Folate-modified beta-acid-loaded chitosan nanoparticle-sodium alginate hydrogel, preparation method and application

Abstract

The application provides a folate-modified beta-acid-loaded chitosan nanoparticle-sodium alginate hydrogel, a preparation method and application, and belongs to the technical field of biological medicines.The application realizes efficient and accurate colorectal cancer targeted treatment.The folate-modified beta-acid-loaded chitosan nanoparticle-sodium alginate hydrogel can combine folate (FA) -mediated active targeting with the colon pH-responsive release of the sodium alginate hydrogel.The FA modification enables the nanoparticles to specifically recognize and enrich on the surface of colorectal cancer cells with overexpressed folate receptors, realizing cell-level targeting;and the hydrogel carrier ensures that the loaded nanoparticles remain stable in the stomach acid environment and dissolve in the colon neutral / weak alkaline environment (liquid phase transition), thereby realizing the positioning release at the lesion site.This double-targeting strategy (organ positioning+cell targeting) greatly improves the concentration of the drug in the tumor tissue and reduces the systemic exposure.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a folic acid-modified chitosan nanoparticle-sodium alginate hydrogel, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, posing a serious threat to human health. Currently, clinical interventions mainly include surgical resection, radiotherapy, and chemotherapy. Among these, chemotherapy is considered an important and promising systemic treatment, especially for unresectable or metastatic tumors. However, first-line chemotherapy drugs, represented by 5-fluorouracil (5-FU), have long faced significant challenges in clinical application, including insufficient efficacy, severe toxic side effects, and the development of intrinsic or acquired drug resistance in tumor cells. Therefore, developing novel, highly effective, and low-toxicity treatment strategies, such as targeted therapy, neoadjuvant chemotherapy, and immunotherapy, has become an urgent need and an important research direction in this field.

[0003] Among various delivery strategies, nanoparticle-based drug delivery systems can significantly improve drug solubility, prolong circulation time, and achieve targeted delivery through functionalization, making them a key technology for enhancing the efficacy of anticancer drugs and reducing side effects. Folic acid (FA), as a crucial nutrient, has its receptor often overexpressed on the surface of various epithelial tumor cells (such as colorectal cancer, ovarian cancer, and lung cancer), with expression levels reaching 100-300 times that of normal cells. It also possesses advantages such as high affinity and non-immunogenicity, making it an ideal active targeting ligand. Modifying the surface of nanoparticles with FA can effectively mediate the specific recognition and internalization of drugs by tumor cells, enhancing drug accumulation at the lesion site.

[0004] The selection of carrier materials is the cornerstone of constructing efficient nanodelivery systems. Chitosan (CS), a natural cationic polysaccharide, is widely considered an ideal material for preparing nanocarriers due to its good biocompatibility, biodegradability, low toxicity, and cost-effectiveness. FA-modified CS nanoparticles can further enhance receptor-mediated targeting, significantly improving drug delivery efficiency. Regarding active ingredients, β-acids, natural compounds derived from hops, have been shown to possess significant antibacterial, antioxidant, and anticancer activities, making them promising therapeutic candidates.

[0005] Despite significant progress in FA-targeted nanosystems, systemic administration still faces challenges such as non-specific distribution and clearance by the reticuloendothelial system. Especially for colorectal cancer, oral administration is a highly attractive local delivery route, but drugs must traverse the complex gastrointestinal environment, making them prone to degradation, premature release, or ineffective accumulation at the tumor site before reaching the lesion. Therefore, developing a highly efficient delivery system that can resist the upper gastrointestinal environment and specifically release drugs at the colon cancer site, and constructing an orally administered, intelligently responsive, and actively targeted delivery system to overcome the limitations of existing chemotherapy and delivery strategies, remains a critical technical challenge in the treatment of colorectal cancer. Summary of the Invention

[0006] The purpose of this invention is to provide a folic acid-modified chitosan nanoparticle-sodium alginate hydrogel, its preparation method, and its application. The invention constructs a stable and targeted nano-formulation of the natural compound β-acid with anti-cancer potential, and further integrates it into an oral colon-targeting hydrogel, which significantly improves its bioavailability and therapeutic effect, providing a feasible delivery solution for the development of natural product-based colorectal cancer treatment drugs.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing folic acid-modified chitosan nanoparticles-sodium alginate hydrogel, comprising the following steps: (1) Folic acid, 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide were dissolved in DMSO and stirred to obtain folic acid active ester; (2) Add folic acid active ester to CS solution to react and obtain FA / CS powder, dissolve it in acetic acid solution, and adjust the pH to obtain FA / CS solution; (3) Add the β-acid ethanol solution to the FA / CS solution, stir, then add the TPP solution and continue stirring to obtain FA / CS / TPP / β-acid; (4) Sodium alginate solution and CaCl2 solution were mixed to obtain Alg hydrogel; (5) FA / CS / TPP / β-acid and Alg hydrogel were mixed to obtain folic acid modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel.

[0008] Preferably, in step (1), the molar ratio of folic acid, 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide is 1:1~2:1~2; and the stirring time is 2~4h.

[0009] Preferably, in step (2), the concentration of the CS solution is 0.5~1g / mL; the volume ratio of the CS solution to the folic acid active ester is 4~6:1.

[0010] Preferably, in step (3), the concentration of the β-acid ethanol solution is 0.04~0.2 mg / mL; the concentration of the FA / CS solution is 5~25 mg / mL; the concentration of the TPP solution is 1~3 mg / mL; and the volume ratio of the β-acid ethanol solution, FA / CS solution and TPP solution is 5~25:90~110:10~30.

