Cell-penetrating peptide-polysaccharide-small molecule antitumor drug polyion complex and preparation method and application thereof

Abstract

The application relates to a cell-penetrating peptide-polysaccharide-small-molecule antitumor drug polyion complex, a preparation method thereof and application, and belongs to the technical field of medicines. The cell-penetrating peptide-polysaccharide-small-molecule antitumor drug polyion complex comprises a cell-penetrating peptide, a polysaccharide and a small-molecule antitumor drug, and the mass ratio of the cell-penetrating peptide, the polysaccharide and the small-molecule antitumor drug is (1-3):(8-10):(1-3). The preparation method is as follows: the cell-penetrating peptide is dissolved in sterilized water for injection to obtain solution A; the polysaccharide is dissolved in sterilized water for injection to obtain solution B; the small-molecule antitumor drug is dissolved in sterilized water for injection to obtain solution C; solution A is mixed with solution B, and then solution C is mixed to obtain the cell-penetrating peptide-polysaccharide-small-molecule antitumor drug polyion complex. The drug efficacy of the cell-penetrating peptide-polysaccharide-small-molecule antitumor drug polyion complex is obviously better than that of a single-drug polyion complex. Not only can the cell-penetrating peptide-polysaccharide-small-molecule antitumor drug polyion complex improve the antitumor effect of the drug through synergistic action, but also can reduce the hemolytic toxicity of the cell-penetrating peptide.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a cell penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex and a preparation method and application thereof. Background Art

[0002] With a deeper understanding of tumor biology and the tumor microenvironment (TME), cancer treatments are becoming more diverse, with approaches such as surgery, radiotherapy, chemotherapy, and anti-tumor drug therapy. Despite recent biomedical advances in cancer treatment, the efficacy of chemotherapy, for example, is limited by severe side effects and multidrug resistance. A single treatment strategy is no longer sufficient to keep pace with disease progression and meet clinical needs. In addition to considering traditional cytotoxic chemotherapy drugs, the selection of anti-tumor drugs is shifting towards new, highly targeted drugs and immunotherapy. Different combination approaches are being developed by considering the different stages of the tumor, tumor burden, different metastatic patterns, and the biological characteristics of different populations.

[0003] In recent years, by designing new anti-tumor therapies that simultaneously deliver two or more therapeutic drugs in a single drug delivery system, synergistic effects of drugs with different therapeutic mechanisms can be achieved. Song et al. (MINGMING SONG. Self-assembled polymeric nanocarrier-mediated co-delivery of metformin and doxorubicin for melanoma therapy[J]. Drug Delivery, 2021. 28(1): 594-606.) prepared self-assembled polymeric nanocarriers and co-delivered metformin and doxorubicin (DOX) to treat melanoma, successfully inducing tumor cell apoptosis and inhibiting the progression of melanoma. Penelope et al. (OTTEWELL PD, LEFLEY DV, CROSS SS, et al. Sustained inhibition of tumor growth and prolonged survival following sequential administration of doxorubicin and zoledronic acid in a breast cancer model [J]. International Journal of Cancer, 2010. 126 (2): 522-532.) Continuous administration of DOX and zoledronic acid in a breast cancer model can continuously inhibit tumor growth and prolong survival. Therefore, the use of two or more chemotherapy drugs in combination chemotherapy kills cancer cells through different sites of action or mechanisms, which can further prolong the life of cancer patients while improving the quality of life. The research on this drug co-delivery system has greatly promoted the development of cancer combination therapy. The clever and reasonable design of the drug co-delivery system can co-deliver two drugs with large differences in physicochemical properties (such as hydrophilicity and molecular weight) to tumor tissue or cells, release the drugs simultaneously or sequentially, and exert a synergistic anti-tumor effect, which has great advantages in the application of cancer combination therapy. Compared with single formulations, combined drug co-delivery systems increase the complexity of carriers and the challenges of carrier application, such as the selection of carriers and therapeutic substances, drug loading, release behavior, physicochemical properties and biocompatibility, etc., which have a significant impact on the success of co-delivery.

[0004] Recent studies have shown that the ability of nanoparticles modified with tumor-specific ligands to accumulate in tumors depends on tumor-associated immune cells, and the extent of immune cell infiltration in the tumor region affects the targeting efficiency of nanoparticles after systemic administration. Actively targeted drug delivery systems designed for the tumor immune microenvironment can effectively deliver drugs to tumors, achieving precise and efficient killing. During tumor progression, monocytes are recruited from the blood circulation and infiltrate into the inflamed area of ​​the tumor, differentiating into tumor-associated macrophages (TAMs), which, under certain conditions, can promote tumor growth and account for up to 50% of the total tumor mass. TAMs are activated in vitro into the anti-tumor M1 type and the tumor-promoting M2 type. TAMs secrete a series of immunosuppressive cytokines to tame infiltrating immune cells. The dynamic interaction between tumor cells and the immune system is crucial to the progression of cancer, making it necessary to develop efficient therapeutic strategies targeting TAMs. Polysialic acid (PSA) is an endogenous negatively charged polysaccharide, a linear, straight-chain polymer composed of negatively charged sialic acid (SA). The degree of polymerization (DP) of PSA ranges from 8 to 400 monomers, with individual SA monomers linked by α-2,8-, α-2,9-, or α-2,8 / α-2,9-glycosidic bonds. Because the two adjacent PSA monomers linked by α-2,8 glycosidic bonds form a structurally stable lactone under weakly acidic conditions, it is widely present in the human body and other organisms. Siglecs, receptors for SA, are highly expressed in monocytes and TAMs in tumor-bearing mice. Chemokines secreted by the TME recruit monocytes to tumors, influencing their growth. Leveraging the concept of "recruitment and homing" and the highly specific Siglec-SA binding, PSA- or SA-modified formulations can effectively kill tumor cells by targeting TAMs.

[0005] Cell penetrating peptides (CPPs) are cationic or amphipathic peptides composed of 20-40 amino acid sequences that can be used as effective cell delivery vehicles for drug delivery. Melittin (MEL / MLT) is a water-soluble, linear, cationic, hemolytic, cytolytic, and amphipathic peptide with a molecular weight of 2846.46. The cationic amino acid residues of MEL preferentially interact with negatively charged membrane surfaces or phospholipids through electrostatic interactions. This interaction causes membrane permeation and disruption, forming "ring-shaped pores." Tumor cells highly express SA on their surfaces, which serves as a key binding site for cationic CPPs to bind to glycoproteins and gangliosides. The electrostatic binding of MEL to tumor cells makes it a tumor-selective CPP and a candidate for future anticancer therapy. Lee et al. (LEE, C., JEONG H, BAEY, et al. Targeting of M2-like tumor-associated macrophages with a melittin-based pro-apoptotic peptide [J]. J Immunother Cancer, 2019. 7(1): 147.) used a hybrid peptide MEL-dKLA to target M2-type TAMs. The study found that the hybrid peptide destroyed the mitochondrial membrane and thus reduced the mitochondrial membrane potential, eliminating the immunosuppressive M2-type TAMs in the tumor stroma, inducing the specific death of M2-type TAMs, and successfully inhibiting tumor proliferation and angiogenesis. This indicates that even after binding to the drug, the property of MEL specifically binding to M2-type TAMs remains unchanged.

[0006] However, due to its membrane-perforating effect, MEL often causes severe hemolysis when injected intravenously, thus affecting its clinical application. The hemolytic mechanism reported so far is mainly based on MEL's cell membrane penetration and electrostatic interactions between MEL and membrane surface components. Studies have reported that after removing SA from the erythrocyte membrane by sialidase hydrolysis, the hemolytic activity is reduced by 40%, indicating that sialic acid molecules on the erythrocyte membrane may be the key to contact between MEL and the erythrocyte membrane, mediating MEL's hemolysis (Shi Wei, Li Caiyun, Chen Yuqing. Analysis of the hemolytic effect and mechanism of melittin on mouse erythrocytes [N]. Journal of Nanjing Normal University, 2015-38(02):86-92.).

[0007] Doxorubicin (DOX), a broad-spectrum anthracycline anticancer drug, is one of the most effective anticancer drugs developed to date. The pKa value of the primary amine group of DOX is 8.2 to 9.9, making it a cationic molecule at acidic and neutral pH. DOX exerts its antitumor effects through antimitotic and cytotoxic activities. It directly inserts into base pairs in a manner independent of cellular metabolism to inhibit DNA and RNA synthesis, thereby preventing nucleic acid replication and transcription in cancer cells. However, DOX can cause serious adverse reactions, including bone marrow hematopoietic dysfunction and impaired cardiac function. Therefore, the rational design of more effective tumor nanomedicines is urgent.

