A chitosan derivative, a preparation method and application thereof

Abstract

The present application relates to a kind of chitosan derivatives, its preparation method and application, belong to biological medical polymer material technical field.The method includes under the action of first catalyst, N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan is dissolved in first solvent, second solvent containing n-hexanoic anhydride is added to the first solvent containing N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan and is reacted, obtains chitosan derivative.The preparation method of the present application is easy to obtain raw material, and reaction condition is mild;And the chitosan derivative obtained carries a large number of positive charge, can be combined to the surface of negative electric tumor cell membrane by electrostatic interaction, promotes the interaction between the hydrophobic part of chitosan derivative and cell membrane phospholipid hydrophobic chain, destroys cell membrane stability and finally causes membrane lysis to make tumor cell necrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials technology, and specifically relates to a chitosan derivative, its preparation method and application. Background Technology

[0002] According to the latest Global Cancer Report released by the World Health Organization, 19.3 million new cancer cases were diagnosed globally in 2020 (with 9.9 million deaths), and the number is projected to increase by 47% to 28.4 million by 2040. Therefore, cancer has become one of the world's major public health problems, posing a significant threat to human health. Over the past few decades, various technologies have been developed for cancer treatment, with chemotherapy remaining one of the most effective methods, especially for chemotherapy-sensitive cancers or advanced cancers that have metastasized. Chemotherapy is the primary treatment and its role is irreplaceable. However, traditional small-molecule chemotherapy drugs typically target specific sites within tumor cells. Faced with these drugs, cancer cells gradually develop drug-resistant phenotypes. Even if most tumor cells are killed, some drug-resistant cancer cells can still grow, leading to incomplete cancer treatment and recurrence. Therefore, finding broad-spectrum anti-breast cancer drugs that are less likely to induce drug resistance and possess novel anti-tumor mechanisms is particularly important.

[0003] Antimicrobial peptides (AMPs) are a collective term for a class of natural polypeptides widely found in microorganisms, animals, and plants, possessing anti-inflammatory, antibacterial, and antiviral activities. Recently, researchers have discovered that many of these peptides also exhibit good anti-breast cancer activity. This is because AMPs typically consist of dozens of amino acid residues, carrying +2 to +9 positive charges and a high proportion of hydrophobic regions. The cationic segments endow AMPs with the ability to interact electrostatically with the negatively charged tumor cell membranes. Subsequently, the hydrophobic segments of the peptide can insert into the phospholipid bilayer of the cell membrane, altering the permeability of the tumor cell membrane and ultimately inducing membrane lysis, leading to rapid necrosis of cancer cells. Normal cells, on the other hand, have fewer positive charges on their cell membrane surface, remaining essentially electroneutrally neutral, thus exhibiting lower toxicity to AMPs. Furthermore, since AMPs primarily target the phospholipid bilayer of the cell membrane, independent of specific intracellular targets, it is difficult to induce drug resistance. However, natural peptide drugs face inherent challenges in clinical applications, such as complex and cumbersome synthesis processes, high costs, easy adsorption to plasma proteins, and poor in vivo stability.

[0004] Chitosan (CS) is a natural cationic polymer that not only possesses excellent biocompatibility, biodegradability, and low immunogenicity, but also exhibits functions such as bioadhesion, antibacterial activity, and antitumor activity. Because its molecular structure contains multiple active functional groups that facilitate chemical modification, surface modification of CS can optimize its physical and chemical properties, playing a positive role in inhibiting tumor growth and prolonging survival. Summary of the Invention

[0005] To address the above-mentioned problems, this invention provides a chitosan derivative, its preparation method, and its applications.

[0006] The first objective of this invention is to provide a chitosan derivative having the structure shown in formula (I):

[0007]

[0008] Wherein, 3000≤n≤6300, 500≤x≤2600, and 540≤y≤2000.

[0009] In specific embodiments of the present invention, n is 3000, 4000, 5000 or 6000; x is 600, 750, 900, 1050 or 1200; y is 550, 650, 750, 850 or 950.

[0010] A second objective of this invention is to provide a method for preparing a chitosan derivative, comprising:

[0011] Under the action of a first catalyst, N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) is dissolved in a first solvent, and a second solvent containing hexanoic anhydride is added to the first solvent containing N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) to react and obtain a chitosan derivative with the structure of formula (I).