[0011] Preferably, in step (3), the stirring time is 20-40 min; the stirring time is 0.8-1.2 h.

[0012] Preferably, in step (4), the concentration of sodium alginate solution is 1-2%; the concentration of CaCl2 solution is 1-2%; and the volume ratio of sodium alginate solution to CaCl2 solution is 1-3:1.

[0013] Preferably, in step (5), the volume ratio of the FA / CS / TPP / β-acid to the Alg hydrogel is 10:2~4.

[0014] The present invention also provides a folic acid-modified chitosan nanoparticle-sodium alginate hydrogel prepared according to the above preparation method.

[0015] The present invention also provides the application of the above-mentioned folic acid-modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel in the preparation of colorectal cancer therapeutic drugs.

[0016] The beneficial effects of this invention compared to the prior art are as follows: (1) This invention achieves efficient and precise targeted therapy for colorectal cancer. The folic acid-modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel of this invention combine folic acid (FA)-mediated active targeting with the colonic pH-responsive release of sodium alginate hydrogel. FA modification enables the nanoparticles to specifically recognize and accumulate on the surface of colorectal cancer cells overexpressing folic acid receptors, achieving cell-level targeting; while the hydrogel carrier ensures that the loaded nanoparticles remain stable in the acidic environment of the stomach and undergo dissolution (liquid phase transition) in the neutral / weakly alkaline environment of the colon, thereby achieving localized release at the lesion site. This dual targeting strategy (organ localization + cell targeting) greatly improves the drug concentration in tumor tissue and reduces systemic exposure.

[0017] (2) The folic acid-modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel of the present invention can effectively protect the active ingredients FA / CS / TPP / β-acid nanoparticles, providing a physical barrier so that they can effectively resist gastric acid degradation and upper gastrointestinal enzyme destruction after oral administration; it can also ensure that the active ingredient β-acid is successfully delivered to the colorectal lesion site in its intact form, overcome the drug delivery barrier, and solve the key problem that oral biological drugs are easily inactivated in the gastrointestinal tract.

[0018] (3) Experiments show that the folic acid-modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel of the present invention exhibits excellent antitumor effects in an in situ colorectal cancer model, with efficacy superior to or equivalent to the positive control drug 5-FU. More importantly, the folic acid-modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel of the present invention also shows excellent safety: the weight of mice in the treatment group remained stable, and no liver or kidney function damage or pathological abnormalities in major organs were observed. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The figures show the FA / CS characterization results in the experimental examples of this invention, where A represents the FA / CS synthesis scheme; B represents the CS... 1 H NMR spectrum; C is FA 1 1H NMR spectrum; D represents the FA / CS conjugate. 1 H NMR spectrum; Figure 2-1 The following are the characterization results of FA / CS / TPP / β-acid in Experimental Example 1 of the present invention, where A is the Fourier transform infrared spectrum of CS, FA, TPP, β-acid, FA / CS, FA / CS / TPP, and FA / CS / TPP / β-acid; B is the scanning electron microscope image of FA / CS / TPP (a1), FA / CS / TPP / 7.5β-acid (b1), FA / CS / TPP / 15β-acid (c1), and FA / CS / TPP / 22.5β-acid (d1). Figure 2-2 The characterization results of FA / CS / TPP / β-acid in Experimental Example 1 of this invention are shown, where C is the X-ray diffraction pattern of CS, β-acid, FA / CS / TPP, and FA / CS / TPP / β-acid; D is the particle size, zeta potential, LC% and EE% of the nanoparticles (n = 6). Figure 3The images show the characterization results of Alg, Alg / FA / CS / TPP, and Alg / FA / CS / TPP / β-acid hydrogels in Experimental Example 1 of this invention. A represents representative macroscopic images of the hydrogels in media with pH values ​​of 1.2 (stomach), 6.8 (small intestine), and 7.4 (colon); B shows the Fourier transform infrared spectra of (a) SA, (b) Alg, (c) Alg / FA / CS / TPP, and (d) Alg / FA / CS / TPP / β-acid; C shows scanning electron microscope images of Alg: (a1) 50 μm, (a2) 2 μm, (a3) ​​1 μm; images of Alg / FA / CS / TPP: (b1) 50 μm, (b2) 2 μm, (b3) 1 μm; images of Alg / FA / CS / TPP / 7.5β-acid: (c1) 50 μm, (c2) 2 μm, (c3) 1 μm; Alg / FA Images of Alg / FA / CS / TPP / 15β-acid: (d1) 50 μm, (d2) 2 μm, (d3) 1 μm; Images of Alg / FA / CS / TPP / 22.5β-acid: (e1) 50 μm, (e2) 2 μm, (e3) 1 μm; D represents the frequency scan results (storage modulus (G′) and loss modulus (G″)) of Alg / FA / CS / TPP hydrogel and Alg / FA / CS / TPP / β-acid hydrogel; E represents the step strain scan of Alg / FA / CS / TPP hydrogel and Alg / FA / CS / TPP / β-acid hydrogel; F represents the time scan results of Alg / FA / CS / TPP hydrogel and Alg / FA / CS / TPP / β-acid hydrogel; G represents the oscillatory strain scan results of Alg / FA / CS / TPP hydrogel and Alg / FA / CS / TPP / β-acid hydrogel. Figure 4 This diagram illustrates the in vitro release of β-acids from nanoparticles and hydrogels in Experimental Example 1 of this invention. A represents FA / CS / TPP / 7.5β-acid; B represents FA / CS / TPP / 15β-acid; C represents FA / CS / TPP / 22.5β-acid; D represents Alg / FA / CS / TPP / 7.5β-acid; E represents Alg / FA / CS / TPP / 15β-acid; and F represents Alg / FA / CS / TPP / 22.5β-acid (n=3). Figure 5The results of CCK-8 detection and scratch assay in Experiment 1 of this invention are shown. A represents the CCK-8 detection of HCT116 cells by β-acid, 5-fluorouracil, FA / CS / TPP, FA / CS / TPP / 7.5β-acid, FA / CS / TPP / 15β-acid, and FA / CS / TPP / 22.5β-acid (n=3); B represents the CCK-8 detection of HCT116 cells by β-acid, 5-fluorouracil, Alg / FA / CS / TPP, Alg / FA / CS / TPP / 7.5β-acid, and Alg / F... A. Detection of CCK8 in HCT116 cells using A / CS / TPP / 15β-acid and Alg / FA / CS / TPP / 22.5β-acid (n=3); C. HCT116 cell scratch assay using 5-fluorouracil, FA / CS / TPP / 22.5β-acid, and Alg / FA / CS / TPP / 22.5β-acid; D. NCM460 cell scratch assay using FA / CS / TPP / 22.5β-acid and Alg / FA / CS / TPP / 22.5β-acid. Figure 6 The results of cell experiments in Experiment Example 1 of this invention are shown, where A represents the effect of different nanoparticles and hydrogels on HCT116 cell apoptosis; and B represents the DAPI fluorescence results. Figure 7 The results of the mouse experiment in Experiment Example 1 of this invention are shown below. A shows the changes in body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, and creatinine over 14 days; B shows the histological assessment of important organs using hematoxylin-eosin staining (scale bar is 50 micrometers); C shows a photograph of the colon and rectum of the mouse; D shows the weight of the colon and rectum; E shows the hematoxylin-eosin staining of colon sections (10x magnification) and tumor tissue (40x magnification) from treated and healthy mice. Figure 8 To demonstrate the safety of the hydrogel in Experiment 1 of this invention, A shows the changes in body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, and creatinine over 14 days; B shows the combined treatment performed on CT-26 tumor-bearing mice over 14 days, as indicated by H&E staining of the major organs (scale bar at 50 micrometers); C shows a photograph of the mouse colon; and D shows the H&E staining results of colon sections (10x magnification) and tumor tissue (40x magnification) from treated and healthy mice. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1 Example 1 of this invention provides a method for preparing Alg / FA / CS / TPP / β-acid, the specific steps of which are as follows: (1) Take 50 mg FA, 17.57 mg EDC and 13.04 mg NHS in a molar ratio of 1:1.5:1.5, mix and dissolve in 40 mL DMSO, stir at 500 rpm for 3 hours in the dark at 30 °C to obtain FA active ester.