[0008] Due to the different in vivo pharmacokinetics and distribution of small molecule chemotherapeutic drugs DOX and CPPs, insufficient tumor selectivity and tumor accumulation, and hemolysis and other side effects caused by intravenous injection, considerable efforts have been invested in developing co-delivery systems to co-encapsulate small molecule chemotherapeutic drugs DOX and CPPs in the same carrier system to achieve optimal synergistic antitumor efficacy, hoping to release more payload at the target site. Summary of the Invention

[0009] The technical problem solved by the present invention is to overcome the defects of the existing technology. By utilizing the electrostatic interaction between cell-penetrating peptides, polysaccharides and small-molecule anti-tumor drugs, a cell-penetrating peptide-polysaccharide-small-molecule anti-tumor drug polyion complex and its preparation method and application are designed and prepared. Based on the tumor targeting of polysaccharides, polysaccharides are degraded in the slightly acidic environment of the tumor, releasing cell-penetrating peptides and small-molecule anti-tumor drugs. The small-molecule anti-tumor drugs are delivered deeply through the membrane-perforating effect of the cell-penetrating peptides. The two act synergistically to jointly kill tumor cells and M2-type TAMs, thereby achieving a highly effective anti-tumor effect. The cell-penetrating peptide-polysaccharide-small-molecule anti-tumor drug polyion complex can improve tumor targeting and reduce the nonspecific toxicity of cell-penetrating peptides and small-molecule anti-tumor drugs, achieving co-delivery of cell-penetrating peptides and small-molecule anti-tumor drugs, and solving the problems of poor efficacy of single anti-tumor drugs, easy hemolysis caused by intravenous injection, and drug resistance of small-molecule anti-tumor drugs.

[0010] The present invention is achieved through the following technical solutions:

[0011] The present invention discloses a cell penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex, which comprises a cell penetrating peptide, a polysaccharide and a small molecule anti-tumor drug, wherein the mass ratio of the cell penetrating peptide:polysaccharide:small molecule anti-tumor drug is (1-3):(8-10):(1-3).

[0012] Furthermore, the average molecular weight of the polysaccharide is 600 to 100,000 Daltons (600 Da to 100 kDa); preferably 3,000 to 80,000 Daltons (3,000 Da to 80 kDa), and more preferably 5,000 to 50,000 Daltons (5,000 Da to 500 kDa).

[0013] Furthermore, the cell-penetrating peptide is a positively charged cell-penetrating peptide with anti-tumor effect, with an amino acid sequence of less than 40 amino acids, and more preferably is melittin.

[0014] Furthermore, the polysaccharide is preferably negatively charged polysialic acid.

[0015] Furthermore, the small molecule anti-tumor drug is a positively charged drug selected from anthracyclines and / or anthraquinones. The anthracycline is selected from one or more of doxorubicin, epirubicin, pirarubicin, and idarubicin; and the anthraquinone anti-tumor drug is selected from pixantrone maleate and / or mitoxantrone hydrochloride.

[0016] The cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex, when the cell-penetrating peptide is melittin, the polysaccharide is polysialic acid, and the small molecule anti-tumor drug is the anthracycline drug doxorubicin, is preferably prepared in a mass ratio of melittin:polysialic acid:doxorubicin of 1:8:3, and the average molecular weight of the polysialic acid is 20,000 to 50,000 Daltons.

[0017] The present invention further provides a method for preparing the cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex, which is to co-load the cell-penetrating peptide, polysaccharide and small molecule anti-tumor drug.

[0018] Specifically, cell-penetrating peptides, polysaccharides and small molecule anti-tumor drugs are self-assembled using their respective electrostatic interactions.

[0019] The method for preparing the cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex specifically comprises the following steps:

[0020] (1) Weigh the raw materials according to the mass ratio of cell penetrating peptide, polysaccharide, and small molecule anti-tumor drug;

[0021] (2) dissolving the cell-penetrating peptide in sterile water for injection to obtain solution A, wherein the mass volume concentration of the cell-penetrating peptide in solution A is 0.1 mg / mL to 10 mg / mL;

[0022] (3) dissolving the polysaccharide in sterile water for injection to obtain solution B, wherein the mass volume concentration of the polysaccharide in solution B is 1 mg / mL to 100 mg / mL;

[0023] (4) dissolving the small molecule anti-tumor drug in sterile water for injection to obtain solution C, wherein the mass volume concentration of the small molecule anti-tumor drug in solution C is 1 mg / mL to 10 mg / mL;

[0024] (5) Solution A is mixed with solution B, and then mixed with solution C, and stirred to react to obtain a cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex.

[0025] In the step (5), the stirring reaction temperature is 18 to 40° C., preferably 20° C., and the stirring reaction time is 1.3 to 2.5 h, preferably 1.5 h.

[0026] The cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex is prepared by co-loading and self-assembling the cell-penetrating peptide, polysaccharide and small molecule anti-tumor drug by utilizing their respective electrostatic interactions.

[0027] The cell penetrating peptide-polysaccharide-small molecule antitumor drug polyion complex of the present invention is used as a drug co-delivery system, and is used to prepare synergistic antitumor drugs, drugs for reducing intravenous hemolysis, and drugs for eliminating multidrug resistance.

[0028] The cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex of the present invention, its preparation method and application have the following advantages:

[0029] ① Although numerous literature reports indicate that the efficacy of a complex prepared by mixing two single drugs is generally superior to that of a co-loaded complex, the cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex of the present invention is not only far superior to the complex formed by a single drug, but also superior to the combined use of two single-drug complexes;

[0030] ② The cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex of the present invention is a co-loaded self-assembled polyion complex that can effectively reduce the hemolytic toxicity of the cell-penetrating peptide and the side effects of the small molecule anti-tumor drug, can efficiently target tumor cells, and exert a toxicity-reducing and synergistic anti-tumor effect;

[0031] ③ The present invention has the characteristics of simple operation, controllable quality and low cost, and has practical application value;

[0032] ④ It was unexpectedly found that the melittin-polysialic acid-doxorubicin polyion complex can significantly enhance the cellular uptake of doxorubicin compared with the polysialic acid-doxorubicin polyion complex;

[0033] ⑤ It was unexpectedly discovered that the bee venom peptide-polysialic acid-doxorubicin polyion complex can exert the synergistic anti-tumor effect of bee venom peptide and doxorubicin, which helps to reduce the multidrug resistance caused by doxorubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 To optimize the process of preparing bee venom peptide-polysialic acid-doxorubicin polyion complex; A: addition order; B: temperature; C: stirring time;

[0035] Figure 2 This is a transmission electron microscopy (TEM) image of the melittin-polysialic acid-doxorubicin polyion complex;

[0036] Figure 3 is the infrared spectrum of the polyion complex of melittin-polysialic acid-doxorubicin; wherein, A: infrared spectrum of the polyion complex of melittin-polysialic acid;

[0037] (a) Melittin, (b) Polysialic acid, (c) Polysialic acid-melittin electrostatic complex, (d) Polysialic acid / melittin physical mixture

[0038] B: IR spectrum of polysialic acid-doxorubicin polyion complex;

[0039] (a) Doxorubicin, (b) polysialic acid, (c) polysialic acid-doxorubicin electrostatic complex, (d) polysialic acid / doxorubicin physical mixture

[0040] C: IR spectrum of melittin-polysialic acid-doxorubicin polyion complex;

[0041] (a) Melittin, (b) Polysialic acid, (c) Doxorubicin, (d) Melittin-polysialic acid-doxorubicin electrostatic complex, (d) Melittin / polysialic acid / doxorubicin physical mixture

[0042] Figure 4 This is the result of polyion complex hemolysis assay;

[0043] Figure 5 In vitro release experiment of polyion complex; A: cumulative release of doxorubicin; B: cumulative release of melittin;

[0044] Figure 6 Long-term storage stability of polyion complexes;

[0045] Figure 7 MTT assay was used to detect the effect of polyion complexes on cell viability; A: survival rate of S180 cells treated with doxorubicin; B: survival rate of RAW264.7 cells treated with doxorubicin; C: survival rate of S180 cells treated with melittin; D: survival rate of RAW264.7 cells treated with melittin;