[0012]

[0013] Wherein, 3000≤n≤6300, 500≤x≤2600, and 540≤y≤2000.

[0014] In a specific embodiment of the present invention, the first solvent is distilled water, the first catalyst is triethylamine, pyridine or sodium carbonate; and the second solvent is methanol, dimethyl sulfoxide or N,N-dimethylformamide.

[0015] In a specific embodiment of the present invention, the molar ratio of the structural unit of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) to n-hexanoic anhydride is 1:0.1-3; the reaction temperature of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) and n-hexanoic anhydride is 60-100°C; and the reaction time is 12-36 h.

[0016] In a specific embodiment of the present invention, the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) is prepared according to the following steps:

[0017] Chitosan having the structure shown in formula (IV) and 2,3-epoxypropyltrimethylammonium chloride were added to a first solvent, and acetic acid was added to aid dissolution. The mixture was then sealed and stirred to carry out the reaction, thereby obtaining N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II).

[0018]

[0019] In a specific embodiment of the present invention, the volume ratio of the first solvent to acetic acid is 1:0.002 to 0.01.

[0020] In a specific embodiment of the present invention, the molar ratio of the chitosan structural unit having the structure of formula (IV) to 2,3-epoxypropyltrimethylammonium chloride is 1:0.3 to 3.

[0021] In a specific embodiment of the present invention, the reaction temperature of the chitosan having the structure of formula (IV) and 2,3-epoxypropyltrimethylammonium chloride is 30-80°C; the reaction time is 12-24 h.

[0022] A third objective of this invention is to provide the application of the chitosan derivatives described above or the chitosan derivatives prepared by the methods described above in the preparation of antitumor drugs.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a chitosan derivative and its preparation method. The chitosan derivative having the structure of formula (I) is synthesized in two steps using chitosan, 2,3-epoxypropyltrimethylammonium chloride and hexanoic anhydride as reaction substrates. The raw materials are readily available and the reaction conditions are mild.

[0025] Furthermore, the chitosan derivative with the structure of formula (I) carries a large number of positive charges, which can bind to the negatively charged tumor cell membrane surface through electrostatic interaction, promoting the interaction between the hydrophobic part of the chitosan derivative and the hydrophobic chain of the cell membrane phospholipid, disrupting the stability of the cell membrane and ultimately causing membrane lysis and tumor cell necrosis. It can inhibit the survival rate of various tumor cells to below 30%.

[0026] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS prepared in Example 3 of this invention 3000 -G 900 The proton NMR spectrum;

[0029] Figure 2 The N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS prepared in Example 4 of this invention 3000 -G 600 The proton NMR spectrum;

[0030] Figure 3 The N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS prepared in Example 6 of this invention 4000 -G 1050 The proton NMR spectrum;

[0031] Figure 4 CS is the chitosan derivative prepared in Example 17 of this invention. 3000 -G 1200 -H 950 The proton NMR spectrum;

[0032] Figure 5 CS is the chitosan derivative prepared in Example 18 of this invention. 3000 -G 1200 -H 850 The proton NMR spectrum;

[0033] Figure 6 CS is the chitosan derivative prepared in Example 19 of this invention. 3000 -G 1200 -H 650 The proton NMR spectrum;

[0034] Figure 7 The chitosan derivative CS prepared in Example 20 of this invention 3000 -G 1200 -H 550 The proton NMR spectrum;

[0035] Figure 8 CS is the chitosan derivative prepared in Example 24 of this invention. 3000 -G 600 -H 950 The proton NMR spectrum;

[0036] Figure 9 CS is the chitosan derivative prepared in Example 26 of this invention. 3000 -G 900 -H 650 The proton NMR spectrum;

[0037] Figure 10 CS is the chitosan derivative prepared in Example 28 of this invention. 3000 -G 900 -H 950 The proton NMR spectrum;

[0038] Figure 11 CS is the chitosan derivative prepared in Example 41 of this invention. 4000 -G 1200 -H 550 The proton NMR spectrum;

[0039] Figure 12 In Example 17, 4T1 mouse breast cancer cells were introduced with chitosan derivative CS. 3000 -G 1200 -H 950 The increase in concentration corresponds to the cell survival rate;

[0040] Figure 13 In Example 17, B16-F10 murine melanoma cells were introduced with chitosan derivative CS. 3000 -G 1200 -H 950 The increase in concentration corresponds to the cell survival rate;

