A ternary composite nanogel, a preparation method, an antitumor drug and application thereof

Abstract

This invention provides a ternary composite nanogel, its preparation method, an antitumor drug, and its applications. The functional nanogel obtained by this invention relies on the non-covalent interaction between cisplatin and small molecules that can chelate with cisplatin. The chelation of cisplatin can be reversed under physiological conditions, thereby achieving sustained release of cisplatin, which can then be used as an antitumor drug. Simultaneously, with the release of cisplatin, small molecules that can chelate with cisplatin are also released, possessing both cisplatin therapeutic function, polysaccharide biological (targeting) function, and the therapeutic function of the small molecules chelated with cisplatin. Depending on the small molecules used, it can be used for combination therapy of tumors. Depending on the polysaccharide used, targeted drug delivery mediated by CD44, P-selectin, and Galectin-3 proteins in tumor cells can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a ternary composite nanogel, its preparation method, anti-tumor drugs, and applications. Background Technology

[0002] Polysaccharides are generally readily available, biodegradable, and highly biocompatible, and have been widely used as biomaterials in the pharmaceutical industry. Furthermore, some polysaccharides possess unique biological activities; for example, hyaluronic acid can be specifically recognized by its receptor (CD44), dextran has macrophage targeting capabilities, and small-molecule pectin has Galectin-3 protein binding ability. Therefore, polysaccharide-based drug delivery carriers have significant application potential for targeted drug delivery in diseases such as tumors.

[0003] The main methods for preparing polysaccharide drug delivery carriers are as follows: (1) A large number of studies and applications use polysaccharides as target molecules, modifying pre-synthesized carriers (micron and nanoparticles, etc.) with polysaccharides to achieve targeted drug delivery (tumor). (2) Another widely used strategy is to chemically modify polysaccharides. By connecting other molecules or groups to polysaccharides, or by directly performing chemical cross-linking, polysaccharide-based nanoparticles, including micelles, nanogels, and other drug delivery carriers, can be formed. (3) Polysaccharides based on ionic cross-linking (including metal chelation) achieve cross-linking between polysaccharide chains through ionic interactions or metal chelation between ions and groups on the side chains of polysaccharides, thereby preparing macroscopic gels or nanoscale gels (nanogels), which have been widely used in the fields of biomaterials and drug delivery.

[0004] Due to the physiological characteristics of tumors, nanomedicines can accumulate locally within tumors through enhanced permeability and retention (EPR) effects, achieving targeted delivery (also known as passive targeting). Furthermore, the binding of nanomedicines to tumor-specific antibodies (antigens) can enhance tumor cell uptake (known as active targeting). In addition, tumors are often heterogeneous, and combination therapy with multiple drugs can kill tumor cells more efficiently through various mechanisms. However, controlling the proportions of different drugs in combination therapy (free drug administration) remains challenging due to their varying pharmacokinetic properties. Simultaneously, the use of multiple free drugs can easily lead to additive toxicity, limiting the dosage in combination therapy. Loading two or more drugs into tumor-targeting nanoparticles allows for better control of drug proportions, achieving targeted tumor delivery while reducing drug toxicity.

[0005] In the process of drug delivery targeting tumors, several bottlenecks exist at the cellular level. First, a sufficient number of drug-loaded nanoparticles need to enter the cell. Second, the drug delivered by the nanoparticles must reach its intracellular site of action to bind to its target and exert a therapeutic effect. Typically, drug-loaded nanoparticles undergo endocytosis, entering endosomes and lysosomes (hereinafter collectively referred to as lysosomes). This process results in the drug molecules carried by the nanoparticles being trapped within the lysosomes. However, the target site for most drug molecules is not lysosomes. Therefore, promoting lysosomal escape of the drug during delivery helps more of the delivered drug reach its intracellular site of action (such as the nucleus and cytoplasm), thereby enhancing its therapeutic effect.

[0006] Cisplatin is a common first-line chemotherapy drug widely used in the treatment of various cancers. Its mechanism of action involves hydration and activation of cisplatin upon entering cells due to the low chloride ion concentration in the cytoplasm. This allows cisplatin to chelate with intracellular DNA, leading to DNA cross-linking and ultimately killing tumor cells. Similar to cisplatin-crosslinked DNA, cisplatin can also chelate with carboxyl-containing polysaccharides (such as hyaluronic acid), sulfonic acid-containing polysaccharides (such as fucoidan), and amino-containing polysaccharides (such as chitosan), mediating polysaccharide cross-linking and preparing cisplatin-crosslinked polysaccharide nanogels. The chelation of cisplatin can be reversed under chloride-containing conditions (such as phosphate-buffered saline), allowing for sustained release of cisplatin in the crosslinked polysaccharide nanogels. As a chemotherapy drug, sustained release of cisplatin prolongs its circulation time in the bloodstream. Furthermore, the application of polysaccharides with tumor-targeting effects can promote cisplatin uptake in tumor cells. This results in a highly effective tumor treatment and reduced toxicity of cisplatin.

[0007] Current technologies utilize cisplatin as a drug, directly adsorbing or chelating it into pre-formed nanoparticles (e.g., silicon nanoparticles and gold nanoparticles). A second method uses cisplatin as a cross-linking agent to form nanoparticles by cross-linking polymers, including polysaccharides, block polymers, and DNA. For example, cisplatin-crosslinked polysaccharide nanogels can co-load drug molecules containing both aromatic rings and positive charges. Other existing technologies involve the combined administration of hyaluronic acid and cisplatin, or require hyaluronic acid aldehyde esterification, or nanoparticles based on hyaluronic acid derivatives, or drug delivery systems co-assembled with another polymer, or nanoparticles composed solely of cisplatin and hyaluronic acid, or nanomedicines requiring albumin and chemically modified hyaluronic acid. Existing technologies utilize... Linker technology utilizes the metal chelating effect of cisplatin to conjugate drug molecules to antibodies. This method is mainly used to prepare antibody-drug conjugates and cannot be used to prepare drug-loaded nanoparticles.

[0008] Therefore, the existing technology has not yet provided a method for cisplatin to simultaneously bind with small molecules and polysaccharides to form ternary composite nanogels, which cannot solve the one-step co-loading and targeted delivery application of cisplatin and small molecules. How to obtain stable and uniform cisplatin chelate nanogels at low cost and quickly has become an urgent problem to be solved. Summary of the Invention

[0009] Therefore, the purpose of this invention is to overcome the defects in the prior art and provide a ternary composite nanogel, its preparation method, an anti-tumor drug, and its application.

[0010] Before describing the content of this invention, the following terms are defined as follows:

[0011] The term "CD44 protein" refers to a multi-molecular membrane-integrated protein with a molecular weight of (85–160) × 10 kDa, and is one of the common marker proteins of tumor stem cells. CD44 has a molecular weight range of 85–250 kDa, mediates cell-cell and cell-extracellular matrix interactions, and is a glycoprotein composed of extracellular, transmembrane, and cytoplasmic parts, with glycan chains of chondroitin sulfate and heparan sulfate.

[0012] The term "LLOMe" refers to L-leucyl-L-leucine methyl ester.

[0013] The term "GSK-J1" refers to a histone demethylase inhibitor with the chemical formula C0. 22 H 23 N5O2, CAS number 1373422-53-7.

[0014] The term "heparosan precursor" refers to a bacterial mucopolysaccharide derived from the Escherichia coli K12 strain, named Heparsan. It consists of regularly repeating 4-β-glucuronic acid-(1-4)-α-N-acetylglucosamine groups and is produced and found in some pathogenic bacteria, such as Escherichia coli K5 and Pasteurella multocida. The main structure of Heparosan is a precursor for heparin synthesis, and bacterial-derived Heparosan itself can serve as a precursor for the synthesis of non-animal-derived heparin products.