[0046] Figure 8The isobol method was used to evaluate the combined effect of polysialic acid-melittin and polysialic acid-doxorubicin polyion complexes; A: S180 cells, B: RAW264.7 cells;

[0047] Figure 9 The morphological analysis of RAW264.7 cells by melittin-polysialic acid-doxorubicin polyion complex; A: no staining B: trypan blue staining (40×10

[0048] Figure 10 Laser confocal microscopy analysis of the in vitro uptake of doxorubicin and melittin by S180 cells; A: doxorubicin, B: melittin, C: co-localization of doxorubicin and melittin;

[0049] Figure 11 Laser confocal microscopy analysis of the in vitro uptake of doxorubicin and melittin by RAW264.7 cells; A: doxorubicin, B: melittin, C: colocalization of doxorubicin and melittin;

[0050] Figure 12 Flow cytometric analysis of doxorubicin uptake by S180 and RAW264.7 cells in vitro; A: S180 cells, B: RAW264.7 cells;

[0051] Figure 13 Flow cytometric analysis of the in vitro uptake of melittin by S180 and RAW264.7 cells; Figure A: S180 cells, B: RAW264.7 cells;

[0052] Figure 14 In vivo imaging of S180 tumor-bearing Kunming mice;

[0053] Figure 15 The tissue distribution and fluorescence quantification of S180 tumor-bearing Kunming mice; A: Cy7, B: PC, C: PMC;

[0054] Figure 16 This is the tumor growth curve of S180 tumor-bearing Kunming mice;

[0055] Figure 17 The changes in body weight and net weight of S180 tumor-bearing Kunming mice;

[0056] Figure 18 is the tumor inhibition index of different polyion complexes on S180 tumor-bearing Kunming mice;

[0057] Figure 19 This is the tumor shedding phenomenon in S180 tumor-bearing Kunming mice;

[0058] Figure 20 Tumor and important tissue sections of S180 tumor-bearing Kunming mice;

[0059] Figure 21 The apoptotic effects of different polyion complexes on S180 tumor cells in Kunming mice;

[0060] Figure 22 The fluorescence intensity of different polyion complexes on the apoptosis of S180 tumor cells in Kunming mice was quantified.

[0061] Figure 23 Immunofluorescence staining of M2 TAMs by different polyion complexes;

[0062] Figure 24 Quantification of immunofluorescence staining of M2 TAMs by different polyion complexes. DETAILED DESCRIPTION

[0063] The present invention will be described in more detail below with reference to the following examples. It should be understood that the present invention is not limited to the following examples, and any modifications and / or variations made to the present invention fall within the scope of protection of the present invention.

[0064] Sources:

[0065] In the present invention, the polysialic acid with an average molecular weight of 30 kDa was purchased from Carbosynth Company (Suzhou) Co., Ltd., UK.

[0066] Example 1

[0067] This embodiment provides a cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex, wherein the cell-penetrating peptide is melittin (molecular weight 2846 Daltons), the polysaccharide is polysialic acid (average molecular weight 30,000 Daltons, degree of polymerization 100), and the small molecule anti-tumor drug is doxorubicin. The melittin-polysialic acid-doxorubicin polyion complex is prepared. The preparation process includes the following steps:

[0068] Accurately weigh melittin, polysialic acid, and doxorubicin into a volumetric flask. Dissolve in sterile water for injection and filter through a 0.22 μm microporous membrane to obtain a 1 mg / mL melittin solution, a 12 mg / mL polysialic acid solution, and a 2 mg / mL doxorubicin solution. Accurately pipette the corresponding volumes of each solution and mix thoroughly. Stir at 30°C for 2 hours using a thermostatic magnetic stirrer. Filter through a 0.22 μm microporous membrane to obtain the melittin-polysialic acid-doxorubicin polyion complex.

[0069] Example 2 Screening of Formulations for Melittin-Polysialic Acid-Doxorubicin Polyion Complexes

[0070] The ratio of melittin, polysialic acid and doxorubicin was screened based on the particle size, drug loading and hemolysis rate of the polyion complex.

[0071] Polysialic acid ratio: Melittin, polysialic acid, and doxorubicin were prepared using ratios of melittin:polysialic acid:doxorubicin of 1:6:1, 1:7:1, 1:8:1, 1:9:1, and 1:10:1 (m / m / m), respectively. The effect of the polysialic acid ratio on particle size, drug loading, and hemolysis rate was investigated. With increasing polysialic acid ratios, the particle size and drug loading of the melittin-polysialic acid-doxorubicin polyion complexes first increased and then gradually decreased. The drug loading was highest at a ratio of melittin:polysialic acid:doxorubicin of 1:8:1 (m / m / m). The optimal mass ratio was: melittin:polysialic acid:doxorubicin = 1:8:1. The results are shown in Table 1.

[0072] Table 1 Screening ratio of polysialic acid in the polyion complex of melittin-polysialic acid-doxorubicin (n=3)

[0073]

[0074] Melittin and doxorubicin ratio: Melittin, polysialic acid, and doxorubicin were prepared using ratios of melittin:polysialic acid:doxorubicin of 1:8:1, 1:8:2, 1:8:3, 2:8:1, and 3:8:1 (m / m / m), respectively. The effect of the ratio of melittin to doxorubicin on particle size, drug loading, and hemolysis rate was investigated. When the ratios of melittin to doxorubicin were 1:1, 1:2, 1:3, 2:1, and 3:1 (m / m / m), the particle size and hemolysis rate first decreased and then increased. Furthermore, when the ratio of melittin to doxorubicin was 1:3 (m / m / m), the drug loading rate was higher, the hemolysis rate was lower, and the particle size was smaller. The results are shown in Table 2.

[0075] Table 2 Screening of the ratio of melittin to doxorubicin in the polyion complex of melittin-polysialic acid-doxorubicin (n=3)

[0076]

[0077]

[0078] In summary, the optimal formula of melittin-polysialic acid-doxorubicin polyion complex is melittin:polysialic acid:doxorubicin=1:8:3 (m / m / m).

[0079] Example 3 Process Optimization of Melittin-Polysialic Acid-Doxorubicin Polyion Complex

[0080] Addition order: Melittin-polysialic acid-doxorubicin polyion complexes were prepared with a ratio of Melittin:polysialic acid:doxorubicin = 1:8:3 (m / m / m). The effects of the addition order on the drug loading of the Melittin-polysialic acid-doxorubicin polyion complexes were investigated. Adding the corresponding solutions in different orders produced different results on the drug loading of the Melittin-polysialic acid-doxorubicin complexes. Figure 1 (A), in the figure, M represents melittin (MEL), D represents doxorubicin (DOX), and P represents polysialic acid (PSA). The corresponding order corresponds to the order of addition. Overall, the drug loading capacity prepared in the order of adding polysialic acid, melittin and doxorubicin is the highest. Therefore, the following experimental preparations all prepare polyion complexes in the order of adding polysialic acid, melittin and doxorubicin.

[0081] Temperature: Melittin-polysialic acid-doxorubicin polyion complex was prepared with a ratio of melittin:polysialic acid:doxorubicin = 1:8:3 (m / m / m). Polysialic acid, melittin and doxorubicin were added in the order of preparation. The temperatures were 0, 20, 30, 40 and 50 °C, respectively. The effect of temperature on the drug loading of melittin-polysialic acid-doxorubicin polyion complex was investigated. Figure 1 (B). The drug loading levels of melittin and doxorubicin in melittin-polysialic acid-doxorubicin varied at different temperatures, with optimal loading levels of melittin exceeding 16% and doxorubicin exceeding 25%. The corresponding temperature range was 18-40°C, with the highest loading level at 20°C. Therefore, a temperature of 20°C was used for subsequent preparations.

[0082] Stirring reaction time: Melittin-polysialic acid-doxorubicin polyion complex was prepared, and the ratio of melittin: polysialic acid: doxorubicin was 1:8:3 (m / m / m). The preparation was carried out in the order of adding polysialic acid, melittin and doxorubicin. The temperature was 20°C, and the stirring time was 0.5h, 1h, 1.5h, 2h, and 2.5h, respectively. The effect of stirring time on the drug loading of melittin-polysialic acid-doxorubicin polyion complex was investigated. The results are shown in FIG. Figure 1 (C). Different stirring times affect the tightness of the binding between the components of the complex and also affect its drug loading. Melittin loading of more than 15% and doxorubicin loading of more than 25% are optimal, corresponding to a time range of 1.3 to 2.5 hours. The time corresponding to the optimal drug loading: 1.5 hours was used as the standard for subsequent experiments.