[0027] (2) After dissolving 1 g of CS in 200 mL of 1% acetic acid solution, the FA active ester was added dropwise to the CS solution at a rate of 1 mL / min, and the reaction was carried out at 25 °C for 24 h. The pH was adjusted to 9.0 with 3 mol / L sodium hydroxide solution, and the mixture was centrifuged at 12000 rpm and 4 °C for 10 min to produce a yellow precipitate. The yellow precipitate was washed with saturated sodium bicarbonate solution and then freeze-dried to obtain FA / CS powder with a yield of 56%.

[0028] (3) Dissolve 500 mg FA / CS powder in 100 mL of 1% acetic acid solution, and adjust the pH to 5.0 with 3 mol / L sodium hydroxide to obtain FA / CS solution. Different volumes of β-acid ethanol solution (1 mg / L) were then added. mL, =7.5, 15, or 22.5) was added dropwise to the FA / CS solution at a rate of 1 mL / min, while stirring at 500 rpm for 30 min. 20 mL of 2 mg / mL TPP solution was added in portions (1 mL / min), and the reaction was stirred at 500 rpm for 1 h. The mixture was centrifuged at 12000 rpm (20 min, 4 °C) and then freeze-dried to obtain FA / CS / TPP / β-acid, yield: 78%.

[0029] (4) Dissolve sodium alginate (Alg) powder in deionized water at 60°C for 30 min to prepare an Alg solution. Mix 1.5% (mass / volume) Alg solution and 1% (mass / volume) CaCl2 solution at a volume ratio of 2:1 to form an Alg hydrogel.

[0030] (5) Mix 30 mg of FA / CS / TPP / β-acid and 9 mL of Alg hydrogel together to obtain folic acid-modified chitosan nanoparticles-sodium alginate hydrogels loaded with β-acid: Alg / FA / CS / TPP / 7.5β-acid, Alg / FA / CS / TPP / 15β-acid and Alg / FA / CS / TPP / 22.5β-acid.

[0031] Experimental Example 1 Experimental Example 1 of this invention characterized the product prepared in Example 1, and the specific steps are as follows: (1) FA / CS characterization Studies have found that the low solubility of folic acid severely affects its bioavailability, but this deficiency can be mitigated by providing methods to increase its solubility or controlling the pH value. Furthermore, folic acid is photosensitive, therefore all reactions are carried out under light conditions. Folic acid contains a highly reactive γ-COOH group in its structure. This invention first activates the γ-COOH group using EDC and NHS, then reacts it with the CS structure on the amino group; the final product is obtained by adjusting the pH value. Figure 1 (A). Because folic acid has an active carboxyl group, a hyaluronic acid derivative was generated by a one-step ester condensation reaction between the carboxyl group at the γ-position of folic acid and the hydroxyl group on hyaluronic acid. Studies have shown that this derivative possesses dual-targeting properties. This invention successfully linked cellulose acid (FA) with chitosan to form different nanoparticles. These nanoparticles exhibit significant bioactivity. The above-mentioned experimental results demonstrate the reproducibility of the method of this invention.