[0015] The term "P-selectin" refers to a glycoprotein with a relative molecular mass of 140,000, which is present on the Weibe-Palade body membrane of vascular endothelial cells and the α-granule membrane of platelets. It is rapidly expressed on the plasma membrane after stimulation by histamine, thrombin, phorbol ester and calcium ion carriers. Hypoxia / re-oxidation or oxygen free radicals can also induce its expression.

[0016] The term "Galectin-3 protein" refers to a β-galactose-binding protein, abbreviated as Gal-3, belonging to the galactolectin family. It is widely distributed in mammalian cells and tissues and has a variety of biological functions. Galectin-3 plays an important role in cell-cell interactions, immune responses, cell proliferation, and apoptosis.

[0017] The term "Ribociclib" refers to a small molecule inhibitor of cyclin-dependent kinases 4 / 6, with the chemical structural formula C. 23 H 30 N8O, CAS number 1211441-98-3.

[0018] The term "HC nanogel" refers to a nanogel composed solely of cisplatin and hyaluronic acid.

[0019] The term "HCL nanogel" refers to a ternary composite nanogel composed of cisplatin, LLOMe, and hyaluronic acid.

[0020] The term "HCP nanogel" refers to a ternary composite nanogel composed of cisplatin, pabistat, and hyaluronic acid.

[0021] The term "HCGd nanogel" refers to a ternary composite nanogel composed of cisplatin, gadolinium, and hyaluronic acid.

[0022] The term "HCAs nanogel" refers to a ternary composite nanogel composed of cisplatin, arsenic, and hyaluronic acid.

[0023] The term "ACR nanogel" refers to a ternary composite nanogel composed of cisplatin, parzopanib, and astragalus polysaccharide.

[0024] The term "MCR nanogel" refers to a ternary composite nanogel composed of cisplatin, ribociclib, and small molecule citrus pectin.

[0025] The term "CRT" refers to calreticulin.

[0026] The term "acidic polysaccharide" refers to a polysaccharide containing negatively charged groups such as carboxyl, carboxymethyl, and sulfonic acid groups.

[0027] The term "alkaline polysaccharide" refers to a positively charged polysaccharide containing groups such as amino and imine groups.

[0028] The term "Chinese herbal polysaccharide" refers to a class of active polysaccharides extracted from Chinese herbal medicines. They generally do not include starch and cellulose and are usually used as bioactive substances to maintain or enhance the body's physiological functions. They have effects such as immune regulation, anti-tumor, antiviral, hypoglycemic, and anti-aging.

[0029] The term "marine polysaccharide" refers to a class of polysaccharides that are isolated and purified from marine and lake organisms.

[0030] The term "DMF" refers to N,N-dimethylformamide.

[0031] To achieve the above objectives, a first aspect of the present invention provides a ternary composite nanogel with a polysaccharide-cisplatin-drug small molecule chelate structure, the ternary composite nanogel comprising:

[0032] Polysaccharides,

[0033] Cisplatin, and

[0034] Small drug molecules chelated with cisplatin; among them,

[0035] The polysaccharide is selected from one or more of the following: acidic polysaccharides, basic polysaccharides, traditional Chinese medicine polysaccharides, and marine polysaccharides, preferably selected from one or more of the following: acidic polysaccharides, basic polysaccharides, and traditional Chinese medicine polysaccharides;

[0036] The drug molecule contains one or more elements selected from the following: phosphorus, nitrogen, sulfur, arsenic, gadolinium, preferably containing one or more elements selected from the following: phosphorus, nitrogen, sulfur, arsenic, and most preferably containing: phosphorus, nitrogen, sulfur and / or arsenic.

[0037] According to a first aspect of the present invention, the ternary composite nanogel is wherein the drug molecule is chelated with the cisplatin through non-covalent interactions and then co-assembled with the polysaccharide to form the ternary composite nanogel.

[0038] According to the first aspect of the present invention, a ternary composite nanogel is provided, wherein,

[0039] The nitrogen-containing drug molecule is selected from one or more of the following: L-leucyl-L-leucine methyl ester, pabisostat, lapatinib, parzopanib, afatinib, olaparib, ibrutinib, praxavir, GSK-J1, Ribociclib, preferably selected from one or more of the following: L-leucyl-L-leucine methyl ester, pabisostat, parzopanib, afatinib, praxavir, more preferably selected from one or more of the following: L-leucyl-L-leucine methyl ester, parzopanib, afatinib, pabisostat;

[0040] The arsenic-containing drug molecule is selected from one or more of the following: arsenic trioxide, arsanoic acid, potassium arsenide, preferably arsenic trioxide and / or arsanoic acid, and most preferably arsenic trioxide;

[0041] The phosphorus-containing drug molecule is selected from one or more of the following: flotinib, zoledronic acid, tilmidronic acid, minodronic acid, risedronic acid, thiamine phosphate, preferably selected from one or more of the following: flotinib, zoledronic acid, tilmidronic acid, minodronic acid, risedronic acid, more preferably flotinib and / or zoledronic acid;

[0042] The sulfur-containing drug molecule is selected from one or more of the following: sulfadiazine, azathioprine, chlorothiazide, hydrochlorothiazide, preferably selected from one or more of the following: sulfadiazine, azathioprine, chlorothiazide, more preferably sulfadiazine and / or azathioprine; and / or

[0043] The gadolinium-containing drug molecule is gadolinium chloride and / or gadolinium nitrate, with gadolinium chloride being the most preferred.

[0044] According to the first aspect of the present invention, the ternary composite nanogel is wherein the polysaccharide is a non-neutral polysaccharide and contains one or more groups selected from the following: carboxyl, sulfonic acid, carboxymethyl, amino, preferably containing one or more groups selected from the following: carboxyl, sulfonic acid, amino.

[0045] According to the first aspect of the present invention, the ternary composite nanogel contains polysaccharides selected from one or more of the following: hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, chitosan, citrus pectin, astragalus polysaccharide, tremella polysaccharide, poria polysaccharide, and ginseng polysaccharide; preferably selected from one or more of the following: hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, chitosan, citrus pectin, astragalus polysaccharide, tremella polysaccharide, and poria polysaccharide; more preferably selected from one or more of the following: hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, chitosan, citrus pectin, astragalus polysaccharide, and tremella polysaccharide; and even more preferably selected from one or more of the following: hyaluronic acid, citrus pectin, and astragalus polysaccharide.

[0046] According to the first aspect of the present invention, a ternary composite nanogel is provided, wherein,

[0047] In the ternary composite nanogel, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 4–35:2–60:1–16, preferably 6–24:3–36:1–10, more preferably 8–20:4–24:1.2–8; and / or

[0048] The hydrated particle size of the ternary composite nanogel is 40-4000 nm, preferably 50-1000 nm, and more preferably 70-550 nm.

[0049] A second aspect of the present invention provides a method for preparing the ternary composite nanogel described in the first aspect, the method comprising the following steps:

[0050] 1) Prepare polysaccharide aqueous solution, cisplatin aqueous solution and drug small molecule solution respectively;

[0051] 2) Mix the cisplatin aqueous solution and the drug small molecule solution prepared in step 1) and heat to obtain a cisplatin drug small molecule mixed solution. Then add the polysaccharide aqueous solution prepared in step 1) and mix well. Heat and incubate, then cool to obtain a cisplatin-drug small molecule-polysaccharide mixed solution.

[0052] 3) Dialyze the cisplatin-drug small molecule-polysaccharide mixed solution prepared in step 3) to remove free drug small molecules, thereby obtaining the ternary composite nanogel.

[0053] Preferably, in step 2), the solvent used for the drug small molecule solution is selected from one or more of the following: deionized water, DMF, dimethyl sulfoxide, and acetonitrile.