[0083] Therefore, the final preparation process of melittin-polysialic acid-doxorubicin polyion complex was determined as follows: precisely pipette 1 mg / mL melittin solution, 12 mg / mL polysialic acid solution and 2 mg / mL doxorubicin solution, and take the corresponding volumes according to the ratio of melittin: polysialic acid: doxorubicin = 1:8:3 (m / m / m), mix them evenly in the order of polysialic acid, melittin and doxorubicin, and stir at 20°C for 1.5 h to obtain melittin-polysialic acid-doxorubicin polyion complex.

[0084] The polysialic acid-melittin and polysialic acid-melittin polyion complexes used in comparative experiments were prepared by the final preparation method.

[0085] Example 4 Characterization and Quality Evaluation of Polyion Complexes

[0086] Particle size determination: The particle size of the polyion complex was determined using a Nicomp-380 particle size analyzer. An appropriate amount of the polyion complex was taken with a pipette, diluted with sterile water for injection, and placed in a sample tank. The optical density was adjusted to between 250 and 350 kHz for measurement.

[0087] Zeta potential measurement: The zeta potential of the polyion complex was measured using a Nicomp-380 Zeta potential meter. An appropriate amount of the polyion complex was pipetted and diluted with sterile water for injection, then placed in a sample well and an electrode was inserted. The measurement conditions were: an electric field strength of 10 V·cm -1 The equilibrium time was set to 180 s, and each sample was measured in parallel for 3 times.

[0088] pH determination: Take an appropriate amount of polyion complex and use a pH meter to measure the pH value of the sample.

[0089] Method for determining the drug loading of melittin and doxorubicin: Take two 100 μL portions of the polyion complex, place one portion directly in a 5 mL volumetric flask, add 1.2 mL of deionized water, and then dilute to the scale with 50% (v / v) methanol aqueous solution, shake well, and after the dilution is passed through a 0.45 μm microporous filter membrane, measure the absorbance at 280 nm (melittin) and 480 nm (doxorubicin), respectively. Another portion was added to the top of the column and centrifuged at 2000 rpm for 4 min. 100 μL of deionized water was added to the top of the column and centrifuged as above. The eluate was combined three times and transferred to a 5 mL volumetric flask. A 50% (v / v) methanol aqueous solution was added (demulsified and diluted to the mark. The dilution was filtered through a 0.45 μm microporous membrane and the absorbance was measured as above. Drug loading (DL) was calculated as follows: DL (%) = (loaded drug mass / total mass of carrier and loaded drug) × 100%.

[0090] Table 3 Characterization of polysialic acid-melittin, polysialic acid-doxorubicin and melittin-polysialic acid-doxorubicin

[0091]

[0092] Morphological characterization: A small amount of polyion complex was dripped onto a copper grid covered with a carbon film, and negatively stained with 2.0% (w / v) phosphotungstic acid. The excess liquid was absorbed with filter paper, and the mixture was allowed to dry naturally at room temperature. The mixture was then observed and photographed under a transmission electron microscope. The transmission electron microscope images are attached. Figure 2 The figure shows that the prepared polyion complex is spherical and has a particle size of about 30 nm, which is consistent with the particle size result measured by the particle size analyzer.

[0093] Infrared spectroscopy (FT-IR) characterization: KBr tableting method was used, i.e., appropriate amounts of freeze-dried powder of polyion complex and corresponding physical mixture were ground and mixed evenly with anhydrous KBr (1:100), pressed using a tablet press (20 MPa, 2 min), and placed in the sample chamber of an infrared spectrometer to obtain an infrared spectrum. The test wave number range was 4000-500 cm -1 See attached infrared spectrum Figure 3 , where polysialic acid / melittin represents a physical mixture of the two, and the others are similar, through Figure 3 (A) can be seen at 3377.26cm -1 The hydroxyl peak of polysialic acid disappeared at 1541.08 cm -1 The weakened amino peak of melittin at the 400 nm position indicates the formation of a polysialic acid-melittin polyion complex. The physical mixture of polysialic acid / melittin contains various characteristic peaks of polysialic acid and melittin, with unchanged peak positions and intensities, indicating that the formation of the polyion complex prepared by the method of the present invention is different from that of the physical mixture alone. Figure 3 (B), it can be seen that at 3377.26cm -1 The hydroxyl peak of polysialic acid disappeared at 1583.51 cm -1 The weakened amino peak of doxorubicin at the 40 nm spectral region indicates the formation of a polysialic acid-doxorubicin polyion complex. Compared with the infrared spectrum of the polysialic acid / doxorubicin physical mixture, the peak position and peak intensity remain unchanged, indicating that the formation of the polyion complex prepared by the method of the present invention is different from that of the physical mixture alone. Figure 3 (C), it can be seen that at 3377.26cm -1 The hydroxyl peak of polysialic acid disappeared at 1541.08 cm -1 The weakened amino peak of melittin at 1583.51 cm -1The weakened amino peak of doxorubicin at the 40 nm spectral region indicates the formation of a melittin-polysialic acid-doxorubicin polyion complex. Compared with the infrared spectrum of the melittin / polysialic acid / doxorubicin physical mixture, the peak position and peak intensity remain unchanged, indicating that the polyion complex prepared by the method of the present invention is different from that formed by the physical mixture alone.

[0094] Example 5 Polyion Complex Hemolysis Assay

[0095] Take about 1 mL of fresh rat blood and transfer it to a centrifuge tube coated with sodium heparin. Centrifuge at 3000 rpm for 5 minutes, discard the upper plasma, add an equal volume of 5% Glu solution, mix thoroughly, and repeat the centrifugation 3 times. Pipette the washed sedimented red blood cells and dilute with 5% Glu solution to prepare a 2% red blood cell suspension (prepare immediately before use). Incubate the red blood cell suspension with the polyion complex at 37±0.5°C for 2 hours, centrifuge at 3000 rpm for 10 minutes, take the supernatant, and measure the absorbance of hemoglobin at a wavelength of 540 nm using an enzyme-linked reader. Use 0.9% NaCl as a negative control and 1% Triton X-100 as a positive control. Calculate the relative hemolysis rate (RH) according to the formula: RH (%) = (A X -A N ) / (A P -A N )×100%, where A X is the absorbance value of melittin at different concentrations, A N is the absorbance value of the negative control, A P The relative hemolysis rate of the polyion complex and the relative hemolysis rate at different pH values ​​(pH = 4.5, pH = 5.5, pH = 6.5, pH = 7.5) were determined. The results are shown in the attached Figure 4 and Table 4.

[0096] Table 4 Polyion complex hemolysis assay

[0097]

[0098] Hemolysis experiments showed that the hemolysis rate of the polyion complex remained below 5%, demonstrating good biocompatibility and facilitating its clinical translation. When different pH values ​​were examined, hemolysis occurred under acidic conditions, with the highest hemolysis rate at pH 4.5. This suggests that after entering the tumor microenvironment, polysialic acid degradation can release melittin, thereby exerting its membrane-perforating effect.

[0099] In addition, the prepared bee venom peptide-polysialic acid-doxorubicin polyion polymer, polysialic acid-bee venom peptide polyion polymer, polysialic acid-doxorubicin polyion polymer, and polysialic acid-bee venom peptide + polysialic acid-doxorubicin mixed polyion polymer all have hemolysis rates lower than 5%, and can be used in subsequent in vivo experiments. Compared with the bee venom peptide solution, they have a lower hemolysis rate under the condition of pH = 7.4.

[0100] Example 6 Investigation of in vitro release of polyion complexes

[0101] FITC-labeled melittin solution (melittin), polysialic acid-melittin (polysialic acid-melittin), and melittin-polysialic acid-doxorubicin (melittin-polysialic acid-doxorubicin) polyion complex with a melittin concentration of 2 mg / mL, as well as 2.0 mL each of doxorubicin solution (doxorubicin), polysialic acid-doxorubicin, and melittin-polysialic acid-doxorubicin polyion complex with a doxorubicin concentration of 1 mg / mL were precisely pipetted and added to dialysis bags (10 kDa) and placed in 10 mmol / L 4% HCl. PBS (pH = 7.4) and citrate buffer (0.1M, pH = 4.5 / 6.5) were placed in a dark-proof chamber at 37 ± 0.5°C with stirring at 100 rpm. 1.0 mL of dialysate was drawn at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h, and an equal volume of isothermal release medium was added. The 1.0 mL of dialysate was measured and calculated using a microplate reader at excitation wavelengths of 494 / 518 nm and emission wavelengths of 470 / 580 nm. The cumulative release of the drug, R, was calculated according to the following formula: n , see attached for release results Figure 5 .