[0032] Further analysis using 1H nuclear magnetic resonance (NMR) Figure 1 The coupling effect of FA / CS was verified by BD. The chemical shift values ​​were 8.65, 8.13, 7.64, 6.95, 6.66, 4.49, 4.33, 2.51, and 2.03 ppm, corresponding to the protons of FA, H-7, H-18, H-13 / 15, H-10, H-12 / 16, H-19, H-19, H-22, and H-21, respectively. After the coupling effect between FA and CS, some absorption peaks of FA and CS overlapped. The CH peak appeared in the range of 3.33 to 4.03 ppm, corresponding to hydrogen atoms 3, 4, 5, and 6 of the amino ring of CS. The signal at 2.35 ppm corresponds to acetamide in CS, further confirming the successful coupling of FA and CS.

[0033] (2) Characterization of FA / CS, FA / CS / TPP, and FA / CS / TPP / β-acid The characteristic absorption peaks of CS, FA, TPP, β-acid, FA / CS, FA / CS / TPP, and FA / CS / TPP / β-acid were first characterized by Fourier transform infrared spectroscopy (FTIR). Figure 2-1 (A). CS shows at 3365cm. -1 A distinct absorption peak is observed at 2876 cm⁻¹, representing the -OH functional group. -1 The absorption peak at [value] corresponds to the asymmetric / symmetric stretching vibration absorption of CH. Subsequently, at 1655 cm⁻¹... -1 1598 cm -1 and 1381 cm -1 The absorption peak at approximately 1194 cm⁻¹ indicates vibrations associated with the O=C-NH (I, II, and III) groups of CS. -1 The value at 3544 cm represents the CO stretching vibration in the CS. FA indicates that at 3544 cm... -1 3324 cm -1 and 3116 cm -1 Absorption peaks associated with stretching vibrations of -NH, -OH, and -CH functional groups are observed. The characteristic peak of FA is mainly located at 1696 cm⁻¹. -1 1605 cm -1 and 1485 cm -1 These peaks are related to carboxylic acid vibrations, amino groups, and C=C or C=N. By comparing the FTIR of FA / CS, FA, and CS, the absorption peak of FA / CS was found to be at 1655 cm⁻¹. -1 and 1598 cm -1 It disappeared at 1512 cm. -1 and 1609cm -1The new absorption peak at 1512 cm⁻¹ is attributed to the stretching vibration of C=C in the aromatic ring backbone. These changes are caused by the chemical reaction between the -OH group in fatty acid (FA) and the -NH₂ group in chitosan (CS). Furthermore, at 1512 cm⁻¹... -1 The absorption peak at 1169 cm⁻¹ is related to the bending vibration of NH (amine II) in FA / CS. This indicates that the -COOH in the fatty acid reacts with the -NH₂ in chitosan to form an amide bond, demonstrating the successful synthesis of the FA / CS linker. -1 and 1213 cm -1 The absorption peak at 900 cm⁻¹ is related to the stretching vibration and P=O vibration of the OPO group in TPP. Furthermore, the absorption peak at 900 cm⁻¹... -1 The peak at this point is related to the stretching vibration of the POP bridge in the TPP. Meanwhile, the peak at 1541 cm⁻¹ in the FA / CS / TPP... -1 The peak at 3224 cm⁻¹ is attributed to the CO-NH stretching vibration. β-acids, as active substances, have a characteristic peak at 3224 cm⁻¹. -1 In addition, 2971 cm -1 The peak at 1665 cm⁻¹ is attributed to the CH vibration of the β-acid. -1 The peak at 3403 cm⁻¹ is attributed to the C=O vibration of the β-acid, consistent with previous studies. The addition of the β-acid altered the FTIR absorption spectrum. The -OH group in the β-acid interacts with the FA / CS / TPP nanoparticles, shifting the -OH absorption peak to 3403 cm⁻¹. -1 This formed a 2971 cm specific to β-acids. -1 The absorption peak.

[0034] like Figure 2-1As shown in Figure B, FA / CS / TPP, FA / CS / TPP / 7.5β-acid, FA / CS / TPP / 15β-acid, and FA / CS / TPP / 22.5β-acid are all spherical or nearly spherical, but all exhibit severe aggregation and adhesion. Although the nanoparticles are not uniform in size, their sizes are all slightly less than 300 nm. With the increase of β-acid content in the nanoparticles, the surface of the nanoparticles becomes smoother. This phenomenon can be attributed to the fact that the presence of β-acid improves the adhesion of the nanoparticles to some extent and increases the aggregation of the nanoparticles. Compared with nanoparticles without added β-acid, the adhesion of FA / CS / TPP nanoparticles is reduced. The morphology of the nanoparticles was further analyzed by TEM. Due to the presence of β-acid, the dispersion of the drug-loaded nanoparticles is severely weakened, resulting in the lack of clear connection boundaries between the nanoparticles. This phenomenon may be explained by the formation of intermolecular hydrogen bonds between FA / CS / TPP particles and β-acid. Studies have suggested that the aggregation of nanoparticles may be due to increased interparticle attraction, stemming from a higher surface area to volume ratio. Furthermore, the core-shell structure of polyethylene glycol / chitosan-stabilized nanoparticles is separated, resulting in a wider distribution of nanoparticles due to the use of polymers. Additionally, some studies indicate that the presence of agglomerated particles in the final sample is due to the particles tightly bonding together and forming clumps during the co-precipitation method used to prepare the sample. In this case, interactions occur between the anions and cations on the surface, leading to mutual attraction and thus clumping.