[0054] A third aspect of the present invention provides a targeted antitumor drug for combination therapy, the targeted antitumor drug comprising the ternary composite nanogel described in the first aspect.

[0055] The fourth aspect of the present invention provides the use of the ternary composite nanogel described in the first aspect in the preparation of targeted antitumor drugs for combination therapy.

[0056] According to the fourth aspect of the present invention, the tumor is selected from one or more of the following: gallbladder cancer, liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, thyroid cancer, non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, cervical cancer, appendicitis, skin cancer, kidney cancer, myeloma, and lymphoma.

[0057] Preferred cancers include one or more of the following: liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, thyroid cancer, non-small cell lung cancer, bladder cancer, squamous cell carcinoma of the head and neck, and cervical cancer.

[0058] More preferably, it is selected from one or more of the following: liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, thyroid cancer, and cervical cancer.

[0059] According to a preferred embodiment of the present invention, the multifunctional nanogel prepared based on cisplatin chelation is formed through the interaction of polysaccharides, the chemotherapeutic drug cisplatin, and small molecules that can chelate with cisplatin. The functional nanogel obtained by the present invention depends on the non-covalent interaction between cisplatin and small molecules that can chelate with cisplatin. The chelation of cisplatin can be reversed under physiological conditions, thereby achieving sustained release of cisplatin, which can act as an anti-tumor drug. Simultaneously, with the release of cisplatin, small molecules that can chelate with cisplatin are also released, possessing both cisplatin therapeutic function, polysaccharide biological (targeting) function, and the therapeutic function of the small molecules chelated with cisplatin. Depending on the small molecules used, it can be used for combined drug therapy for tumors. The polysaccharides used can be hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, small molecule citrus pectin, astragalus polysaccharide, and tremella polysaccharide, etc. Furthermore, depending on the polysaccharides used, targeted drug delivery mediated by CD44, P-selectin, and Galectin-3 proteins in tumor cells can be achieved.

[0060] Studies on the antitumor mechanism of cisplatin have shown that cisplatin can chelate with intracellular DNA, proteins, and other components. This chelation process mainly involves cisplatin chelating with groups containing phosphorus, nitrogen, sulfur, and other elements. This invention proposes that small molecules capable of metal chelation with cisplatin can be used to form chelates, which are then co-assembled with polysaccharides to create multifunctional nanogels. The ability of small molecule drugs to chelate with cisplatin is a prerequisite for the formation of multifunctional nanogels. Depending on the small molecule drug used, the multifunctional nanogel can be targeted to tumor cells, achieving tumor-targeted delivery, and can be used for combination drug therapy and immunotherapy for tumors.

[0061] Novel polysaccharide nanogels were prepared by utilizing the interactions between polysaccharides and cisplatin, as well as small molecules that can chelate with cisplatin. The characteristics of the small molecules that can co-assemble with polysaccharides and cisplatin, and their preparation methods, were identified. Based on the characteristics of the selected small molecules, the resulting multifunctional nanogels can simultaneously promote immunogenic cell death in solid tumor cells after cell entry, thereby activating anti-tumor immunity.

[0062] The functionalized nanogel of this invention enables the co-loading of small molecules with cisplatin within the nanogel. This process does not involve complex chemical reactions and is simple to prepare. It retains the tumor cell-targeting delivery capability of hyaluronic acid.

[0063] On the one hand, the small molecules in the functionalized nanogel can chelate with cisplatin, achieving drug co-loading. Therefore, compared with HC nanogel, the functionalized nanogel of this invention can form more cisplatin-DNA conjugates, mediating a stronger tumor therapeutic effect. On the other hand, direct damage to lysosomes can induce lysosome-associated cell death, stimulating an anti-tumor immune response.

[0064] Compared with existing technologies, the ternary composite nanogel, its preparation method, anti-tumor drugs, and applications of the present invention may have, but are not limited to, the following beneficial effects:

[0065] 1. The preparation method of this invention can synthesize nanogels containing both cisplatin and small molecules in one step, and the hydrated particle size can be controlled by adjusting the heating time. The entire synthesis process is simple and rapid, with mild conditions that are easy to scale up, and can improve the water solubility of small molecules.

[0066] 2. Compared with HC nanogels containing cisplatin alone, the ternary composite nanogel of the present invention can promote cisplatin delivery and carry out combined drug therapy, thus having a better tumor treatment effect.

[0067] 3. Compared with free drugs, the ternary composite nanogel of the present invention can reduce their toxic side effects.

[0068] 4. Since hyaluronic acid can be recognized by its receptor CD44, the ternary composite nanogel of the present invention has the CD44 targeting function, which can mediate the immunogenic death of tumor cells and be used to achieve tumor targeted delivery and combination therapy. Attached Figure Description

[0069] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0070] Figure 1 The preparation process of the ternary composite nanogel of the present invention is shown.

[0071] Figure 2 The hydrated particle size of the ternary composite nanogel prepared in Example 1 is shown.

[0072] Figure 3 Electron microscopy images of the ternary composite nanogel prepared in Example 1 are shown.

[0073] Figure 4 The 1H NMR spectrum of the ternary composite nanogel prepared in Example 1 is shown. This demonstrates the co-loading of cisplatin and LLoMe dipeptide molecules in the ternary composite nanogel.

[0074] Figure 5The drug release curve of cisplatin in the ternary composite nanogel prepared in Example 1 is shown. This demonstrates that the ternary composite nanogel has a sustained-release effect of cisplatin.

[0075] Figure 6 The effect of the ternary composite nanogel prepared in Example 1 on the percentage of mononuclear cells in mouse blood is shown, demonstrating that the ternary composite nanogel can improve the blood toxicity of LLoMe dipeptide molecules.

[0076] Figure 7 The co-localization of the ternary composite nanogel prepared in Example 1 in HeLa cell lysosomes is shown.

[0077] Figure 8 The targeted uptake of the ternary composite nanogel prepared in Example 1 in HeLa cells is shown.

[0078] Figure 9 The cytotoxicity evaluation of the ternary composite nanogel prepared in Example 1 in HeLa cells is shown.

[0079] Figure 10 The image shows an immunofluorescence staining pattern of the ternary composite nanogel *Galectin-3 prepared in Example 1, indicating that the ternary composite nanogel can cause lysosomal damage.

[0080] Figure 11 The immunofluorescence staining image of the ternary composite nanogel*cisplatin-DNA conjugate prepared in Example 1 is shown, indicating that the ternary composite nanogel can promote lysosomal escape of cisplatin.

[0081] Figure 12 The diagram shows a comparison of the average fluorescence intensity of CRT in HeLa cells after treatment with the ternary composite nanogel prepared in Example 1, HC nanogel, free cisplatin, LLoMe, and free cisplatin + LLoMe. Compared with HC nanogel, the ternary composite nanogel of this invention can induce an increase in the CRT of calreticulin, an immunogenic cell death marker, in HeLa cells.

[0082] Figure 13 The comparison of blood circulation time of the ternary composite nanogel prepared in Example 1 with HC nanogel and free cisplatin + LLoMe shows that the ternary composite nanogel has a longer blood circulation time than free cisplatin.

[0083] Figure 14 The tumor-inhibiting effect of the ternary composite nanogel prepared in Example 1 on the U14 mouse subcutaneous tumor model is shown.

[0084] Figure 15The comparison of immunofluorescence staining patterns of cisplatin-DNA conjugates at the tumor site in U14 subcutaneous tumor mice after treatment with ternary composite nanogel prepared in Example 1, HC nanogel, free cisplatin, LLoMe, and free cisplatin + LLoMe is shown, illustrating that the ternary composite nanogel induces lysosomal damage in tumor cells at the in vivo level.

[0085] Figure 16 The diagram shows the changes in CD4 and CD8 positive cells induced by the ternary composite nanogel prepared in Example 1 in U14 mouse tumors, indicating that the ternary composite nanogel can induce stronger anti-tumor immunity.

