured after 24 hours of incubation in the dark for the dark toxicity group; for the phototoxicity group, cells were incubated for 4 hours and then irradiated with an 808 nm laser (1.0 W / cm²) for 5 minutes, followed by 24 hours of further culture before measuring cell viability. Cells were stained using the Calcein-AM / PI live / dead cell staining kit and observed under a fluorescence microscope. Apoptosis was detected by flow cytometry using the Fluor Violet 450 / 7-AAD double staining method.

[0083] Figure 11 China A- Figure 11 The results from the B-cell assay showed that the difference in cell viability between the Mel group and the preheated Mel group determined whether high-temperature treatment affected the antitumor activity of melittin. The results indicated that the activity of melittin was not significantly reduced after heating to 60 °C, demonstrating its good thermal stability. This provides experimental evidence that melittin can maintain its activity even after PDA shell degradation during subsequent photothermal therapy. The cell viability of the MPHA NPs combined with laser irradiation group decreased to 28.5%, significantly lower than that of the MPHA NPs alone group, indicating that the MPHA NPs + 808 nm group exhibited the strongest tumor cell killing effect. Live / dead staining and flow cytometry results were consistent with this finding.

[0084] Example 8

[0085] In vivo targeting evaluation of MPHA NPs: 4T1 cells (3.5 × 10⁻⁶) were used. 5 (Number) of xenografts were subcutaneously injected into both sides of the back of BALB / c mice to establish a subcutaneous xenograft model. When the tumor volume reached 60–80 mm... 3Tumor-bearing mice were randomly divided into three groups: a control group, a Cy5-labeled MP NPs group (5.45 mg / kg), and a Cy5-labeled MPHA NPs group (6.06 mg / kg). The drugs were administered via tail vein injection, and fluorescence imaging was performed 4 hours later using a small animal in vivo imaging system. The mice were then sacrificed, and major organs and tumors were dissected for ex vivo fluorescence imaging.

[0086] Figure 12 The results showed that the fluorescence signal intensity at the tumor site in the MPHA NPs group was significantly stronger than that in the MP NPs group, indicating that hyaluronic acid modification enables MPHA NPs to have active tumor targeting ability. Figure 12 In vitro organ imaging further confirmed that the accumulation of MPHA NPs in tumor tissue was significantly higher than that in the MP NPs group.

[0087] Example 9

[0088] In vivo photothermal imaging evaluation of MPHA NPs: Tumor-bearing mice were randomly divided into three groups (n = 3): control group (intravenous injection of phosphate buffer), MP NPs-Cy5 group (intravenous administration of 5.45 mg / kg) - ¹) and the MPHA NPs-Cy5 group (intravenous dose of 6.06 mg kg) - ¹). All formulations were administered via tail vein injection. Four hours post-injection, a power density of 0.5 W / cm² was used. - ² Laser irradiation was applied to the tumor sites of mice. The real-time temperature rise of the tumor sites was continuously recorded using an infrared thermal imager until the local temperature reached a stable plateau.

[0089] Figure 13 D- Figure 13 The results from the study showed that the average tumor temperature in the MPHA NPs group reached 39.4 °C, while that in the MPNPs group it was only 34.3 °C, indicating that MPHA NPs have better active tumor targeting and enrichment capabilities, thus producing a stronger photothermal effect.

[0090] Example 10

[0091] Evaluation of the in vivo antitumor efficacy of MPHA NPs: When the tumor volume reaches 60-80 mm... 34T1 breast cancer-bearing mice were randomly divided into five groups for in vivo treatment experiments: control group (I), Mel group (II) (0.4 mg / kg), MPHANPs monotherapy group (III) (1.52 mg / kg), PHA NPs + 808 nm laser group (IV) (1.12 mg / kg), and MPHA NPs + 808 nm laser group (V) (1.52 mg / kg). Drugs were administered via tail vein injection on days 0, 2, 4, 6, 8, 10, and 12. Four hours after drug administration, the MPHA NPs + 808 nm group and the PHA NPs + 808 nm group received 3-minute irradiation of the tumor site with an 808 nm laser (0.5 W / cm²). Tumor volume was measured every two days. After treatment, the mice were sacrificed, the tumors and major organs were dissected, photographed, and subjected to H&E staining and TUNEL apoptosis staining.

[0092] Figure 14 A- Figure 14 The results showed that the tumor growth inhibition rate of the MPHA NPs+808 nm group reached 76.8%, which was significantly better than that of other groups.

[0093] Figure 15 The tumor tissue in this group showed extensive tumor cell necrosis and lysis, disordered cell arrangement, and significant interstitial edema. TUNEL staining revealed a significant increase in the number of apoptotic cells in this group. No significant abnormal fluctuations in body weight were observed in any of the treatment groups, and no abnormal behaviors such as hair loss or diarrhea were observed.