[0041] Figure 14 In Example 18, 4T1 mouse breast cancer cells were treated with chitosan derivative CS. 3000 -G 1200 -H 850 The increase in concentration corresponds to the cell survival rate;

[0042] Figure 15 In Example 19, 4T1 mouse breast cancer cells were introduced with chitosan derivative CS. 3000 -G1200 -H 650 The increase in concentration corresponds to the cell survival rate;

[0043] Figure 16 In Example 20, 4T1 mouse breast cancer cells were treated with chitosan derivative CS. 3000 -G 1200 -H 550 The increase in concentration corresponds to the cell survival rate;

[0044] Figure 17 CS is the chitosan derivative prepared in Example 17 of this invention. 3000 -G 1200 -H 950 Fluorescence inverted microscope image after co-culturing with 4T1 mouse breast cancer cells for 6 hours;

[0045] Figure 18 CS is the chitosan derivative prepared in Example 17 of this invention. 3000 -G 1200 -H 950 SEM image of 4T1 mouse breast cancer cells after 6 hours of co-culture. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] A method for preparing a chitosan derivative according to an embodiment of the present invention includes:

[0048] Under the action of a first catalyst, N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) is dissolved in a first solvent, and then hexanoic anhydride with the structure of formula (III) is dissolved in a second solvent. The second solvent containing hexanoic anhydride is added to the first solvent containing N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) to react and obtain a chitosan derivative with the structure of formula (I).

[0049]

[0050]

[0051] Wherein, 3000≤n≤6300, 500≤x≤2600, and 540≤y≤2000.

[0052] In some embodiments of the present invention, the first solvent is distilled water, the first catalyst is triethylamine, pyridine or sodium carbonate; and the second solvent is methanol, dimethyl sulfoxide or N,N-dimethylformamide.

[0053] In some embodiments of the present invention, the molar ratio of the structural unit of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) to n-hexanoic anhydride is 1:0.1 to 3. Exemplarily, the molar ratio of the structural unit of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan to n-hexanoic anhydride is specifically 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2.

[0054] In some embodiments of the present invention, the molar ratio of the first catalyst to hexanoic anhydride is 1:0.1 to 3, and the molar ratio of the first catalyst to N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan is 1:0.8 to 1.2.

[0055] In some embodiments of the present invention, the reaction temperature of the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) and the hexanoic anhydride is 60-100°C; the reaction time is 12-36 h; the reaction temperature of the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan and the hexanoic anhydride is exemplary to be 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C, and the specific reaction time is exemplary to be 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h or 36 h.

[0056] In some embodiments of the present invention, when the first solvent is distilled water and the second solvent is methanol, the volume ratio of the distilled water to the methanol is 1:0.9 to 1.5.

[0057] In some embodiments of the present invention, the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) is prepared according to the following steps:

[0058] Chitosan having the structure shown in formula (IV) and 2,3-epoxypropyltrimethylammonium chloride having the structure of formula (V) were added to a first solvent, and acetic acid was added to aid dissolution. The mixture was sealed and stirred to carry out the reaction, thereby obtaining N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II).

[0059]

[0060] In some embodiments of the present invention, the volume ratio of the first solvent to acetic acid is 1:0.002 to 0.01.

[0061] In some embodiments of the present invention, the molar ratio of the chitosan structural unit having the structure of formula (IV) to 2,3-epoxypropyltrimethylammonium chloride is 1:0.3 to 3. Exemplarily, the molar ratio of the chitosan structural unit to 2,3-epoxypropyltrimethylammonium chloride is specifically 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2.

[0062] In some embodiments of the present invention, the reaction temperature of the chitosan having the structure of formula (IV) and 2,3-epoxypropyltrimethylammonium chloride is 30-80°C; the reaction time is 12-24 h. Exemplarily, the specific values ​​of the reaction temperature of the chitosan and 2,3-epoxypropyltrimethylammonium chloride are 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C, and the specific values ​​of the reaction time are 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h or 24 h.

[0063] In some embodiments of the present invention, the reaction of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan and hexanoic anhydride in first and second mixed solvents, and the reaction of chitosan and 2,3-epoxypropyltrimethylammonium chloride in the first solvent, are simultaneously stirred. The present invention does not impose any particular limitation on the stirring method; any stirring method well known to those skilled in the art can be used.