[0086] Figure 17 The diagram shows a comparison of the ternary composite nanogel prepared in Example 1 with HC nanogel and the treatment groups of free cisplatin, LLoMe, and free cisplatin + LLoMe. Compared with free cisplatin, the ternary composite nanogel of the present invention has less cisplatin-DNA conjugate formation in the kidney and lower nephrotoxicity.

[0087] Figure 18 Transmission electron microscopy images of the ternary composite nanogel prepared in Example 2 are shown.

[0088] Figure 19 The image shown is a scanning electron microscope image of the ternary composite nanogel prepared in Example 2.

[0089] Figure 20 The elemental distribution of the ternary composite nanogel prepared in Example 2 is shown, demonstrating that the ternary composite nanogel can achieve co-loading of cisplatin and arsenic trioxide.

[0090] Figure 21 Transmission electron microscopy images of the ternary composite nanogel prepared in Example 3 are shown.

[0091] Figure 22 The UV-Vis absorption spectrum of the ternary composite nanogel prepared in Example 3 is shown. The characteristic absorption of pabisostat indicates drug loading in the ternary composite nanogel.

[0092] Figure 23 The drug release curve of the ternary composite nanogel prepared in Example 3 is shown, indicating that both cisplatin and pabisostat in the ternary composite nanogel can achieve sustained release.

[0093] Figure 24 The hydrated particle size of the ternary composite nanogel prepared in Example 4 is shown.

[0094] Figure 25 The zeta potential of the ternary composite nanogel prepared in Example 4 is shown.

[0095] Figure 26 The ternary composite nanogel prepared in Example 4 is shown in comparison with the HC nanogel, which exhibits magnetic resonance T1 contrast signal of gadolinium.

[0096] Figure 27 The hydrated particle size distribution of the ternary composite nanogel prepared in Example 5 is shown.

[0097] Figure 28 The Galectin-3 targeting ability of the ternary composite nanogel prepared in Example 5 is shown. In HeLa cells, the cellular uptake of the ternary composite nanogel increased significantly after the extracellular addition of recombinant human Galectin-3 protein.

[0098] Figure 29 A transmission electron microscope image of the ternary composite nanogel prepared in Example 6 is shown.

[0099] Figure 30 The co-localization of lysosomes in 4T1 cells is shown in the ternary composite nanogel prepared in Example 6.

[0100] Figure 31 The concentration-dependent toxicity of the ternary composite nanogel prepared in Example 6 in 4T1 cells is shown.

[0101] Figure 32 The tumor-suppressive effect of the ternary composite nanogel prepared in Example 6 in the 4T1 mouse tumor model is shown. Compared with free cisplatin and parzopanib, the ternary composite nanogel exhibits better tumor inhibition. Detailed Implementation

[0102] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0103] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0104] The reagents and instruments used in the following examples are as follows:

[0105] Reagents:

[0106] Cisplatin and arsenic trioxide were purchased from Sigma-Aldrich; hyaluronic acid was purchased from Bloomage Biotech; LLoMe, praxaviva, futtatinib, GSK-J1, and ribociclib were purchased from Cayman; astragalus polysaccharide, pabisostat, parzopanib, and gadolinium chloride were purchased from Aladdin; CRT antibody was purchased from Santa Cuz; Cis-DNA antibody, CD4, and CD8 were purchased from Abcam; U14, HeLa, and 4T1 cells were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; and mice were purchased from Vital Rivers.

[0107] instrument:

[0108] Transmission electron microscope (JEOL, JEM 2100F); Scanning electron microscope (ZEISS, Gemini SEM 300); UV-Vis spectrophotometer (Hitachi, UH5300); Fluorescence microscope (Thermo Fisher Scientific, EVOS M7000); Small animal in vivo imaging system (Xenogen, IVIS); Flow cytometer (BD, Accuri C6); Dynamic light scattering system (Malvin, Nano ZS90); Inductively coupled plasma mass spectrometer (Agilent, 7800(MS)).

[0109] Example 1

[0110] This embodiment is an exemplary description of the ternary composite nanogel and its preparation method of the present invention.

[0111] The small molecule drug used in this embodiment is the dipeptide molecule L-leucyl-L-leucine methyl ester (LLoMe), and the polysaccharide is hyaluronic acid (molecular weight 10kDa-1000kDa). This embodiment takes hyaluronic acid with a molecular weight of 1000kDa as an example.

[0112] (1) Weigh 100mg of hyaluronic acid and add double-distilled water to prepare an aqueous solution of hyaluronic acid with a concentration of 5mg / ml.

[0113] (2) At 85±5℃, a cisplatin aqueous solution with a concentration of 10mg / mL was heated until the cisplatin dissolved.

[0114] (3) Weigh 100 mg of LLoMe dipeptide and dissolve it in an aqueous solution to obtain an LLoMe dipeptide aqueous solution with a concentration of 20 mg / mL.

[0115] (4) Mix 300 μL of cisplatin solution with 300 μL of LLoMe dipeptide aqueous solution at a mass ratio of 1:2, heat for 2 ± 0.5 minutes to obtain a small molecule cisplatin drug mixed solution, then add 800 μL of hyaluronic acid solution and mix quickly, and incubate at 85 ± 5℃ for 4 hours.

[0116] (5) Cool on ice for 10 minutes.

[0117] (6) Transfer the product to a dialysis bag and dialyze it in double-distilled water for 24 hours to remove free drug molecules.

[0118] (7) Store the obtained hyaluronic acid nanogel at 4°C for later use.

[0119] In the ternary composite nanogel prepared in this embodiment, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 4:1:2.

[0120] The hydration particle size, electron micrograph, and 1H NMR spectrum of the ternary composite nanogel prepared in this embodiment are as follows: Figure 2-4 As shown, this demonstrates the co-loading of cisplatin and LLoMe dipeptide molecules in the ternary composite nanogel.

[0121] Example 2

[0122] This embodiment is another exemplary illustration of the ternary composite nanogel and its preparation method of the present invention.

[0123] The drug used in this embodiment is arsenic trioxide, and the polysaccharide is hyaluronic acid (molecular weight 10kDa-1000kDa). This embodiment takes hyaluronic acid with a molecular weight of 200kDa as an example.

[0124] (1) Weigh 100mg of hyaluronic acid and add double-distilled water to prepare an aqueous solution of hyaluronic acid with a concentration of 5mg / ml.

[0125] (2) Heat a cisplatin aqueous solution with a concentration of 10 mg / mL at 80±5℃ until the cisplatin dissolves.

[0126] (3) Weigh 50 mg of arsenic trioxide, dissolve the arsenic trioxide in an aqueous solution, heat at 80±5℃ until dissolved, and obtain an aqueous solution of arsenic trioxide with a concentration of 8 mg / mL.

[0127] (4) Mix 200 μL of cisplatin solution with 150 μL of arsenic trioxide at a mass ratio of 10:6, heat for 2 minutes to obtain a small molecule cisplatin drug solution, then add 800 μL of hyaluronic acid solution and mix quickly, and incubate at 80±5℃ for 3 hours.

[0128] (5) Cool on ice for 5 minutes.

[0129] (6) Transfer the product to a dialysis bag and dialyze it in double-distilled water for 24 hours to remove free drug molecules.

[0130] (7) Store the obtained hyaluronic acid nanogel at 4°C for later use.

[0131] In the ternary composite nanogel prepared in this embodiment, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 10:5:3.

[0132] Transmission electron microscopy images of the ternary composite nanogels prepared in this embodiment are shown below. Figure 18 As shown, the scanning electron microscope image is as follows: Figure 19 As shown. Figure 20 As shown, this demonstrates that ternary composite nanogels can achieve co-loading of cisplatin and arsenic trioxide.