[0102]

[0103] Where V0 is the volume of the release medium, C n is the concentration at the time of sampling for the nth time, C n-1 is the concentration at the time of sampling (n-1), V is the sampling volume, M t is the total drug concentration.

[0104] In vitro release experiments showed that the melittin and doxorubicin solution groups released the most rapidly, releasing nearly all of the melittin / doxorubicin within 4 hours. The release rates of the polyion complex groups were significantly lower than those of the solution groups. The melittin-polysialic acid-doxorubicin polyion complex released faster than the polysialic acid-melittin polyion complex and the polysialic acid-doxorubicin polyion complex in the medium due to ion exchange. The cumulative release rate of melittin / doxorubicin at pH 7.4 was also lower than that at pH 4.5. This is due to strong electrostatic interactions between the negative charge of the polysialic acid carboxyl group and the positive charge of melittin / doxorubicin at pH 7.4, which hindered the release of doxorubicin. Furthermore, the release rate of all complexes was fastest at pH 4.5, indicating that they can be rapidly released in the tumor microenvironment, thereby exerting a highly effective antitumor effect.

[0105] Example 7 Long-term storage stability of polyion complex

[0106] The polyion complex was sealed with nitrogen and stored at 4±2℃ away from light. Samples were taken at 0, 1, 3, and 6 months to observe the state of each preparation and measure its particle size. The storage stability results are shown in the attached Figure 6 ,pass Figure 6 It can be seen that after 6 months of storage, the particle size of each group of preparations did not change significantly (p>0.05), indicating good storage stability.

[0107] Example 8 In vitro cytotoxicity test of polyion complexes

[0108] The prepared polyion complexes polysialic acid-melittin, polysialic acid-doxorubicin, melittin-polysialic acid-doxorubicin, melittin solution (melittin), and doxorubicin solution (doxorubicin) were filtered through a 0.22 μm microporous filter membrane and placed into sterile centrifuge tubes. The drugs were diluted with RMPI 1640 culture medium to prepare melittin concentrations of 0.0032, 0.032, 0.064, 0.32, and 3.2 μg / mL, and doxorubicin concentrations of 0.01, 0.1, 0.2, 1.0, and 10.0 μg / mL.

[0109] The cells were digested with trypsin for 3 minutes and then quenched by adding RPMI 1640 medium containing 10% fetal bovine serum. S180 and RAW264.7 cells were collected in a 10 mL sterile centrifuge tube and centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum to adjust the cell density to 5 × 10 4 cells·mL -190 μL of cell suspension was plated per well in a 96-well plate, with five replicates. 200 μL of PBS was added to the edge wells to prevent evaporation. The 96-well plate was incubated overnight in a 37°C, 5% CO2 incubator until the cells were completely adhered to the wall before the experiment was performed. Zero wells, control wells, and experimental wells were set up. 10 μL of drug dilution medium was added to the zero wells and control wells, and the corresponding concentration of drug was added to the experimental wells. The replicates were 5. After drug addition, the 96-well plate was incubated in a 37°C, 5% CO2 incubator for a specified period of time. 10 μL of MTT staining solution (5 mg / mL) was added to the 96-well plate and incubated for another 4 hours to reduce the MTT to formazan crystals. After 4 hours, 100 μL of triple lysis solution (10% SDS / 5% isobutanol / 0.012 mol / L HCl, w / v / v) was added and the plate was incubated in the incubator until the crystals dissolved. The light absorption value (OD value) of each well was measured using a microplate reader at a wavelength of 570 nm. The average value of the replicate wells was used as the OD value of the cells in that group, and the cell survival rate was calculated (see the attached cell survival rate results). Figure 7 ), the calculation formula is as follows:

[0110] Cell viability = (OD value of experimental well - OD value of blank well) / (OD value of control well - OD value of zero-adjusted well) × 100%

[0111] Half maximal inhibitory concentration (IC 50 ) refers to the drug concentration required when the number of surviving cells is reduced by half after drug administration. By measuring 5 or more drug concentrations and the corresponding cell inhibition rate, the inhibition rate-concentration curve can be fitted and calculated. As a quantitative indicator reflecting the efficacy of the drug, it is widely used in the screening of various drugs. The cell growth inhibition rate of S180 and RAW264.7 cells under various concentration gradients was calculated, and the inhibition rate-concentration curve was drawn. The above experimental data were fitted to obtain the IC 50 The results of the values ​​are shown in Tables 5 and 6.

[0112] Table 5 Doxorubicin polyion complex IC 50 value

[0113]

[0114] Table 6 Melittin polyion complex IC 50 value

[0115]

[0116]

[0117] Whether the concentration of doxorubicin or melittin was used as the standard, the inhibitory effect of melittin-polysialic acid-doxorubicin on S180 and RAW264.7 cells was greater than that of polysialic acid-melittin and polysialic acid-doxorubicin alone, indicating that the combination of melittin and doxorubicin has a better inhibitory effect. 50 It is lower, indicating that it has stronger cytotoxicity, indicating that polysialic acid has monocyte targeting, which provides a theoretical basis for indirectly achieving TAMs targeting.

[0118] Example 9 Evaluation of Polyion Complex Combination Therapy

[0119] Polysialic acid-melittin and polysialic acid-doxorubicin were directly mixed at various concentrations in non-equal proportions. Nine groups were selected, each containing three concentrations with cell inhibition rates of approximately 50% or less, to facilitate visualization of the combined effect. The cells were incubated for 48 hours, and the cell growth inhibition was observed. The results are shown in Table 7.

[0120] Table 7 Inhibitory effect of polysialic acid-melittin and polysialic acid-doxorubicin polyion complexes on S180 and RAW264.7 cells

[0121]

[0122] The inhibition rate of polysialic acid-melittin and polysialic acid-doxorubicin when used in combination was obtained through the MTT experiment. Then, the isobol line method, King's formula method and Chou-Talalay method were used to evaluate the combined effects of polysialic acid-melittin and polysialic acid-doxorubicin mixed group (mixed) and co-loaded (melittin-polysialic acid-doxorubicin).

[0123] The isobol method is to first calculate the IC values ​​of two drugs A (melittin) and B (doxorubicin) when used alone. 50 , plotted on the x-axis and y-axis respectively, with coordinates (IC 50，A ,0) and (0,IC 50，B ), the line connecting these two points is called the superposition line. Then, a fixed dose of A is combined with a series of doses of B to calculate the IC when combined with B. 50 , with this IC 50 The IC of the combined application is plotted as the ordinate and the fixed dose of A as the abscissa. 50 point, that is, fixed dose method. If IC 50 If the point is at the lower left of the superposition line, it shows a synergistic effect; if it is at the upper right of the superposition line, it shows an antagonistic effect; and if it is above the line, it shows an additive effect.

[0124] Tables 8 and 9 list the results of the electrostatic complex combination of polysialic acid-melittin and polysialic acid-doxorubicin, that is, the IC values ​​corresponding to the combination of polysialic acid-melittin when the doxorubicin concentration in polysialic acid-doxorubicin is 0, 0.1, 0.2, and 1 μg / mL, respectively. 50 .

[0125] Table 8 IC values ​​of S180 cells treated with different concentrations of doxorubicin and polysialic acid-melittin 50 value

[0126]

[0127] Table 9 IC values ​​corresponding to polysialic acid-melittin in RAW264.7 cells treated with different concentrations of doxorubicin 50 value

[0128]

[0129] The above data are plotted into an isobologram. Figure 8 In S180 cells and RAW264.7, the curve of the mixed group of polysialic acid-melittin and polysialic acid-doxorubicin (mixed) can be seen above the right side of the superimposed line, indicating that there is a certain antagonistic effect between polysialic acid-melittin and polysialic acid-doxorubicin in the mixed group.