[0064] In some embodiments of the present invention, after the reaction of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan and hexanoic anhydride in first and second mixed solvents, and the reaction of chitosan and 2,3-epoxypropyltrimethylammonium chloride in the first solvent, the product solution is further subjected to dialysis and lyophilization to obtain N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II), and chitosan derivatives having the structure shown in formula (I). The present invention does not impose any particular limitation on the dialysis and lyophilization methods; any dialysis and lyophilization methods well known to those skilled in the art can be used.

[0065] In this embodiment of the invention, there are no special restrictions on the source of the hexanoic anhydride, 2,3-epoxypropyltrimethylammonium chloride and chitosan, which can be commercially available.

[0066] In some embodiments of the present invention, in the chitosan derivative having the structure of formula (Ⅰ), 3000≤n≤6300; in some embodiments of the present invention, n is 3000, 4000, 5000 or 6000.

[0067] In some embodiments of the present invention, in the chitosan derivative having the structure of formula (Ⅰ), 500≤x≤2600, 540≤y≤2000; in some embodiments of the present invention, x is 600, 750, 900, 1050 or 1200; y is 550, 650, 750, 850 or 950.

[0068] In the embodiments of this invention, the chitosan derivative with the structure of formula (I) carries a large amount of positive charge, which can bind to the negatively charged tumor cell membrane surface through electrostatic interaction. This promotes the interaction between the hydrophobic portion of the chitosan derivative and the hydrophobic chains of the cell membrane phospholipids, disrupting the stability of the tumor cell membrane and ultimately causing membrane lysis and tumor cell necrosis. It can inhibit the survival rate of various tumor cells to below 30%, exhibiting a broad-spectrum anti-tumor effect. Therefore, this invention seeks protection for the use of the chitosan derivative described above or the chitosan derivative prepared by the method described above in the preparation of anti-tumor drugs.

[0069] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a chitosan derivative, its preparation method, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0070] The raw materials used in the following examples are all commercially available.

[0071] The model tumor cells were selected from B16-F10 murine melanoma cells and 4T1 murine breast cancer cells. The B16-F10 murine melanoma cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the 4T1 murine breast cancer cells were purchased from Shanghai Bogu Biotechnology Co., Ltd.

[0072] Preparation of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with structure (II):

[0073] Example 1

[0074] Preparation of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with n=3000 and x=1200 in formula (II):

[0075] Weigh 2.00 g of chitosan (CS, 12.4 mmol by molar amount of structural unit) and dissolve it in 80 mL of distilled water. Stir for 10 min and observe for dissolution. Then add 400 μL of acetic acid and stir for 30 min. Next, add 2.29 g of 2,3-epoxypropyltrimethylammonium chloride (GTMAC, 15.10 mmol) in three equal portions, 2 h apart, to the above chitosan 2,3-epoxypropyltrimethylammonium chloride solution. Stir and heat to 60 °C. After the last addition, seal and react for 18 hours. After the reaction is complete, centrifuge the reaction solution (4000 rpm, 10 min), filter, and retain the filtrate. Transfer the filtrate to a dialysis bag and dialyze in distilled water for three days. Freeze-dry the purified conjugate solution for 2 days to prepare N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS. 3000 -G 1200 .

[0076] Examples 2-16 all describe the preparation of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with formula (II). The preparation steps are exactly the same as those in Example 1, except for the amount of raw materials added and the values ​​of n and x in formula (II). The specific correspondence is shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] The N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) obtained in Examples 3, 4, and 6 was subjected to nuclear magnetic resonance (NMR) detection, and the resulting proton NMR spectra are shown below. Figure 1-3 As shown, Figure 1 The corresponding example is the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS obtained in Example 3, which has the structure of formula (II). 3000 -G 900 The proton NMR spectrum, Figure 2 The corresponding example is the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS obtained in Example 4, which has the structure of formula (II). 3000 -G 600 The proton NMR spectrum, Figure 3 The corresponding example is the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS obtained in Example 6, which has the structure of formula (II). 4000 -G 1050 The proton NMR spectrum;

[0081] from Figure 1It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride to obtain N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS with the structure of formula (II). 3000 -G 900 .

[0082] from Figure 2 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride to obtain N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS with the structure of formula (II). 3000 -G 600 .

[0083] Figure 3 The image shows the 1H NMR spectrum of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan prepared in Example 6 of this invention. Figure 3 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride to obtain N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan CS with the structure of formula (II). 4000 -G 1050 .