[0133] Example 3

[0134] This embodiment is yet another exemplary illustration of the ternary composite nanogel and its preparation method of the present invention.

[0135] The drug molecule used in this embodiment is parbistat, and the polysaccharide is hyaluronic acid (molecular weight 10kDa-1000kDa). This embodiment takes hyaluronic acid with a molecular weight of 100kDa as an example.

[0136] (1) Weigh 50mg of hyaluronic acid and add double-distilled water to prepare an aqueous solution of hyaluronic acid with a concentration of 5mg / ml.

[0137] (2) At 85±5℃, a cisplatin aqueous solution with a concentration of 10mg / mL was heated until the cisplatin dissolved.

[0138] (3) Weigh 20 mg of Pabistat and dissolve it in DMF to obtain a Pabistat solution with a concentration of 10 mg / mL.

[0139] (4) Mix 200 μL of cisplatin solution with 150 μL of pabistat solution at a mass ratio of 4:3, heat for 3 ± 0.5 minutes to obtain a small molecule cisplatin drug solution, then add 800 μL of hyaluronic acid solution and mix quickly, and incubate at 85 ± 5 °C for 3 hours.

[0140] (5) Cool on ice for 5 minutes.

[0141] (6) Transfer the product to a dialysis bag and dialyze it in double-distilled water for 24 hours to remove free drug molecules.

[0142] (7) Store the obtained hyaluronic acid nanogel HCP at 4℃ for later use.

[0143] In the ternary composite nanogel prepared in this embodiment, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 8:4:3.

[0144] Electron micrographs, UV-Vis absorption spectra, and drug release curves of the ternary composite nanogel prepared in this embodiment are shown below. Figure 21-23 As shown, this demonstrates the co-loading and sustained drug release of cisplatin and pabisostat in the ternary composite nanogel.

[0145] Example 4

[0146] This embodiment is yet another exemplary illustration of the ternary composite nanogel and its preparation method of the present invention.

[0147] The small molecule drug used in this embodiment is gadolinium chloride, a contrast agent that can be used for magnetic resonance imaging, and the polysaccharide is hyaluronic acid (molecular weight 10kDa-1000kDa). This embodiment takes hyaluronic acid with a molecular weight of 200kDa as an example.

[0148] (1) Weigh 50mg of hyaluronic acid and add double-distilled water to prepare an aqueous solution of hyaluronic acid with a concentration of 5mg / ml.

[0149] (2) At 90±5℃, a cisplatin aqueous solution with a concentration of 15mg / mL was heated until the cisplatin dissolved.

[0150] (3) Weigh 20 mg of gadolinium chloride and dissolve it in water to obtain a gadolinium chloride solution with a concentration of 10 mg / mL.

[0151] (4) Mix 250 μL of cisplatin solution with 40 μL of gadolinium chloride solution at a mass ratio of 15:1.6, heat for 3 ± 0.5 minutes to obtain a small molecule cisplatin drug solution, then add 800 μL of hyaluronic acid solution and mix quickly, and incubate at 90 ± 5 °C for 6 hours.

[0152] (5) Cool on ice for 10 minutes.

[0153] (6) Transfer the product to a dialysis bag and dialyze it in double-distilled water for 24 hours to remove free drug molecules.

[0154] (7) Store the obtained hyaluronic acid nanogel at 4°C for later use.

[0155] In the ternary composite nanogel prepared in this embodiment, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 16:15:1.6.

[0156] The hydration particle size, Zeta potential, and T1 magnetic resonance imaging of the ternary composite nanogel prepared in this embodiment were analyzed. Figure 24-26 As shown, the addition of gadolinium to the ternary composite nanogel demonstrates that magnetic resonance T1 imaging capability was obtained.

[0157] Example 5

[0158] This embodiment is yet another exemplary illustration of the ternary composite nanogel and its preparation method of the present invention.

[0159] The small molecule drug used in this embodiment is Ribociclib, and the polysaccharide is small molecule citrus pectin (molecular weight 10kDa-200kDa). This embodiment takes small molecule citrus pectin with a molecular weight of 20kDa as an example.

[0160] (1) Weigh 50mg of small molecule citrus pectin and add it to double-distilled water to prepare a hyaluronic acid aqueous solution with a concentration of 2.5mg / ml.

[0161] (2) At 80±5℃, heat an aqueous solution of cisplatin with a concentration of 15mg / mL until the cisplatin dissolves.

[0162] (3) Weigh 10 mg of Ribociclib and dissolve it in DMF to obtain a Ribociclib solution with a concentration of 10 mg / mL.

[0163] (4) Mix 100 μL of cisplatin solution with 100 μL of parzopanib at a mass ratio of 3:2, heat for 4 ± 0.5 minutes to obtain a small molecule cisplatin drug mixed solution, then add 400 μL of small molecule citrus pectin and mix quickly, and incubate at 80 ± 5℃ for 1 hour.

[0164] (5) Cool on ice for 10 minutes.

[0165] (6) Transfer the product to a dialysis bag and dialyze it in double-distilled water for 24 hours to remove free drug molecules.

[0166] (7) Store the obtained small molecule citrus pectin nanogel at 4°C for later use.

[0167] In the ternary composite nanogel prepared in this embodiment, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 4:6:4.

[0168] The hydration particle size and cellular uptake of the ternary composite nanogel prepared in this embodiment are as follows. Figure 27-28 As shown, this demonstrates the successful synthesis of the ternary composite nanogel and its Galectin-3 targeting ability in HeLa cells.

[0169] Example 6

[0170] This embodiment is yet another exemplary illustration of the ternary composite nanogel and its preparation method of the present invention.

[0171] The small molecule drug used in this embodiment is parzopanib, and the polysaccharide is astragalus polysaccharide (molecular weight 20kDa-100kDa). This embodiment uses astragalus polysaccharide with a molecular weight of 40kDa as an example.

[0172] (1) Weigh 50mg of Astragalus polysaccharide and add it to double-distilled water to prepare a hyaluronic acid aqueous solution with a concentration of 5mg / ml.

[0173] (2) Heat a cisplatin aqueous solution with a concentration of 10 mg / mL at 90±5℃ until the cisplatin dissolves.

[0174] (3) Weigh 10 mg of parzopanib and dissolve it in dimethyl sulfoxide to obtain a parzopanib solution with a concentration of 10 mg / mL.

[0175] (4) Mix 200 μL of cisplatin solution with 150 μL of parzopanib at a mass ratio of 4:3, heat for 2 ± 0.5 minutes to obtain a small molecule cisplatin drug solution, then add 800 μL of astragalus polysaccharide solution and mix quickly, and incubate at 90 ± 5 °C for 6 hours.

[0176] (5) Cool on ice for 10 minutes.

[0177] (6) Transfer the product to a dialysis bag and dialyze it in double-distilled water for 24 hours to remove free drug molecules.

[0178] (7) Store the obtained Astragalus polysaccharide nanogel at 4℃ for later use.

[0179] In the ternary composite nanogel prepared in this embodiment, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 8:4:3.

[0180] Transmission electron microscopy images of the ternary composite nanogel prepared in this embodiment, lysosomal localization in cells, concentration-dependent cytotoxicity, and in vivo tumor suppression experiments. Figures 29-32 demonstrate the synthesis and antitumor effects of the ternary composite nanogel.

[0181] Examples 7-22

[0182] Examples 7-22 are other exemplary descriptions of the ternary composite nanogel and its preparation method of the present invention.

[0183] The ternary composite nanogels prepared in Examples 7-22 were prepared in the same way as those in Example 1, except for the selection of the contents listed in Table 1 below.

[0184] Table 1. Ternary composite nanogels prepared in Examples 7-22

[0185]

[0186]

[0187] Experimental Example 1

[0188] This experimental example illustrates the drug release effect of the ternary composite nanogel of the present invention.