[0130] Golden formula: Q=E a+b / (E a +E b -E a ×E b ), calculate the Q value and evaluate the combined effect. In the formula, E a is the effect of a alone, E b is the effect of b alone, E a+b = Q = the effect of half the dose of drugs a and b combined. A Q value < 0.55 indicates significant antagonism, 0.55-0.85 indicates antagonism, 0.85-1.15 indicates simple addition, and > 1.15 indicates enhancement. Substituting the inhibition rates of polysialic acid-melittin, polysialic acid-doxorubicin, the mixed group, and the melittin-polysialic acid-doxorubicin group into the formula, the combined effect is as follows:

[0131] Table 10 Q values ​​of the two polyion complexes combined for S180 and RAW264.7 cells

[0132]

[0133] It can be seen from the table that the Q value of the melittin-polysialic acid-doxorubicin group is greater than 1.15, showing an enhancing effect, while the mixed group exhibits an antagonistic effect or an additive effect at high doses. It can be inferred that the co-loaded melittin-polysialic acid-doxorubicin group has a better inhibitory effect.

[0134] When determining the effects of individual and combined treatments on cells, the Chou-Talalay method and the principle of neutralization are used to evaluate whether the two drugs are synergistic or antagonistic. If the CI is less than 1, the two drugs are considered synergistic, CI = 1 indicates additive effect, and CI > 1 indicates antagonism.

[0135] Table 11 CI values ​​of two polyion complexes combined for S180 and RAW264.7 cells

[0136]

[0137] It can be seen from the table that the CI value of the melittin-polysialic acid-doxorubicin group is <1, showing a synergistic effect, while the mixed group shows a certain antagonistic effect. It can be inferred that the co-loaded melittin-polysialic acid-doxorubicin group has a better inhibitory effect.

[0138] Example 10: Morphological determination of RAW264.7 cells by polyion complexes

[0139] Collect RAW264.7 cells from the culture flask, add appropriate amount of fresh culture medium and gently blow the cells to make a cell suspension, and prepare a concentration of 1×10 4 Cell suspension of 100 μg / mL was inoculated into 6-well culture plates at 2 mL of cell suspension / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. After removing the culture plate, bee venom peptide, polysialic acid-bee venom peptide polyion complex, doxorubicin, polysialic acid-doxorubicin polyion complex, and bee venom peptide-polysialic acid-doxorubicin polyion complex (the final concentration of bee venom peptide was 20 / 40 μg / mL, and the final concentration of doxorubicin was 25 / 50 μg / mL) were added and incubated in a 37°C, 5% CO2 incubator for 2 hours. The supernatant of group A was discarded, and group B was stained with trypan blue at a final concentration of 0.04% for 3 minutes. Observation was performed under an optical microscope, and the results are shown in the attached figure. Figure 9 Live cells were shiny and showed no staining, while dead cells were dull and darkly stained with trypan blue. Treatment with melittin, polysialic acid-melittin polyion complex, and melittin-polysialic acid-doxorubicin polyion complex resulted in cell fragmentation and dark staining of the interior. In the doxorubicin and polysialic acid-doxorubicin polyion complex groups, cell morphology was intact and staining was lighter even at high concentrations, indicating that melittin has a certain membrane-perforating effect. A melittin concentration of 20 mg / mL and a doxorubicin concentration of 25 mg / mL were selected as the basis for subsequent cell experiments.

[0140] Example 11 Polyion Complex Uptake Assay

[0141] Confocal assay: S180 and RAW264.7 cells were plated at 1×10 6Cells were seeded at a density of 10 cells / well into a 6-well culture plate covered with a coverslip. 2 mL of culture medium was added to each well and cultured in a 37°C, 5% CO2 incubator for 24 hours. After removing the culture plate, the culture medium in the wells was carefully aspirated and doxorubicin, polysialic acid-doxorubicin polyion complex, melittin-polysialic acid-doxorubicin polyion complex, FITC-labeled melittin (melittin), FITC-labeled polysialic acid-melittin polyion complex (polysialic acid-melittin), and FITC-labeled melittin-polysialic acid-doxorubicin polyion complex (melittin-polysialic acid-doxorubicin) were added (final concentrations of doxorubicin: 25 μg / mL, final concentration of melittin: 20 μg / mL). After incubation at 37°C, 5% CO2 for 2 hours, the cells were gently washed three times with PBS to remove any unabsorbed material. Add 700 μL of 4% paraformaldehyde solution to each well to cover the coverslip, fix the cell sample at room temperature for 20 minutes, absorb the paraformaldehyde, and gently wash 3 times with PBS. Add 700 μl of DAPI (10 μg / mL) staining solution to cover the coverslip, incubate in the dark for 15 minutes at room temperature, remove the DAPI staining solution, and gently wash 3 times with PBS. Subsequently, drop 10 μL of anti-fluorescence quenching mounting agent on the slide, remove the coverslip from the culture plate, seal the slide (avoid bubbles), and image and photograph under a laser confocal microscope. The results are shown in the attached figure. Figure 10 and attached Figure 11 .

[0142] Attachment Figure 10 and attached Figure 11 The cellular uptake of different polyion complexes by S180 and RAW264.7 cells is shown. It has been reported that S180 and RAW264.7 cells, respectively, highly express Siglecs or Selectins, both of which are sialic acid receptors present on the cell surface. DAPI stains the cell nucleus, resulting in a blue signal. Doxorubicin can enter the cell nucleus, so it also stains the nucleus, resulting in a red signal. Melittin interacts primarily with the cell membrane, where it accumulates primarily. The intensity of the superimposed signals can be used to assess the cellular uptake of each agent. The results show that the order of uptake intensity for each agent by S180 / RAW264.7 cells is doxorubicin > melittin-polysialic acid-doxorubicin polyion complex > polysialic acid-doxorubicin polyion complex, and melittin > polysialic acid-melittin polyion complex. The co-localization of melittin and doxorubicin showed that they act on the cell membrane and cell nucleus respectively, further confirming that the membrane perforation effect of melittin can increase the uptake of doxorubicin.

[0143] Flow cytometry assay: S180 and RAW264.7 cells were collected from the culture flask, and an appropriate amount of fresh culture medium was added to gently pipette the cells to form a cell suspension at a concentration of 2×10 4A cell suspension of 1 μg / mL was plated into 6-well culture plates at 2 mL per well and cultured in a 37°C, 5% CO2 incubator for 24 h. After removing the culture plates, doxorubicin, polysialic acid-doxorubicin polyion complex, melittin-polysialic acid-doxorubicin polyion complex, FITC-labeled melittin (melittin), FITC-labeled polysialic acid-melittin polyion complex (polysialic acid-melittin), and FITC-labeled melittin-polysialic acid-doxorubicin polyion complex (melittin-polysialic acid-doxorubicin) were added (final concentration of doxorubicin: 25 μg / mL, final concentration of MEL: 20 μg / mL) and incubated in a 37°C, 5% CO2 incubator for 2 h. After trypsinization, the cells were harvested, centrifuged at 1200 rpm for 5 min, and the supernatant discarded. Resuspend the cells in PBS and wash them. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 200 μL PBS to disperse the cells again, and detect the fluorescence intensity of the samples by flow cytometry PE and FITC channels. Collect 1×10 4 The data were analyzed using FlowJo 7.6.1 software to obtain the mean fluorescence intensity. Figure 12 and attached Figure 13 Attached Figure 12 and attached Figure 13 In vitro cellular uptake experiments showed that the order of uptake intensity of each preparation by S180 and RAW264.7 cells was doxorubicin > melittin-polysialic acid-doxorubicin polyion complex > polysialic acid-doxorubicin polyion complex, and melittin > polysialic acid-melittin polyion complex. Polysialic acid-melittin, polysialic acid-doxorubicin, and melittin-polysialic acid-doxorubicin polyion complexes specifically bound to RAW264.7 cells that overexpress Siglec-1. Quantitative analysis of uptake revealed that doxorubicin uptake was higher in the melittin-polysialic acid-doxorubicin polyion complex group than in the polysialic acid-doxorubicin polyion complex group, further demonstrating that the addition of melittin increased doxorubicin uptake in the melittin-polysialic acid-doxorubicin polyion complex.