[0084] Preparation of chitosan derivatives having the structure shown in formula (Ⅰ):

[0085] Using N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) obtained in Examples 1-16 as a reaction substrate, chitosan derivatives with the structure shown in formula (I) were prepared.

[0086] Example 17

[0087] Preparation of chitosan derivatives with the structure of formula (Ⅰ):

[0088] 100 mg of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan (0.448 mmol by molar amount of structural unit) was dissolved in 10 mL of distilled water, and 205 μL of hexanoic anhydride was dissolved in 10 mL of methanol. Then, under stirring, the anhydride dissolved in methanol was added to the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan solution. 62 μL of triethylamine was then added to catalyze the reaction, and the mixture was refluxed at 80 °C for 24 h. After the reaction was complete, the methanol was removed by rotary evaporation (40 °C, 100 rpm) for 30 min followed by vacuum pumping for 1 h. The resulting solution was then transferred to a dialysis bag and dialyzed three times in water at pH 8–9, twice in water at pH 4–5, and six times in distilled water, with fresh dialysate every 2 h. The resulting solution was then freeze-dried for 2 days to prepare the chitosan derivative CS. 3000 -G 1200 -H950 (The molecular results of this chitosan derivative are corroborated by the subsequent NMR spectrum results).

[0089] Examples 17-76 are all preparations of chitosan derivatives with the structure shown in Formula (Ⅰ). Compared with Example 17, the preparation steps are exactly the same. The difference lies in the N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of Formula (ⅠⅠ) and the amount of each raw material added. For details, please refer to Tables 2-4. Table 2 shows the amount of each raw material added in Examples 17-36, Table 3 shows the amount of each raw material added in Examples 37-56, and Table 4 shows the amount of each raw material added in Examples 57-76.

[0090] Table 2

[0091]

[0092]

[0093]

[0094] Table 3

[0095]

[0096]

[0097] Table 4

[0098]

[0099]

[0100] Molecular structure characterization of chitosan derivatives having the structure shown in formula (Ⅰ):

[0101] Nuclear magnetic resonance (NMR) analysis was performed on the chitosan derivatives obtained in Examples 17, 18, 19, 20, 24, 26, 28, and 41. The results are as follows: Figures 4-7 As shown, Figure 4 The image shows the 1H NMR spectrum of the chitosan derivative obtained in Example 17. Figure 5 The image shows the 1H NMR spectrum of the chitosan derivative obtained in Example 18. Figure 6 The image shows the 1H NMR spectrum of the chitosan derivative obtained in Example 19. Figure 7 The above is the 1H NMR spectrum of the chitosan derivative obtained in Example 20; Figure 8 The above is the 1H NMR spectrum of the chitosan derivative obtained in Example 24; Figure 9 The 1H NMR spectrum of the chitosan derivative obtained in Example 26; Figure 10The above is the 1H NMR spectrum of the chitosan derivative obtained in Example 28; Figure 11 The above is the 1H NMR spectrum of the chitosan derivative obtained in Example 41;

[0102] from Figure 4 It can be seen that the amino groups on the chitosan side chains of the chitosan derivative obtained in Example 17 successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G 1200 -H 950 .

[0103] from Figure 5 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G 1200 -H 850 ;

[0104] from Figure 6 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G 1200 -H 650 .

[0105] from Figure 7 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G 1200 -H 550 .

[0106] from Figure 8 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G 600 -H 950 .

[0107] from Figure 9 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G 900 -H 650 .

[0108] from Figure 10 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 3000 -G900 -H 950 .

[0109] from Figure 11 It can be seen that the amino groups on the chitosan side chains successfully reacted with glycidyltrimethylammonium chloride and hexanoic anhydride to obtain the chitosan derivative CS with the structure of formula (I). 4000 -G 1200 -H 550 .

[0110] The antitumor activity of chitosan derivatives was detected by cytotoxicity assays:

[0111] The chitosan derivatives prepared in Example 17 were co-cultured with 4T1 mouse breast cancer cells and evaluated using the MTT assay.