[0094] The present invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. A bee venom peptide nanocomposite targeting tumors, characterized in that... The melitoxic peptide nanoparticles are formed by the self-assembly of melitoxic peptides under the salting-out effect; MP NPs are obtained by coating the surface of the melitoxic peptide nanoparticles with a polydopamine shell; finally, hyaluronic acid is modified on the surface of the polydopamine shell to obtain the melitoxic peptide nanocomposite MPHA NPs; the salting-out effect is achieved by dissolving melitoxic peptides in water, adding dipotassium hydrogen phosphate and tris(hydroxymethyl)aminomethane (Tris), and stirring to induce the self-assembly of melitoxic peptides under the salting-out effect to form melitoxic peptide nanoparticles.

2. The bee venom peptide nanocomposite targeting tumors according to claim 1, characterized in that, The salting-out effect utilizes the regulation of the hydration layer and surface charge of melitoxin Mel by salt ions to controllably and reversibly induce Mel to self-assemble into nanoparticles. The melitoxin molecules approach and aggregate to form nanoscale self-assembled particles, namely Mel NPs.

3. The bee venom peptide nanocomposite targeting tumors according to claim 1, characterized in that, The K2HPO4 concentration is 0.67 mg / mL, the average particle size of the MPHA NPs nanocomposite is 211.5 nm, the Zeta potential is -32.2 mV, the encapsulation efficiency of meliotide in the MPHA NPs nanocomposite is 76.3%, and the drug loading of meliotide is 0.26 mg / mg.

4. The method for preparing the tumor-targeting bee venom peptide nanocomposite according to claim 1, characterized in that, Includes the following steps: (1) Dissolve bee venom peptide in water, add dipotassium hydrogen phosphate and Tris(hydroxymethylaminomethane), stir to allow bee venom peptide to self assemble into bee venom peptide nanoparticles under the induction of salting-out effect. (2) Add dopamine hydrochloride to the solution obtained in step (1) and carry out an oxidative self-polymerization reaction under alkaline conditions to coat the surface of the bee venom peptide nanoparticles with polydopamine. The polydopamine-coated bee venom peptide nanoparticles are obtained by centrifugation. (3) After activating hyaluronic acid, it is reacted with the polydopamine-coated bee venom peptide nanoparticles obtained in step (2) in a buffer solution, so that hyaluronic acid is modified on the surface of the polydopamine shell through amide bonds, and the bee venom peptide nanocomplex targeting tumors is obtained after purification.

5. The preparation method according to claim 4, characterized in that, Dissolve 5 mg of melitin in 30 mL of deionized water and stir until completely dissolved. Add 20 mg of dipotassium hydrogen phosphate (K2HPO4) and 18 mg of tris(hydroxymethyl)aminomethane (Tris) and stir for 5 minutes to allow the melitin to self-assemble into melitin nanoparticles under the salting-out effect.

6. The preparation method according to claim 5, characterized in that, Add 40 mg of dopamine hydrochloride to the solution obtained in step (1) and stir at room temperature for 8 hours. After the reaction is completed, centrifuge the obtained suspension at 11,000 rpm for 5 minutes and wash the precipitate twice with deionized water to obtain polydopamine-coated bee venom peptide nanoparticles MP NPs.

7. The preparation method according to claim 6, characterized in that, The activation method of hyaluronic acid in step (3) is as follows: 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI is dissolved in 50 mL of pure water, 85 mg of hyaluronic acid is added, and the mixture is activated for 0.5 hours. Then, 35 mg of N-hydroxysuccinimide NHS is added, and the mixture is stirred continuously for 12 hours to obtain activated hyaluronic acid.

8. The preparation method according to claim 7, characterized in that, The modification method of hyaluronic acid in step (3) is as follows: 1 mL of activated hyaluronic acid solution is mixed with 10 mg MP NPs in 5 mL PBS buffer and reacted for 8 hours. The resulting product is washed 3 times with deionized water and collected by centrifugation at 11000 rpm for 5 min.

9. The application of the tumor-targeting bee venom peptide nanocomposite according to claim 1 in the preparation of drug delivery and tumor treatment drugs.

10. The application of the tumor-targeting bee venom peptide nanocomposite according to claim 1 in the preparation of drug delivery and tumor therapeutic drugs, characterized in that, By using polydopamine to coat melitoxin nanoparticles, the hemolytic toxicity of melitoxin was effectively shielded. The resulting nanocomposite had a hemolysis rate of only 1.54%, which significantly reduced the systemic toxicity of melitoxin. This achieved safe, targeted delivery, photothermal synergy, and controlled release of melitoxin, thereby improving tumor targeting and anti-tumor efficacy.

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