[0144] Example 12 In vivo imaging and tissue distribution of polyion complexes

[0145] When the tumor volume increases to 300 mm 3 The mice were randomly divided into three groups, with 5 mice in each group, and injected intravenously with Cy7, polysialic acid-Cy7 complex (PC), and polysialic acid-melittin-Cy7 complex (PMC) (1.0 mg Cy7·kg -1 ), and IVIS Lumina III small animal in vivo imaging was used to observe and take fluorescence and white light photos at 1, 4, 8, 12, and 24 hours, as shown in the attached Figure 1424 hours after injection, mice were killed, and the heart, liver, spleen, lung, kidney, and tumor were removed. After washing with saline, they were blotted dry with filter paper for fluorescence and white light imaging. The conditions for taking fluorescence photos were: ex =749nm,λ em =776nm, exposure time is 10s, as shown in the attached Figure 15 As shown, the tissue distribution of the preparation was semi-quantitatively analyzed using the ROI (Region of Interest) function of the in vivo imaging software, and the average fluorescence intensity of each tissue was calculated. The heart, liver, spleen, lung, kidney, and tumor of the tumor-bearing mice in the above-mentioned dosing group were weighed, and then the tissue samples were placed in 7 mL EP tubes. The tissue samples were processed and the fluorescence intensity was measured as F. The measured fluorescence intensity F was subtracted from the F0 measured for the blank tissue to obtain ΔF. The Cy7 concentration in each tissue sample was calculated from the standard curve, and the Cy7 content in the tissue was calculated. The results are shown in the attached figure. Figure 16 .

[0146] By the attached Figure 15 In vivo imaging reveals that the distribution of different Cy7 fluorescent probes within tumor-bearing mice varies. In tumor-bearing mice injected with Cy7 solution, near-infrared fluorescence signals were primarily localized in the spleen or liver, reaching peak activity at 4 hours, followed by a decrease in fluorescence, indicating that small molecule dyes are rapidly metabolized due to their lack of targeting. Fluorescence from PC and PMC prepared using polysialic acid gradually accumulated at the tumor site, reaching a peak at 12 hours, further demonstrating the advantage of polysialic acid as a carrier in actively targeting tumors. Compared with the PC group, PMC, as a nanocomplex, possesses a compact spatial structure, enabling better accumulation at the tumor site. Tumors and other isolated organs were collected for fluorescence imaging and ROI quantification to examine the biodistribution of the agents within tumor-bearing mice. The results demonstrated that polysialic acid-modified PC and PMC exhibited excellent tumor targeting.

[0147] Example 13 Antitumor Pharmacodynamics Experiment of Polyion Complex

[0148] Dosage regimen: 49 S180 tumor-bearing mice were randomly divided into seven groups, namely control group (5 mL / kg 5% Glu), polysialic acid-melittin (1.6 mg melittin·kg -1 ), polysialic acid-doxorubicin (5 mg doxorubicin·kg -1 ), polysialic acid-melittin + polysialic acid-doxorubicin (mixed-1, 0.8 mg melittin·kg -1 , 2.5mg doxorubicin·kg -1 ), polysialic acid-melittin + polysialic acid-doxorubicin (mixed-2, 1.6 mg melittin·kg -1 , 5mg doxorubicin·kg -1), melittin-polysialic acid-doxorubicin-1 (0.8 mg melittin·kg -1 , 2.5mg doxorubicin·kg -1 ) and melittin-polysialic acid-doxorubicin-2 (1.6 mg melittin·kg -1 , 5mg doxorubicin·kg -1 ), with seven mice per group. Each group of mice received the drug via tail vein injection starting on the fourth day after inoculation, with dosing every three days for a total of five doses (on days 4, 7, 10, 13, and 16 after inoculation). Data such as tumor volume, body weight, and mortality were recorded throughout the pharmacodynamic study.

[0149] Tumor volume: During the 26-day observation period, the long diameter (a) and short diameter (b) of the tumor, as well as the mouse body weight, were measured every other day using a vernier caliper. The formula V = 0.5 × a × b 2 Calculate the tumor volume and draw the tumor growth curve. The results are shown in the attached Figure 16 . The tumor volume of the tumor-bearing mice in the control group (5% Glu) continued to grow. Compared with the control group, the tumor volume of each group showed two trends during the 26-day drug treatment after tumor bearing: the tumor volume of the polysialic acid-melitin polyion complex, polysialic acid-doxorubicin polyion complex and mixed-1 group showed an overall upward trend, indicating that although they can inhibit tumor growth to a certain extent compared with the control group, the treatment effects of the three are still weak; the tumor volume of the mixed-2, bee venom-polysialic acid-doxorubicin-1, and bee venom-polysialic acid-doxorubicin-2 groups showed an overall downward trend, which can better inhibit tumor growth.

[0150] Body weight and net body weight: During the 26-day efficacy experiment, the body weight of tumor-bearing mice was measured and recorded every other day. In order to eliminate the influence of tumor size differences on the body weight evaluation of tumor-bearing mice, the "net body weight" was used to truly reflect the toxic and side effects of the single treatment group and the combination group on tumor-bearing mice. Assuming the tumor density is 1g / cm 3 Calculate the tumor mass. The net body mass of tumor-bearing mice is the body mass of tumor-bearing mice minus the tumor mass. The results are shown in the attached Figure 17 The body weight (A) and lean body weight (B) of each group showed an overall upward trend during the 26-day efficacy treatment after tumor loading. The body weight of the polysialic acid-melittin group and the polysialic acid-doxorubicin group was lower than that of the control group, but higher than that of the melittin-polysialic acid-doxorubicin-2 combination group at the same dose. Analysis may be that the anti-tumor effect of melittin-polysialic acid-doxorubicin-2 was weaker when administered alone. The body weight and lean body weight of the polysialic acid-doxorubicin group, the mixed-2 group, the melittin-polysialic acid-doxorubicin-1 group, and the melittin-polysialic acid-doxorubicin-2 group all showed a trend of first decreasing and then increasing. Analysis may be that the melittin-polysialic acid-doxorubicin-2 group played a cytotoxic role in killing tumor cells in the early stage and mainly played an immunomodulatory role in the later stage, killing residual tumor cells while slowly restoring the body's functions.

[0151] Tumor inhibition index: In order to fully compare the effectiveness and targeting of the preparations, taking into account the inhibition of the preparations on tumor cells and the non-specific damage to the body, our research group proposed a new evaluation index "Tumor-inhibition index (TI) index )”, the larger the tumor inhibition index, the better the overall treatment effect. The results are shown in the attached Figure 18 The order of tumor inhibition index of each group of mice on the 26th day after tumor loading was: melittin-polysialic acid-doxorubicin-2 > melittin-polysialic acid-doxorubicin-1 > mixed-2 > polysialic acid-doxorubicin ≈ polysialic acid-melittin ≈ mixed-1. By comparing the tumor inhibition index of each group, the bee venom peptide-polysialic acid-doxorubicin-2 group was much greater than the other groups, indicating that bee venom peptide-polysialic acid-doxorubicin-2 not only has a good tumor inhibition effect, but also has less non-specific toxicity to the body and the best overall therapeutic effect; the bee venom peptide-polysialic acid-doxorubicin-1 group and the mixed-2 group also have a certain tumor inhibition effect, but the bee venom peptide-polysialic acid-doxorubicin group that encapsulates bee venom peptide and doxorubicin can achieve better efficacy than the mixed injection group even if the dose is halved; it is worth noting that although the tumor inhibition index of the mixed-2 group is lower than that of the bee venom peptide-polysialic acid-doxorubicin-1 and bee venom peptide-polysialic acid-doxorubicin-2 groups, it is higher than that of the polysialic acid-melitin and polysialic acid-doxorubicin groups, indicating the advantage of the combination of bee venom peptide and doxorubicin.

[0152] Tumor shedding: During the entire experiment, the tumor growth status of the tumor-bearing mice was observed every day, and the process of tumor "shedding" and wound healing in the melittin-polysialic acid-doxorubicin group was recorded. The results are attached. Figure 19 As can be seen in the figure, the melittin-polysialic acid-doxorubicin group experienced tumor shedding during the drug's efficacy period, beginning on the 14th day of tumor loading. This means that the majority of the tumor tissue, along with the skin covering it, had "separated" from the tumor site. The wounds from which the tumor had shedding had essentially healed by the 24th day, with hair regrowth and a consistency consistent with the surrounding skin. The tumor shedding and wound healing observed in immunocompetent Kunming mice is strong evidence that the immune system is clearing the tumor.