[0112] The specific process is as follows:

[0113] First, cells were seeded in 96-well plates at a density of 4000 cells / well (suspended in 100 μL of 1640 medium) and incubated at 37°C for 24 h. The next day, the corresponding chitosan derivatives were dissolved in fresh 1640 medium and serially diluted to different concentrations. The previous day's medium was discarded, and 1640 medium containing different concentrations of chitosan derivatives was added. Incubation continued at 37°C for another 24 h. Then, MTT solution (final concentration 0.5 mg / mL) was added to each well, and incubation continued at 37°C for 4 h. The liquid in the plate was discarded, and 100 μL of DMSO was added to each well to dissolve the purple formazan crystals. Finally, the absorbance at 490 nm was measured using a microplate reader (Tecan, Switzerland). Cell viability was calculated by comparing the absorbance values ​​of the experimental group with those of the control group.

[0114] The evaluation results of the above MTT experiment are as follows: Figure 12 As shown, Figure 12 In Example 17, 4T1 mouse breast cancer cells were introduced with chitosan derivative CS. 3000 -G 1200 -H 950 The increase in concentration corresponds to a higher cell survival rate. From Figure 12 It can be seen that chitosan derivative CS 3000 -G 1200 -H 950 It has a strong anti-breast cancer effect, suppressing the survival rate of 4T1 mouse breast cancer cells to below 25%.

[0115] Similarly, the chitosan derivative prepared in Example 17 of this invention was co-cultured with B16-F10 mouse melanoma cells, and the cell viability was calculated according to the above method. The cell viability test results are as follows: Figure 13 As shown. From Figure 13 It can be seen that chitosan derivative CS 3000 -G 1200 -H 950 It has a strong anti-melanoma cell effect, suppressing the survival rate of B16-F10 mouse melanoma cells to below 30%.

[0116] The chitosan derivatives prepared in Example 18 were co-cultured with 4T1 mouse breast cancer cells, and the cell viability was evaluated using the MTT assay. The cell viability is shown in the figure below. Figure 14 As shown, from Figure 14 It can be seen that chitosan derivative CS 3000 -G 1200 -H 850 It has a strong anti-breast cancer effect, suppressing the survival rate of 4T1 mouse breast cancer cells to below 30%.

[0117] The chitosan derivatives prepared in Example 19 were co-cultured with 4T1 mouse breast cancer cells, and the cell viability was evaluated using the MTT assay. The cell viability is shown in the figure below. Figure 15 As shown, from Figure 15 It can be seen that chitosan derivative CS 3000 -G 1200 -H 650 It has a strong anti-breast cancer effect, suppressing the survival rate of 4T1 mouse breast cancer cells to below 30%.

[0118] The chitosan derivatives prepared in Example 20 were co-cultured with 4T1 mouse breast cancer cells, and the cell viability was evaluated using the MTT assay. The cell viability is shown in the figure below. Figure 16 As shown, from Figure 16 It can be seen that chitosan derivative CS 3000 -G 1200 -H 550 It has a strong anti-breast cancer effect, suppressing the survival rate of 4T1 mouse breast cancer cells to below 30%.

[0119] Co-culture experiments with 4T1 mouse breast cancer cells were conducted on Examples 21-31, 34-35, 37, 39, 42, 45, 47, 50, 52, 54, 57-58, and 60-61. The cancer cell survival rates obtained are shown below:

[0120] Chitosan derivative material CS prepared in Example 213000 -G 600 -H 550 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 25%, and the anti-breast cancer effect is similar to that of Example 17.

[0121] Example 22 Chitosan derivative material CS prepared 3000 -G 600 -H 650 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 25%, and the anti-breast cancer effect is similar to that of Example 17.

[0122] Example 23: Chitosan derivative material CS 3000 -G 600 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 18.

[0123] The chitosan derivative material prepared in Example 24 can CS 3000 -G 600 -H 950 As an anti-tumor drug, the survival rate of the obtained 4T1 mouse breast cancer cells was less than 30%, and the anti-breast cancer effect was similar to that of Example 18.

[0124] Example 25: Chitosan derivative material CS 3000 -G 900 -H 550 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 18.

[0125] Example 27: Chitosan derivative material CS 3000 -G 900 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 18.

[0126] Example 28: Chitosan derivative material CS 3000 -G 900 -H 950 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 17.

[0127] Example 29: Chitosan derivative material CS 3000 -G 1050 -H 550It can be used as an anti-tumor drug and has a similar effect to the anti-breast cancer drug in Example 18.

[0128] Chitosan derivative material CS prepared in Example 30 3000 -G 1050 -H 650 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 18.