[0035] The crystal structure of drug-loaded nanoparticles is typically characterized by X-ray diffraction (XRD). This invention can provide information for the subsequent release of drugs from nanoparticles. Figure 2-2(C). It has been reported that some drugs, when loaded into nanoparticles, exist in amorphous or solid forms. The X-ray diffraction pattern of CS shows a distinct diffraction peak at 2θ = 21°, but this peak is not sharp, indicating that CS mainly exists in an amorphous state. The peaks of β-acid at 2θ = 8.8°, 9.2°, and 30.1° indicate that it is essentially crystalline. The X-ray diffraction peaks of TPP as a crosslinking agent are mainly located at 2θ = 19.7°, 20.3°, 20.8°, 26.0°, 35.0°, 35.7°, and 36.2°. When FA and CS are crosslinked through TPP, the diffraction peak formed by the blank nanoparticles is located at 2θ = 22.2°, and this peak is broad, indicating that the nanoparticles formed by crosslinking FA-modified CS with TPP are in an amorphous state. When β-acids were added to the FA / CS / TPP system, the diffraction peaks of the drug-loaded nanoparticles reappeared at approximately 8.7° 2θ compared to the blank nanoparticles (FA / CS / TPP), indicating that the β-acids were successfully loaded but existed in an amorphous form.

[0036] These drug-loaded nanoparticles are amorphous. Analysis of the preparation process revealed that chitosan dissolved in the acidic solution may carry a positive charge, as do the stable polymer nanoparticles formed by crosslinking chitosan after FA modification with TPP. FA / CS / TPP interacts with β-acids to form FA / CS / TPP / β-acid, with the positive charge decreasing upon the addition of β-acid. All these ionic interactions play a crucial role in the crosslinking and subsequent formation of drug-loaded nanoparticles, enabling rapid crosslinking in solution and the formation of stable polymers. β-acids are small molecules that rapidly bind to blank nanoparticles during drug-loaded nanoparticle formation, thus existing in an amorphous form after incorporation into the polymer.

[0037] In the determination of nanoparticle size, it was found that although the content of β-acid gradually increased, there was no significant difference compared with FA / CS / TPP. The particle size distribution was as follows: 286.67±4.63, 266±16.06, 286±12.03 and 265±8.22 nm, respectively. Figure 2-2The nanoparticle size observed by particle size analysis was larger than that observed by TEM / SEM, which can be explained by the expansion of the nanoparticles in water. In contrast, the nanoparticles were prepared by direct drying using TEM / SEM. However, measuring their size in body fluids by TEM / SEM does not accurately reflect their actual size in physiological body fluids. Furthermore, complex components in body fluids can adsorb onto the surface of nanoparticles, thus increasing their overall size. Assessing particle size in solution—especially in media supplemented with serum—is crucial for simulating physiological body fluids. The nanoparticle size measured by DLS was within an acceptable range, indicating successful preparation of nanoparticles using ionogel technology. During the determination of zeta potential, the potential of the FA / CS / TPP nanoparticles was found to be +12.73 mV ( Figure 2-2 (D). β-Acids are negatively charged molecules. The high positive charge on the surface of nanoparticles promotes their binding to negatively charged cell membranes, making them ideal drug delivery platforms. Ionogelation is a common strategy for drug encapsulation. β-Acids, hydrophobic drugs, are stabilized through electrostatic interactions and hydrogen bonding. The encapsulation efficiencies of different drug-loaded nanoparticles were 20.97±0.89%, 20.79±1.04%, and 32.19±1.11%, respectively, and the encapsulation efficiencies were 44.61±1.80%, 45.31±1.49%, and 43.41±1.31%, respectively. Figure 2-2 (D). Encapsulation efficiency and encapsulation percentage efficiency are parameters related to the ability of different raw materials to encapsulate drugs and the quality of the formulation. These parameters mainly depend on the type of raw material, drug polarity, and the preparation method of the nanoparticles.

[0038] (3) Hydrogel characterization Sodium alginate is an anionic polysaccharide composed of mannuronic acid (M) and guluronic acid (G). In aqueous solution, it dissociates sodium ions and stretches its molecular chains; upon the addition of calcium ions, ion exchange occurs, and Ca... 2+Precisely embedded in the spatial grooves of high-rigidity GG chain segments, intermolecular calcium bridges are constructed through ionic and coordination bonds based on the egg-box model, achieving physical cross-linking of multiple polymer chains, building a dense hydrophilic three-dimensional network and firmly locking in water to rapidly form a hydrogel. The higher the G unit content, the greater the gel strength. This cross-linking process is mild and controllable at room temperature, and the resulting gel exhibits thermally irreversible properties, only capable of being broken down by chelating agents or high-concentration sodium salts. Alg / FA / CS / TPP without β-acid was prepared following the steps in Example 1. To characterize Alg, Alg / FA / CS / TPP, Alg / FA / CS / TPP / 7.5β-acid, Alg / FA / CS / TPP / 15β-acid, and Alg / FA / CS / TPP / 22.5β-acid, and to evaluate their interactions with cells, the sol-gel-sol transition of the hydrogel was studied in vitro. The morphological changes of the hydrogel under simulated pH conditions in the stomach (1.2), small intestine (6.8), and large intestine (7.4) were studied to determine whether it exhibited a sol-gel-sol transition. Figure 3 In the mixture A: (I) Alg; (II) Alg / FA / CS / TPP; (III) Alg / FA / CS / TPP / 7.5β-acids; (IV) Alg / FA / CS / TPP / 15β-acids; (V) Alg / FA / CS / TPP / 22.5β-acids. When the hydrogel is exposed to pH 1.2, it immediately forms a gel. Furthermore, this gel structure does not decompose upon exposure to a medium at pH 6.8 and retains its non-flowing semi-solid properties. On the other hand, when exposed to a medium at pH 7.4, the gel completely decomposes and transforms back into a sol. These experimental results confirm that the hydrogel prepared in this invention exhibits sol-gel-sol transition characteristics.