[0189] The following experiments were conducted using the ternary composite nanogels prepared in Examples 1 and 3.

[0190] (1) Take 1 mL of the ternary composite nanogel obtained in Examples 1 and 3 and seal it in a dialysis bag.

[0191] (2) Soak the dialysis bag in 60 mL of phosphate buffer (pH 7.4) and incubate at 37 °C.

[0192] (3) Take 1 mL of the solution outside the dialysis bag at a fixed time point and place it at 4℃ for later use. At the same time, add an equal volume of phosphate buffer to the outside of the dialysis bag.

[0193] (4) The absorbance of Pabistat at 279 nm was used to quantify the Pabistat released in the collected solution and plot its sustained-release curve.

[0194] (5) Inductively coupled plasma mass spectrometry was used to monitor and collect the cisplatin released from the solution and plot the sustained-release curve.

[0195] like Figure 5 As shown, the drug release curves of cisplatin and dipeptide molecules in the ternary composite nanogel prepared in Example 1 demonstrate that the ternary composite nanogel has a sustained-release effect of cisplatin.

[0196] like Figure 23 As shown, both cisplatin and pabisostat in the HCP nanogel prepared in Example 3 exhibit sustained-release effects, indicating that the prepared ternary composite nanogel has a sustained-release effect of cisplatin.

[0197] The ternary composite nanogels prepared in Examples 2, 4-22 also contain cisplatin chelate products. Under the condition of chloride ions, the cisplatin chelation can be reversed, and they also have the drug sustained-release effect of cisplatin.

[0198] Experimental Example 2

[0199] This experimental example illustrates the blood toxicity effect of the ternary composite nanogel of the present invention.

[0200] The following experiments were conducted using the ternary composite nanogel prepared in Example 1.

[0201] (1) Mice were injected with HCL nanogel and LLoMe solution via tail vein, with LLoMe dose being 12 mg / kg.

[0202] (2) After 10 min, 30 min and 60 min of injection, cut off about 2 mm of the tail tip of the mouse, collect 10 μL of peripheral blood using a capillary stenography needle, and add 240 μL of pre-diluted solution containing EDTA.

[0203] (3) After thoroughly mixing the above blood samples, use a blood analyzer for testing.

[0204] like Figure 6 As shown in Example 1, after treatment with HCl nanogel, the proportion of mononuclear cells in peripheral blood fluctuated less, while LLoMe treatment caused a significant increase in the proportion of mononuclear cells, proving that the ternary composite nanogel can improve the blood toxicity caused by LLoMe directly contacting blood cells.

[0205] The ternary composite nanogels prepared in Examples 2-22, because they also contain nanogel structures, can prevent the loaded drugs from directly contacting blood cells and can also improve the blood toxicity of the loaded drugs.

[0206] Experimental Example 3

[0207] This experimental example illustrates the co-localization and targeted uptake effect of the ternary composite nanogel of the present invention in HeLa cell lysosomes.

[0208] The following experiments were conducted using the ternary composite nanogel prepared in Example 1.

[0209] (1) After HeLa cells were seeded in culture chambers and adhered overnight, they were co-incubated with green fluorescent dextran for 12 hours to label lysosomes.

[0210] (2) The cells were further co-incubated with Cy5 fluorescently labeled HCl nanogel for 48 hours.

[0211] (3) After washing the cells with PBS, stain them with nuclear labeling dye for 2 minutes, and then transfer them to phenol red-free medium for fluorescence microscopy observation.

[0212] (4) To observe the competitive binding of HCl nanogel to hyaluronic acid, HeLa cells were co-incubated with Cy5-labeled HCl nanogel or Cy5-labeled HCl nanogel with 1 mg / mL free HA after adhesion. After 10, 30 and 60 minutes, the old culture medium was discarded, the cells were collected, and the intracellular Cy5 fluorescence was measured by flow cytometry.

[0213] like Figure 7As shown, the ternary composite nanogel prepared in Example 1 can be localized to lysosomes in HeLa cells. Meanwhile, as... Figure 8 As shown, the ternary composite nanogel HCL prepared in Example 1 retains the targeting ability of HA during cell uptake, that is, it has CD44 targeted delivery.

[0214] The ternary composite nanogels prepared in Examples 2-22, also containing polysaccharides, can be localized to lysosomes in cells based on the characteristics of polysaccharide metabolism in intracellular lysosomes. Furthermore, based on the biological functions of the polysaccharides in the ternary composite nanogels, they exhibit tumor-targeting drug delivery effects, such as targeted delivery of hyaluronic acid to CD44, targeted delivery of small molecule pectin to Galectin-3, and targeted delivery of dextran to tumor-associated macrophages.

[0215] Test Example 4

[0216] This experimental example illustrates the cytotoxicity evaluation of the ternary composite nanogel of the present invention.

[0217] The following experiments were conducted using the ternary composite nanogel prepared in Example 1.

[0218] (1) HeLa cells were seeded at 8000 cells / well in 96-well plates.

[0219] (2) Cells were co-incubated with different concentrations of cisplatin, HC nanogel and HCl nanogel for 48 hours.

[0220] (3) After washing the cells once with PBS, cell viability was detected using a CCK-8 assay kit, such as... Figure 9 As shown, the ternary composite nanogel HCl prepared in Example 1 exhibits significant concentration-dependent cytotoxicity against HeLa cells, and its cytotoxicity is stronger than that of HC nanogel.

[0221] The ternary composite nanogels prepared in Examples 2-22 also contain cisplatin. As cisplatin and small molecules that can chelate with cisplatin are released from the ternary composite nanogels, they also exhibit significant concentration-dependent cytotoxicity to HeLa cells, and their cytotoxicity is stronger than that of HC nanogels.

[0222] Experimental Example 5

[0223] This experimental example illustrates the effect of the ternary composite nanogel of the present invention on cell lysosome damage.

[0224] The following experiments were conducted using the ternary composite nanogel prepared in Example 1.

[0225] (1) HeLa cells were seeded in culture chambers and cultured overnight.

[0226] (2) HCl nanogel, HC nanogel, cisplatin, LLoMe and cisplatin + LLoMe mixture were added to the cells respectively.

[0227] (3) After co-culturing the cells for 24 hours, they were fixed with paraformaldehyde and labeled with Galectin-3 protein by immunofluorescence staining.

[0228] like Figure 10 As shown, the ternary composite nanogel HCl prepared in Example 1 can significantly enhance the bright spot signal of Galectin-3 protein in HeLa cells, indicating that it can induce lysosomal damage more effectively than HC nanogel.

[0229] The ternary composite nanogels prepared in Examples 2-22, because they also contain cisplatin and small molecules that can chelate with cisplatin, can mediate drug accumulation in lysosomes and can also cause lysosomal damage. Depending on the structural differences of the small molecules that can chelate with cisplatin, they can cause varying degrees of lysosomal damage.

[0230] Experimental Example 6

[0231] This experimental example illustrates the effect of cisplatin lysosomal escape in the ternary composite nanogel of the present invention.

[0232] The following experiments were conducted using the ternary composite nanogel prepared in Example 1.

[0233] (1) HeLa cells were seeded in culture chambers and cultured overnight.

[0234] (2) HCl nanogel, HC nanogel, cisplatin, LLoMe and cisplatin + LLoMe mixture were added to the cells respectively.

[0235] (3) After co-culturing the cells for 24 hours, they were fixed with paraformaldehyde and labeled with immunofluorescence staining for cisplatin-DNA conjugates, such as... Figure 11 As shown, the ternary composite nanogel HCl prepared in Example 1 can induce the formation of more cisplatin-DNA conjugates than HC nanogel, indicating that the loading of LLoMe in HCl nanogel can promote the lysosomal escape of cisplatin.