[0129] Chitosan derivative material CS prepared in Example 31 3000 -G 1050 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 19.

[0130] Example 34: Chitosan derivative material CS 4000 -G 600 -H 650 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 18.

[0131] Chitosan derivative material CS prepared in Example 35 4000 -G 600 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 30%, and the anti-breast cancer effect is similar to that of Example 19.

[0132] Example 37: Chitosan derivative material CS 4000 -G 900 -H 950 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 28%, and the anti-breast cancer effect is similar to that of Example 19.

[0133] Chitosan derivative material CS prepared in Example 39 4000 -G 900 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 28%, and the anti-breast cancer effect is similar to that of Example 19.

[0134] Example 42: Chitosan derivative material CS 4000 -G 1200 -H 950 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 28%, and the anti-breast cancer effect is similar to that of Example 19.

[0135] Example 45: Chitosan derivative material CS 4000 -G 1050 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 28%, and the anti-breast cancer effect is similar to that of Example 19.

[0136] Example 47: Chitosan derivative material CS 4000 -G 1050 -H 650 It can be used as an anti-tumor drug and has a similar effect to the anti-breast cancer drug in Example 19.

[0137] Chitosan derivative material CS prepared in Example 50 5000 -G 1050 -H 550 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 28%, and the anti-breast cancer effect is similar to that of Example 19.

[0138] Example 52: Chitosan derivative material CS 5000 -G 1050 -H 650 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 28%, and the anti-breast cancer effect is similar to that of Example 19.

[0139] Example 54: Chitosan derivative material CS 5000 -G 600 -H 850 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 27%, and the anti-breast cancer effect is similar to that of Example 20.

[0140] Example 57: Chitosan derivative material CS 5000 -G 900 -H 650 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 27%, and the anti-breast cancer effect is similar to that of Example 20.

[0141] Example 58: Chitosan derivative material CS 5000 -G 900 -H 950 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 27%, and the anti-breast cancer effect is similar to that of Example 20.

[0142] Chitosan derivative material CS prepared in Example 60 5000 -G 900 -H 550It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 27%, and the anti-breast cancer effect is similar to that of Example 20.

[0143] Chitosan derivative material CS prepared in Example 61 5000 -G 1200 -H 550 It can be used as an anti-tumor drug, with the survival rate of the obtained 4T1 mouse breast cancer cells being less than 27%, and the anti-breast cancer effect is similar to that of Example 20.

[0144] Cell morphology observation using a fluorescence inverted microscope:

[0145] Inoculate 1.2 × 10⁻⁶ cells into 6-well plates. 5 Four T1 cells per well (suspended in 2 mL of 1640 medium) were incubated in a cell culture incubator at 37°C for 24 hours. The next day, the corresponding chitosan derivatives were dissolved in fresh 1640 medium and serially diluted to different concentrations. The previous day's medium was discarded, and 1640 medium containing different concentrations of chitosan derivatives (prepared in Example 17) was added. Incubation continued for 6 hours at 37°C in a cell culture incubator. The liquid in the wells was then discarded, and the cells were washed twice with PBS. Finally, the cells were observed using a fluorescence inverted microscope. The resulting images are shown below. Figure 17 As shown.

[0146] Figure 17 The results showed that after treatment with the chitosan derivative obtained in Example 17, the 4T1 breast cancer cells underwent cell membrane rupture, cell contents flowed out, and the cells shrank, collapsed, and died completely.

[0147] Cell morphology was observed using scanning electron microscopy.

[0148] Inoculate 2.5 × 10⁻⁶ cells into 24-well plates. 4 Four T1 cells / well (suspended in 0.5 mL of 1640 medium) were incubated in a cell culture incubator at 37°C for 24 hours. The next day, the corresponding chitosan derivative (prepared in Example 17) was dissolved in fresh 1640 medium and serially diluted to different concentrations. The previous day's medium was discarded, and 1640 medium containing different concentrations of chitosan derivative was added. Incubation continued at 37°C for 6 hours. The liquid in the culture dish was then discarded, and the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes. Dehydration was then performed sequentially with 10%, 30%, 50%, 70%, and 100% ethanol. Finally, the cells were observed using a scanning electron microscope (Zeiss Merlin, Germany). The results are shown below. Figure 18 As shown.