[0039] Fourier transform infrared spectroscopy (FTIR) was used to characterize the hydrogel. First, the FTIR characteristic peak of sodium alginate includes 3428 cm⁻¹. -1 The OH stretching vibration peak at 1612 cm⁻¹ -1 CO stretching vibration peak at 1417 cm⁻¹ and 1417 cm⁻¹ -1 The CO stretching vibration peak at this location indicates the presence of a -COOH group in sodium alginate (Alg). When sodium alginate reacts with Ca... 2+ When the hydrogel was successfully crosslinked and formed, it was at 1598 cm⁻¹ -1 and 1416 cm -1 Characteristic absorption peaks will appear at these locations. Studies have shown that these absorption peaks are related to the stretching vibrations of -COO-, with most of them related to Ca. 2+ A three-dimensional cross-linked network forms the hydrogel. Compared with the FTIR of sodium alginate, its wavenumber shifts to the lower frequency region (1598 cm⁻¹). -1This further demonstrates the relationship between -COO- and Ca. 2+ There may be interactions between them. Sodium alginate interacts with calcium ions, and a similar shift occurs in FTIR, following the interaction with Ca. 2+ The infrared characteristic peak changes with the content. Adding blank nanoparticles to the hydrogel did not result in a significant shift in the infrared characteristic peak. However, after adding β-acid, the characteristic absorption peak of β-acid (2974 cm⁻¹) changed. -1 1758 cm -1 The β-acid's active groups interact with the post-hydrogel molecules, causing changes in the infrared absorption peaks. Specifically, the OH stretching vibration peak appears at 3402 cm⁻¹. -1 The CO stretching vibration peak appears at 1598 cm⁻¹. -1 The CO stretching vibration peak appears at approximately 1420 cm⁻¹. -1 The above experimental results demonstrate that drug-loaded nanoparticles have been successfully loaded into sodium alginate hydrogel. Scanning electron microscopy (SEM) results of the hydrogel are shown below. Figure 3 As shown in C. It consists of sodium alginate and Ca. 2+ Cross-linked hydrogels exhibit a sparsely porous and wrinkled surface structure. Adding blank nanoparticles reduces surface wrinkles but introduces smaller structures. With increasing drug-loaded nanoparticle content, the hydrogels exhibit a more pronounced three-dimensional network structure. These hydrogels share similar morphological characteristics, exhibiting a significant three-dimensional network structure and large pore sizes.

[0040] exist Figure 3 In the angular frequency sweep test (1-100 radians / second) of D, both hydrogels exhibited frequency-dependent viscoelastic responses: all experimental hydrogels showed significant elastic behavior (G'>G''), with the storage modulus (G') exceeding the loss modulus (G''). The storage modulus G' of the Alg / FA / CS / TPP hydrogel was higher than that of the Alg / FA / CS / TPP / β-acid hydrogel, and the introduction of β-acid was mainly due to the enhancement of the cross-linking network, which increased G'. Furthermore, the shear recovery properties of the hydrogel microcapsules were investigated by applying alternating high-shear and low-shear strain cycles. Figure 3 Rheological analysis showed that both the blank hydrogel and the drug-loaded hydrogel exhibited good mechanical compatibility. Figure 3 (F). Within a strain range of 0.01% to 1000%, the storage modulus G' is dominant and remains stable, indicating that these hydrogels are in a solid state. Figure 3 (G). Subsequently, when G'' exceeds G', the hydrogel transitions to a quasi-fluid state rather than a solid gel.

[0041] (4) In vitro release of nanoparticles and hydrogels The release of β-acids from nanoparticles was investigated. In most cases, the pH of human tumors is approximately 6.8, while the pH of normal tissues is approximately 7.4. The cumulative release percentage of β-acids from nanoparticles is shown below. Figure 4 As shown. The release curves were similar under three different pH conditions. A biphasic kinetic pattern was observed in the release of the β-acid: initially rapid release followed by slow release. In the initial rapid release phase, the β-acid was released quickly, while in the subsequent steady phase, it was released slowly through degradation maintained by diffusion. Figure 4 (AC). The nanoparticle group exhibits a two-stage release pattern. The initial rapid drug release is likely due to the dissolution of drug adsorbed on the nanoparticle surface and increased drug diffusion on the medium surface due to the large surface area of ​​the nanoparticles. The slow drug release stage is likely due to its encapsulation in the polymer matrix. The cumulative release of β-acid decreases with decreasing pH, and the nanoparticles degrade faster in alkaline media. Experiments show that the nanoparticles are more stable under acidic conditions. Under alkaline conditions, the diffusion of β-acid increases significantly. This may be due to the deprotonation of amino groups on the chitosan chains under alkaline conditions, leading to a weakening or disappearance of the ionic interaction between cationic chitosan and anionic TPP, thereby accelerating the release of β-acid. For example, in the initial rapid release stage at pH 7.8, the release percentages of nanoparticles with different β-acid loadings were 17.5% (FA / CS / TPP / 7.5β-acid), 16% (FA / CS / TPP / 15β-acid), and 21% (FA / CS / TPP / 22.5β-acid). In the subsequent stable release phases, the concentrations were 18% (FA / CS / TPP / 7.5β-acid), 16.5% (FA / CS / TPP / 15β-acid), and 22% (FA / CS / TPP / 22.5β-acid). During stable release, both the cumulative release amount and release rate of β-acid decreased significantly, likely due to buffer permeation into the nanoparticles and subsequent disintegration. Ultimately, the β-acid diffused from the dissolved polymer. As previously mentioned, the in vitro release of the nanoparticles was pH-dependent.