[0153] Tissue sections: To investigate the organ toxicity of each polyion complex and the proliferation of tumor cells, the heart, liver, spleen, lung, kidney and tumor tissues of each group of tumor-bearing mice were collected and fixed with 4% paraformaldehyde solution for more than 24 hours and embedded in paraffin. The embedded specimens were cut into 5 μm sections by ultrathin microtome, heated at 70°C to remove paraffin, and stained with H&E. The sections were observed under an inverted microscope and images were taken. The results are shown in the attached figure. Figure 20Observation of organ pathological sections in each group revealed certain pathological changes in various organs in the control and mixed-1 groups, primarily manifested by myocardial cell edema (arrows), splenic fragmentation (arrowheads), and eosinophilic mucus secretion in the local bronchial lumens of the lungs (yellow arrows). The greatest difference among the other formulation groups was their tumor-killing effects (circles), with the order being: melittin-polysialic acid-doxorubicin-2 > melittin-polysialic acid-doxorubicin-1 > mixed-2 > polysialic acid-doxorubicin > polysialic acid-melittin > mixed-1. This suggests that the melittin-polysialic acid-doxorubicin polyion complex possesses highly effective tumor-killing ability and good biosafety.

[0154] Apoptosis Assay and Immunofluorescence: To further examine the effects of each formulation on tumor cell apoptosis and TAM depletion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect tumor cell apoptosis. Immunofluorescence analysis was performed on tumor tissues from each group, using CD68 (a protein expressed on the surface of monocytes and macrophages) as a marker for TAMs, and CD206 as a marker for M2 TAMs. Tumors from tumor-bearing mice in each group (n=3) were dissected and fixed in 4% paraformaldehyde solution. Wuhan Seville Company embedded, sectioned, stained (PE-coupled CD206 antibody, FITC-coupled CD68 antibody), and photographed (green fluorescence: PE-coupled CD206 antibody, red fluorescence: FITC-coupled CD68 antibody). Image-pro plus 6.0 was used to analyze the fluorescence intensity of the antibodies in the images (the ratio of the fluorescence intensity of M2 TAMs to TAMs is equal to the colocalization fluorescence intensity of CD206 and CD68 divided by the fluorescence intensity of CD68). The proportions of TUNEL-positive and TAMs-positive cells were calculated, respectively. The results are shown in the attached figure. Figure 21 、 Figure 22 、 Figure 23 and Figure 24 shown.

[0155] Apoptosis assay: Count the number of TUNEL-positive cells in three different fields of view and take photos of representative fields of view. Calculate the percentage of apoptotic cells based on the ratio of apoptotic cells to the total number of cells. TUNEL staining results indicate the apoptosis of tumor cells induced by the preparation. The more green, the more TUNEL-positive cells, and the more significant the induction of apoptosis. Figure 21As can be seen, a large number of green TUMEL-positive cells were distributed in the tumor tissue of the experimental group, while there was no obvious green distribution in the control group, indicating that the tumor tissue of the experimental group had a large number of apoptotic cells. Both the polysialic acid-melittin and polysialic acid-doxorubicin groups induced a certain degree of apoptosis, but the apoptosis results in the combination treatment group were more significant (p < 0.01), indicating that the combination of melittin and doxorubicin can promote apoptosis of tumor cells. In particular, the melittin-polysialic acid-doxorubicin-2 group showed a large area of ​​TUNEL-positive staining, indicating that the removal of TAMs promoted tumor cell apoptosis and inhibited tumor growth. This may be because when melittin and doxorubicin are co-encapsulated in polysialic acid, the membrane perforation effect of melittin can deliver doxorubicin into the tumor, greatly promoting doxorubicin-induced apoptosis.

[0156] Immunofluorescence: The slides were placed under a fluorescence microscope for observation and image collection. At a 400x field of view, randomly selected areas of each tissue point were photographed, and the fluorescence intensity of the positive areas in each photo was analyzed to obtain the fluorescence intensity of CD206 and CD68, respectively. The fluorescence intensity of the CD206-positive area was divided by the fluorescence intensity of the CD68-positive area to obtain the ratio of M2 TAMs to TAMs. The results are shown in the attached figure. Figure 22 . CD68 (macrophage sialoprotein) is a heavily glycosylated transmembrane protein expressed by monocytes and macrophages and often used as their marker. CD68 and CD206 are markers for TAMs and M2 TAMs, respectively. The fluorescence intensity of CD206 decreases, and the number of M2 TAMs decreases. Both polysialic acid-melittin and polysialic acid-doxorubicin polyion complexes can kill TAMs and M2 TAMs to a certain extent. The mixed-2 in the combined group also has a killing effect on TAMs, while the melittin-polysialic acid-doxorubicin-2 group can significantly reduce the number of TAMs and M2 TAMs (p<0.01), indicating that the combination of melittin and doxorubicin, and co-loading them in the same carrier, can maximize the combined effect of the two.

[0157] The bee venom peptide-polysialic acid-doxorubicin polyion complex achieves the co-loading of bee venom peptide and doxorubicin, maximizing the advantages of combined chemotherapy, solving the problems of nonspecific killing effects and multidrug resistance that are prone to occur when using a single drug, reducing damage to normal cells and tissues, and specifically targeting and killing tumor cells.

[0158] We hypothesize that deep drug delivery occurs primarily through three stages: ① Polysialic acid binds to Siglec-1 in TAMs, directing the melittin-polysialic acid-doxorubicin polyion complex to the tumor site. In in vivo and tissue distribution experiments, PMCs were clearly observed to accumulate in the tumor, suggesting a tumor-targeting effect. ② The slightly acidic pH of the tissue membrane (TME) degrades polysialic acid, releasing melittin and doxorubicin. In vivo release experiments demonstrated that the melittin-polysialic acid-doxorubicin polyion complex can be released at pH 4.5, indicating drug release in response to the slightly acidic TME environment. ③ Melittin perforates the membrane, promoting deep delivery of doxorubicin. Confocal microscopy revealed increased doxorubicin uptake in the melittin-polysialic acid-doxorubicin group, with melittin and doxorubicin localized to distinct cellular locations.

[0159] The main mechanisms of action are: ① Direct cell-killing effects based on melittin and doxorubicin: Both melittin and doxorubicin are cytotoxic drugs that rapidly destroy cell membranes and induce cell necrosis; ② Specific killing of M2 TAMs: Literature reports indicate that melittin can specifically kill M2 TAMs. Immunofluorescence staining results in this experiment indicate that the melittin-polysialic acid-doxorubicin polyion complex has the specific ability to kill M2 TAMs. ③ Synergistic inhibitory effect: The diffusion of melittin into surrounding tumor cells and cells in the TME promotes the formation of pores in the membrane, increases the cellular uptake of loaded doxorubicin, and enhances anticancer efficacy. Deep penetration activates the systemic immune response to effectively prevent tumor recurrence.

Claims

1. A method for preparing a cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex, characterized in that: The cell-penetrating peptide is melittin, the polysaccharide is polysialic acid, and the small-molecule anti-tumor drug is doxorubicin. The mass ratio of melittin:polysialic acid:doxorubicin is 1:8:

3. The average molecular weight of polysialic acid is 20,000 to 50,000 Daltons. The cell-penetrating peptide, polysaccharide, and small-molecule anti-tumor drug are self-assembled using their respective electrostatic interactions. Follow these steps: (1) Weigh the raw materials according to the mass ratio of cell penetrating peptide, polysaccharide, and small molecule anti-tumor drug; (2) dissolving the cell-penetrating peptide in sterile water for injection to obtain solution A, wherein the mass volume concentration of the cell-penetrating peptide in solution A is 0.1 mg / mL to 10 mg / mL; (3) dissolving the polysaccharide in sterile water for injection to obtain solution B, wherein the mass volume concentration of the polysaccharide in solution B is 1 mg / mL to 100 mg / mL; (4) dissolving the small molecule anti-tumor drug in sterile water for injection to obtain solution C, wherein the mass volume concentration of the small molecule anti-tumor drug in solution C is 1 mg / mL to 10 mg / mL; (5) Solution A was added to solution B and mixed, and then solution C was added and mixed, and the mixture was stirred for reaction at a temperature of 20-30°C and a reaction time of 1.3-2.5 h to obtain a cell-penetrating peptide-polysaccharide-small molecule anti-tumor drug polyion complex.

2. Use of the polyion complex prepared by the method for preparing the cell-penetrating peptide-polysaccharide-small molecule antitumor drug polyion complex according to claim 1 in preparing a drug co-delivery system or preparing a synergistic antitumor drug.

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