[0149] Figure 18The results showed that 4T1 breast cancer cells treated with the chitosan derivative obtained in Example 17 shrank, and the cell membrane ruptured, resulting in complete necrosis.

[0150] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chitosan derivative, characterized by, Account: （Ⅰ）； For example,3000≤n≤6300,500≤x≤2600,540≤y≤2000? If n,x,y are assigned to 44, n=3000,x=1200,y=950,n=3000, 200,y=850;n=3000,x=1200,y=650;n=3000,x=1200,y=550;n=300 0,x=600,y=550(n=3000,x=600,y=650(n=3000),x=600,y=850,n=3 000,x=600,y=950(n=3000,x=900,y=550(n=3000),x=900,y=850(n =3000,x=900,y=950;n=3000,x=1050,y=550;n=3000,x=1050,y=6 50;n=3000,x=1050,y=850;n=3000,x=1050,y=950;n=4000,x=600 ,y=550;n=4000,x=600,y=650;n=4000,x=600,y=850;n=4000,x=6 00,y=950;n=4000,x=900,y=950;n=4000,x=900,y=550;n=4000,x= 900,y=850;n=4000,x=900,y=650;n=4000,x=1200,y=550;n=4000 ,x=1200,y=950(n=4000,x=1200,y=650(n=4000),x=1200,y=850(n =4000,x=1050,y=850;n=4000,x=1050,y=550;n=4000,x=1050,y= 650(n=4000,x=1050,y=950(n=5000),x=1050,y=950(n=5000,x=10 50,y=550;n=5000,x=1050,y=850;n=5000,x=1050,y=650;n=5000 ,x=600,y=950(n=5000,x=600,y=850(n=5000),x=600,y=550,n=50 00,x=600,y=650(n=5000,x=900,y=650(n=5000),x=900,y=950(n= 5000,x=900,y=850;n=5000,x=900,y=550;n=5000,x=1200,y=550? Under the action of a first catalyst, N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) is dissolved in a first solvent, and a second solvent containing hexanoic anhydride is added to the first solvent containing N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) to react and obtain a chitosan derivative with the structure of formula (I). （Ⅱ）。 2. The method for preparing a chitosan derivative according to claim 1, characterized in that, include: Under the action of a first catalyst, N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) is dissolved in a first solvent, and a second solvent containing hexanoic anhydride is added to the first solvent containing N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with the structure of formula (II) to react and obtain a chitosan derivative with the structure of formula (I). （Ⅱ）； （Ⅰ）； Wherein, 3000≤n≤6300, 500≤x≤2600, and 540≤y≤2000.

3. The method for preparing a chitosan derivative according to claim 2, characterized by, The first solvent is distilled water, and the first catalyst is triethylamine, pyridine, or sodium carbonate; the second solvent is methanol, dimethyl sulfoxide, or N,N-dimethylformamide.

4. The method for preparing a chitosan derivative according to claim 2, characterized by, The molar ratio of the structural unit of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with formula (II) to hexanoic anhydride is 1:0.1~3; the reaction temperature of N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan with formula (II) and hexanoic anhydride is 60~100℃; the reaction time is 12~36h.

5. The method for preparing a chitosan derivative according to any one of claims 2 to 4, characterized in that, The N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II) is prepared according to the following steps: Chitosan having the structure shown in formula (IV) and 2,3-epoxypropyltrimethylammonium chloride were added to a first solvent, and acetic acid was added to aid dissolution. The mixture was then sealed and stirred to carry out the reaction, thereby obtaining N-(2-hydroxypropyl)-3-trimethylammonium chloride chitosan having the structure of formula (II). (IV).

6. The method for preparing a chitosan derivative according to claim 5, wherein, The volume ratio of the first solvent to acetic acid is 1:0.002~0.

01.

7. The method for preparing a chitosan derivative according to claim 5, wherein The molar ratio of the chitosan structural unit with the structure of formula (IV) to 2,3-epoxypropyltrimethylammonium chloride is 1:0.3~3.

8. The method for preparing a chitosan derivative according to claim 5, wherein, The reaction temperature of the chitosan with the structure of formula (IV) and 2,3-epoxypropyltrimethylammonium chloride is 30~80℃; the reaction time is 12~24h.

9. The use of the chitosan derivative according to claim 1 or the chitosan derivative prepared by any of the preparation methods according to claims 2 to 8 in the preparation of antitumor drugs.

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