[0042] (5) Drug release from the hydrogel.

[0043] Nanoparticles / hydrogel (5 mg) were placed in a dialysis cellulose membrane (MWCO: 3500 Da, Beijing Solab Technology Co., Ltd.) and immersed in 30 mL of PBS with different pH values ​​(1.2, 6.8, 7.8). In vitro drug release was studied by shaking at 37 °C and 220 rpm / min. At fixed time points, 2 mL of the release solution was aspirated for absorbance measurement, and fresh PBS (2 mL) was added simultaneously.

[0044] The results showed that the release curve of β-acids in hydrogels also exhibited a sudden and sustained release process. At pH 1.2, the release amounts of artificial intestinal fluid from different drug-loaded hydrogels were 19.6% (Alg / FA / CS / TPP / 7.5β-acid), 21.0% (Alg / FA / CS / TPP / 15β-acid), and 24.3% (Alg / FA / CS / TPP / 22.5β-acid), respectively. At pH 6.8, the cumulative release amounts of β-acids were approximately 20.3% (Alg / FA / CS / TPP / 7.5β-acid), 24.1% (Alg / FA / CS / TPP / 15β-acid), and 24.1% (Alg / FA / CS / TPP / 22.5β-acid), respectively. At pH 7.8, the release rates of the active ingredient β-acid were 24.2% (Alg / FA / CS / TPP / 7.5β-acid), 26.5% (Alg / FA / CS / TPP / 15β-acid), and 32.1% (Alg / FA / CS / TPP / 22.5β-acid), respectively. This observation indicates that the total drug release reaches its maximum at pH 7.4 in the presence of a hydrogel, suggesting that the drug-loaded hydrogel can accelerate the release of β-acid from the nanoparticles. Therefore, reducing direct drug contact with intestinal cells during actual administration helps overcome drug side effects. In vivo release is expected to differ compared to in vitro conditions.

[0045] After establishing the mathematical model, the release mechanism of acidic substances can be explained more clearly (see Table 1). Three mathematical models (first-order model, Higuchi model, and Ritger-Peppas model) were selected to adapt the release of β-acids in nanoparticles and hydrogels. The first-order, Higuchi, and Ritger-Peppas models were chosen because they are a classic combination for fitting in vitro drug release in pharmaceuticals, and can comprehensively cover the mainstream release behavior of drug-loaded nanoparticles: the first-order model characterizes concentration gradient-driven dissolution-type release, the Higuchi model describes the Fick diffusion-controlled release characteristics of drugs within the nanoframework, and the Ritger-Peppas model can accurately distinguish diffusion, swelling, dissolution, or mixing mechanisms through the release index n; their mathematical forms are simple and easy to fit, the physical meaning of the parameters is clear, the reliability of the results can be mutually verified, and they are universal standard methods in the field, which can systematically elucidate the release law and intrinsic controlled release mechanism of nanoparticles. According to R 2 The test results show that the R² of the Rittg-Peppas model is better than that of the first-order model and the Sigurd model. The diffusion index n in the Rittg-Peppas model is less than 0.45, indicating that the release mechanism of β-acids in nanoparticles and hydrogels follows the Fick diffusion process.

[0046] Table 1. Parameters for fitting the β-acid release curve using the model.

[0047] (6) To investigate the effects of nanoparticles and hydrogels on the proliferation of HCT116 cells, the CCK-8 assay was used to determine cell viability. Figure 5 (A and B in the middle).

[0048] The results showed that β-acid, 5-FU, drug-loaded nanoparticles, and hydrogels all exhibited concentration-dependent effects on the proliferation of target cells. Weighing nanoparticles containing the same drug concentration, the corresponding experimental results indicated that drug-loaded nanoparticles at concentrations greater than 40 μg / mL showed better inhibitory effects on HCT116 cells than β-acid and 5-FU. For HCT116 cells, the inhibitory effect of drug-loaded nanoparticles on cell proliferation can be attributed to the targeting effect of FA by the nanoparticles, allowing β-acid to enter the cancer cells and exert its effects. Similar experiments also confirmed that when the concentration of nanoparticles in the hydrogel was 80 μg / mL, the drug-loaded hydrogel showed superior inhibitory effects on HCT116 cells compared to β-acid and 5-FU.

[0049] The study also showed that FA-grafted-chitosan oligosaccharide-chitosan nanoparticles loaded with cytarabine enhanced cytotoxicity against cancer cells (MCF-7). This enhanced toxicity may be due to receptor-mediated endocytosis, which enhances the endocytosis of cytarabine and improves the penetration and retention rate (EPR) in MCF-7 cells. Tumor cells have a gap of approximately 50 to 500 nanometers surrounding their endothelial cells, allowing molecules smaller than 500 nanometers to penetrate and accumulate. FA-modified nanoparticles improve the bioavailability of the loaded drug through active targeting and the EPR effect. Furthermore, the sustained drug release from these nanoparticles prolongs drug exposure time, thereby enhancing their cytotoxic activity.