[0236] The ternary composite nanogels prepared in Examples 2-22, also containing cisplatin and small molecules that can chelate with cisplatin, also induce the formation of cisplatin-DNA conjugates upon the release of cisplatin, indicating that the loading of LLoMe in HCl nanogels can promote lysosomal escape of cisplatin. Depending on the structural differences of the small molecules that can chelate with cisplatin, varying degrees of lysosomal escape of cisplatin can be induced.

[0237] Experimental Example 7

[0238] This experimental example illustrates the effect of the ternary composite nanogel of the present invention in inducing immunogenic cell death, and compares its effect with that of HC nanogel, cisplatin monomer, dipeptide monomer, cisplatin and dipeptide.

[0239] This experimental example uses the ternary composite nanogel, HC nanogel, cisplatin monomer, dipeptide monomer, cisplatin and dipeptide molecule prepared in Example 1 to conduct the following experiments.

[0240] (1) HeLa cells were seeded in 6-well plates and cultured overnight.

[0241] (2) HCl nanogel, HC nanogel, cisplatin, LLoMe and cisplatin + LLoMe mixture were added to the cells respectively.

[0242] (3) After co-culturing the cells for 24 hours, the cells were collected and labeled with fluorescently labeled CRT antibody by flow cytometry. The average fluorescence intensity of the cells was detected by flow cytometer.

[0243] like Figure 12 As shown, compared with HC nanogel, cisplatin monomer, dipeptide monomer, cisplatin and dipeptide, the ternary composite nanogel HCl prepared in Example 1 can significantly induce CRT expression, indicating that HCl nanogel is more effective in inducing cell immunogenic death than HC nanogel.

[0244] The ternary composite nanogels prepared in Examples 2-22, because they also contain cisplatin and small molecules that can chelate with cisplatin, can also induce immunogenic cell death. Depending on the structural differences of the small molecules that can chelate with cisplatin, they can induce varying degrees of immunogenic cell death.

[0245] Experimental Example 8

[0246] This experimental example illustrates the blood circulation time of the ternary composite nanogel of the present invention, and compares its effect with that of HC nanogel and free cisplatin + LLoMe mixed solution.

[0247] This experimental example uses the ternary composite nanogel, HC nanogel, and free cisplatin + LLoMe mixed solution prepared in Example 1 to conduct the following experiments.

[0248] (1) Balb / c mice (7 weeks old) were injected via tail vein with HCl nanogel, HC nanogel and cisplatin + LLoMe mixture, respectively.

[0249] (2) 10 μL of blood was drawn from the tail tip at 2 minutes, 10 minutes, 30 minutes, 1 hour, 4 hours, 8 hours and 24 hours after injection.

[0250] (3) After acid hydrolysis, the platinum content in the blood samples was detected by inductively coupled plasma mass spectrometry, and the pharmacokinetic curve of cisplatin was plotted. Figure 13 As shown, compared with HC nanogel and free cisplatin + LLoMe mixed solution, the ternary composite nanogel prepared in Example 1 has a longer blood drug circulation time.

[0251] Table 2 Comparison of blood circulation times of free cisplatin, HC nanogel, cisplatin and dipeptide molecules with the ternary composite nanogels prepared in Examples 2-6 of this invention.

[0252]

[0253]

[0254] The ternary composite nanogels prepared in Examples 7-22, being polysaccharide nanogel structures, exhibit similar size effects and also have longer blood drug circulation times.

[0255] Experimental Example 9

[0256] This experimental example illustrates the tumor-suppressing effect of the functionalized nanogel of the present invention and compares its effect with that of HC nanogel and cisplatin + LLoMe mixture.

[0257] The functionalized nanogel prepared in Example 1 was used to conduct the following experiments in this experimental example.

[0258] (1) Balb / c mice (6 weeks old) were injected subcutaneously with 106 U14 cells. Once the tumor volume reached about 100 mm3, drug treatment was started.

[0259] (2) After the tumor-bearing mice were grouped, they were injected with HCL nanogel, HC nanogel and cisplatin + LLoMe mixture via the tail vein.

[0260] (3) Monitor the tumor volume in mice in real time and plot the tumor volume as a tumor growth curve, such as Figure 14 As shown, the functionalized nanogel HCl prepared in Example 1 has a better tumor inhibition effect compared with HC nanogel and cisplatin + LLoMe mixed solution.

[0261] The ternary composite nanogels prepared in Examples 2-22, which also contain cisplatin and small molecules that can chelate with cisplatin, exhibit better tumor inhibition effects than HC nanogels due to the co-delivery of the two drug molecules.

[0262] Experimental Example 10

[0263] This experimental example illustrates the lysosomal damage effect of the ternary composite nanogel of the present invention in tumor tissue, and compares its effect with that of HC nanogel and cisplatin + LLoMe mixture.

[0264] This experimental example uses the ternary composite nanogel, HC nanogel, and cisplatin + LLoMe mixture prepared in Example 1 to conduct the following experiments.

[0265] (1) After the mouse tumor suppression experiment, the tumors of mice in the 10mg / kg dose treatment group were obtained.

[0266] (2) Tumor tissue was fixed with paraformaldehyde.

[0267] (3) Immunofluorescence staining was used to label the cisplatin-DNA conjugate, and the results were observed under a fluorescence microscope, such as... Figure 15 As shown, compared with HC nanogel and cisplatin + LLoMe mixture, the ternary composite nanogel HCl prepared in Example 1 can induce more cisplatin-DNA conjugate staining signals in tumor tissue than HC nanogel, indicating that HCl nanogel can promote the lysosomal escape of delivered cisplatin and its binding to DNA in tumor cells in vivo.

[0268] The ternary composite nanogels prepared in Examples 2-22, due to their similar passive tumor-targeting effect and polysaccharide-targeted delivery effect, can also promote the accumulation of delivered cisplatin at the tumor site in in vivo tumor cells. Based on the structural differences in the small molecules that can chelate with cisplatin, they can induce varying degrees of lysosomal escape and DNA binding.

[0269] Experimental Example 11

[0270] This experimental example illustrates the effect of the ternary composite nanogel of the present invention in activating anti-tumor immunity, and compares its effect with that of HC nanogel and cisplatin + LLoMe mixture.

[0271] This experimental example uses the ternary composite nanogel, HC nanogel, and cisplatin + LLoMe mixture prepared in Example 1 to conduct the following experiments.

[0272] (1) After the tumor suppression experiment, the tumors of mice in the 10mg / kg dose treatment group were obtained.

[0273] (2) Tumor tissue was fixed with paraformaldehyde.

[0274] (3) Immunofluorescence staining was used to label CD4 and CD8 and then observed under a fluorescence microscope.

[0275] like Figure 16 As shown, compared with HC nanogel and cisplatin + LLoMe mixture, the ternary composite nanogel HCl prepared in Example 1 can increase the number of CD4 and CD8 positive T cells more than HC nanogel and cisplatin + LLoMe mixture, indicating that the immunogenic death induced by HCl nanogel can activate anti-tumor immunity.

[0276] The ternary composite nanogels prepared in Examples 2-22, which also contain cisplatin and small molecules that can chelate with cisplatin, can also activate anti-tumor immunity by inducing lysosomal damage.

[0277] Experimental Example 12

[0278] This experimental example illustrates the effect of the ternary composite nanogel of the present invention in reducing nephrotoxicity, and compares its effect with that of HC nanogel and cisplatin + LLoMe mixture.

[0279] This experimental example uses the ternary composite nanogel, HC nanogel, and cisplatin + LLoMe mixture prepared in Example 1 to conduct the following experiments.

[0280] (1) After the tumor suppression experiment, the tumors of mice in the 10mg / kg dose treatment group were obtained.

[0281] (2) Tumor tissue was fixed with paraformaldehyde.

[0282] (3) Immunofluorescence staining was used to label CD4 and CD8, and the results were observed under a fluorescence microscope. Figure 17 As shown, compared with HC nanogel and cisplatin + LLoMe mixture, the ternary composite nanogel HCL prepared in Example 1 had less accumulation of cisplatin-DNA conjugates in the kidneys than the cisplatin + LLoMe mixture group, indicating that HCL nanogel can reduce nephrotoxicity.

[0283] The ternary composite nanogels prepared in Examples 2-22 also contain polysaccharide nanogel structures. Through the size effect of nanoparticles, they reduce the accumulation of cisplatin in the kidneys and also have less accumulation of cisplatin-DNA conjugates in the kidneys, which can reduce nephrotoxicity.

[0284] While the effects of some embodiments have been shown above, those skilled in the art should understand that, based on the concept of the invention, other embodiments not specifically shown or other technical solutions of the invention not shown in the embodiments can also achieve the same technical effects as those claimed in the summary section:

[0285] 1. The preparation method of this invention can synthesize nanogels containing both cisplatin and small molecules in one step, and the hydrated particle size can be controlled by adjusting the heating time. The entire synthesis process is simple and rapid, with mild conditions that are easy to scale up, and can improve the water solubility of small molecules.

[0286] 2. Compared with HC nanogels containing cisplatin alone, the ternary composite nanogel of the present invention can promote cisplatin delivery and carry out combined drug therapy, thus having a better tumor treatment effect.

[0287] 3. Compared with free drugs, the ternary composite nanogel of the present invention can reduce their toxic side effects.

[0288] 4. Since hyaluronic acid can be recognized by its receptor CD44, the ternary composite nanogel of the present invention has the CD44 targeting function, which can mediate the immunogenic death of tumor cells and be used to achieve tumor targeted delivery and combination therapy.

[0289] Although the invention has been described to a certain extent, it is apparent that appropriate variations can be made to the various conditions without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent substitutions for each of the elements.

Claims

1. A ternary composite nanogel with a polysaccharide-cisplatin-drug small molecule chelate structure, characterized in that, The ternary composite nanogel comprises: Polysaccharides, Cisplatin, and Small drug molecules chelated with cisplatin; among them, The polysaccharide is selected from one or more of the following: acidic polysaccharides, basic polysaccharides, traditional Chinese medicine polysaccharides, and marine polysaccharides, preferably selected from one or more of the following: acidic polysaccharides, basic polysaccharides, and traditional Chinese medicine polysaccharides; The drug molecule contains one or more elements selected from the following: phosphorus, nitrogen, sulfur, arsenic, gadolinium, preferably containing one or more elements selected from the following: phosphorus, nitrogen, sulfur, arsenic, and most preferably containing: phosphorus, nitrogen, sulfur and / or arsenic.

2. The ternary composite nanogel according to claim 1, characterized in that, The drug molecule chelates with cisplatin through non-covalent interactions, and then co-assembles with the polysaccharide to form the ternary composite nanogel.

3. The ternary composite nanogel according to claim 1 or 2, characterized in that, The nitrogen-containing drug molecule is selected from one or more of the following: L-leucyl-L-leucine methyl ester, pabisostat, lapatinib, parzopanib, afatinib, olaparib, ibrutinib, praxavir, GSK-J1, Ribociclib, preferably selected from one or more of the following: L-leucyl-L-leucine methyl ester, pabisostat, parzopanib, afatinib, praxavir, more preferably selected from one or more of the following: L-leucyl-L-leucine methyl ester, parzopanib, afatinib, pabisostat; The arsenic-containing drug molecule is selected from one or more of the following: arsenic trioxide, arsanoic acid, potassium arsenide, preferably arsenic trioxide and / or arsanoic acid, and most preferably arsenic trioxide; The phosphorus-containing drug molecule is selected from one or more of the following: flotinib, zoledronic acid, tilmidronic acid, minodronic acid, risedronic acid, thiamine phosphate, preferably selected from one or more of the following: flotinib, zoledronic acid, tilmidronic acid, minodronic acid, risedronic acid, more preferably flotinib and / or zoledronic acid; The sulfur-containing drug molecule is selected from one or more of the following: sulfadiazine, azathioprine, chlorothiazide, hydrochlorothiazide, preferably selected from one or more of the following: sulfadiazine, azathioprine, chlorothiazide, more preferably sulfadiazine and / or azathioprine; and / or The gadolinium-containing drug molecule is gadolinium chloride and / or gadolinium nitrate, with gadolinium chloride being the most preferred.

4. The ternary composite nanogel according to any one of claims 1 to 3, characterized in that, The polysaccharide is a non-neutral polysaccharide and contains one or more groups selected from the following: carboxyl, sulfonic acid, carboxymethyl, amino, preferably containing one or more groups selected from the following: carboxyl, sulfonic acid, amino.

5. The ternary composite nanogel according to claim 4, characterized in that, The polysaccharide is selected from one or more of the following: hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, chitosan, citrus pectin, astragalus polysaccharide, tremella polysaccharide, poria polysaccharide, and ginseng polysaccharide. It is more preferably selected from one or more of the following: hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, chitosan, citrus pectin, astragalus polysaccharide, tremella polysaccharide, and poria polysaccharide. It is more preferably selected from one or more of the following: hyaluronic acid, heparin precursor, heparan sulfate, fucoidan, chitosan, citrus pectin, astragalus polysaccharide, and tremella polysaccharide. It is even more preferably selected from one or more of the following: hyaluronic acid, citrus pectin, and astragalus polysaccharide.

6. The ternary composite nanogel according to any one of claims 1 to 5, characterized in that: In the ternary composite nanogel, the mass ratio of polysaccharide, cisplatin, and small drug molecules is 4–35:2–60:1–16, preferably 6–24:3–36:1–10, more preferably 8–20:4–24:1.2–8; and / or The hydrated particle size of the ternary composite nanogel is 40-4000 nm, preferably 50-1000 nm, and more preferably 70-550 nm.

7. A method for preparing ternary composite nanogels according to any one of claims 1 to 6, characterized in that, The method includes the following steps: 1) Prepare polysaccharide aqueous solution, cisplatin aqueous solution and drug small molecule solution respectively; 2) Mix the cisplatin aqueous solution and the drug small molecule solution prepared in step 1) and heat to obtain a cisplatin drug small molecule mixed solution. Then add the polysaccharide aqueous solution prepared in step 1) and mix well. Heat and incubate, then cool to obtain a cisplatin-drug small molecule-polysaccharide mixed solution. 3) Dialyze the cisplatin-drug small molecule-polysaccharide mixed solution prepared in step 3) to remove free drug small molecules, thereby obtaining the ternary composite nanogel. Preferably, in step 2), the solvent used for the drug small molecule solution is selected from one or more of the following: deionized water, DMF, dimethyl sulfoxide, and acetonitrile.

8. A targeted antitumor drug for combination therapy, characterized in that, The targeted anti-tumor drug includes the ternary composite nanogel according to any one of claims 1 to 6.

9. The use of the ternary composite nanogel according to any one of claims 1 to 6 in the preparation of targeted antitumor drugs for combination therapy.

10. The targeted antitumor drug according to claim 8 or the application according to claim 9, characterized in that: The tumor is selected from one or more of the following: gallbladder cancer, liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, thyroid cancer, non-small cell lung cancer, bladder cancer, squamous cell carcinoma of the head and neck, cervical cancer, appendicitis, skin cancer, kidney cancer, myeloma, and lymphoma. Preferred cancers include one or more of the following: liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, thyroid cancer, non-small cell lung cancer, bladder cancer, squamous cell carcinoma of the head and neck, and cervical cancer. More preferably, it is selected from one or more of the following: liver cancer, lung cancer, stomach cancer, intestinal cancer, breast cancer, colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, thyroid cancer, and cervical cancer.

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