[0050] To further investigate the effects of drug-loaded nanoparticles (FA / CS / TPP / 22.5β-acid) on cell migration, a scratch assay was performed. The results showed that the proliferation of HCT116 cells was effectively inhibited in the presence of drug-loaded nanoparticles, with effects superior to 5-FU and similar to those observed with CCK-8. Figure 5 (C). Furthermore, this study analyzed the effects of different concentrations of nanoparticles (0-40 μg / mL) on normal cell proliferation to demonstrate their non-toxicity to normal cells and their potential as therapeutic agents. The study found that even at a concentration of 40 μg / mL, co-culturing nanoparticles with NCM460 cells for 24 hours had no effect on cell proliferation, indicating that the drug-loaded nanoparticles possess good cell safety characteristics. Figure 5 (D).

[0051] Multiple studies have shown that nanocarriers play a crucial role in delivering sufficient anticancer drugs to cancer cells, which helps reduce drug side effects. To investigate the targeting effect of the nanoparticles prepared in this study on the FA receptor, coumarin-6-loaded nanoparticles were co-cultured with HCT116 cells and analyzed using confocal microscopy. Previous studies have shown that the FA receptor is a well-known tumor marker, highly expressed on the cell membrane surface of colorectal cancer cells, and that both free FA and FA-modified nanomaterials can be recognized and tightly bind to the FA receptor. Furthermore, flow cytometry apoptosis detection revealed that both drug-loaded nanoparticles and drug-loaded hydrogels accelerated apoptosis in HCT116 cells. Figure 6 (A). Furthermore, even when drug-loaded nanoparticles were loaded into hydrogels, the inhibitory effect of the hydrogels on HCT116 cells was similar to that of β-acids. Confocal microscopy images clearly showed strong DID red fluorescence and C6 green fluorescence in HCT116 cells after co-incubation of coumarin-6-loaded nanoparticles. This indicates that C6-loaded nanoparticles can be significantly taken up by HCT116 cells via FA receptor-mediated endocytosis. In addition, HCT116 cells were pretreated with free FA to block FA receptors, and then C6-loaded nanoparticles were co-incubated with these cells to further confirm their recognition ability. These results indicate that FA compound-modified nanoparticles promote FA receptor-mediated endocytosis, and these nanoparticles hold promise as targeted nanocarriers for FA receptor-positive cancers.

[0052] Furthermore, the uptake of drug-loaded nanoparticles by HCT116 cells was investigated when these nanoparticles were loaded into hydrogels. Cell images obtained by CLSM were obtained after HCT116 cells were co-incubated for 1 hour with FA / CS / TPP / Coumarin-6, Alg / FA / CS / TPP / Coumarin-6, Alg / FA / CS / TPP / Coumarin-6+FA, and Alg / CS / TPP / Coumarin-6, respectively.

[0053] Similar to the cellular uptake experiments of drug-loaded nanoparticles, loading nanoparticles modified with compound A increased cellular uptake. Figure 6 (Middle B). From left to right, the images show DAPI fluorescence (blue), Coumarin-6 (C6) fluorescence (green), and a superposition of the three. The images were obtained at 40x magnification. Scale bar: 10 micrometers.

[0054] (7) In vivo biosafety and anticancer therapy of nanoparticles and hydrogels Twenty-one mice were administered nanoparticles via gavage (100, 200, 400 mg / kg) to investigate their potential toxicity. Weight and behavioral changes in the mice were monitored 14 days post-administration. All animals appeared healthy and active, exhibiting no abnormal symptoms (e.g., hyperactivity / hypoactivity, abnormal breathing, lethargy, etc.). All animals survived the entire study period; no deaths were observed. There were no significant differences in body weight between the experimental and control groups.

[0055] Following the gavage procedure, further investigation was conducted to determine whether the drug carrier / nanomedicine could alter the blood levels of biomarkers of tissue damage or inflammation. Figure 7 (A) First, changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured to determine whether the nanoparticles caused hepatotoxicity. Although oral administration of different doses of the carrier / nanoparticles increased changes in ALT and AST levels, these doses remained within the normal range. Furthermore, total protein (TP) levels were measured to reflect changes in liver function. The 14-day intake results showed that the nanocarrier / nanoparticles did not affect TP changes, therefore, nanoparticle intake did not alter liver function. Additionally, renal function was assessed by measuring urea (UREA) and creatinine (CREA) levels. After 14 days of administration, no significant differences were found between the control and experimental groups; all these indicators were below the toxicity threshold.

[0056] After 14 days of treatment with different doses, no changes were observed in any of the organs, including the heart, liver, spleen, lungs, and kidneys, compared to the control group, as determined by visual observation. Figure 7 (B) No signs of atrophy, hyperplasia, necrosis, or inflammation were observed. To further investigate changes in microstructure, histological examination with H&E staining was performed on the aforementioned tissues. Compared to the control group, the microstructure of the heart, liver, spleen, lungs, and kidneys showed no signs of inflammation or fibrosis. These results indicate that oral ingestion of nanoparticles does not affect organ function.

[0057] In further investigating the therapeutic effect of FA / CS / TPP / 22.5β-acid nanoparticles on colorectal cancer (CRC), an orthotopic CRC model was constructed using MC38 cells. Starting from day 6 after MC38 cell implantation, mice were intraperitoneally injected with 5-FU every other day and orally administered FA / CS / TPP / 22.5β-acid (400 mg / kg) for 7 consecutive days. Tumor-bearing mice were sacrificed on day 14, and the colon was removed to assess the drug's effect on the tumor. Figure 7(C). Experimental results showed that the colon of healthy mice was in a normal state (PBS group), while the colon of the control group had tumors, indicating that the modeling using MC38 was successful. After treatment with FA / CS / TPP / 22.5β-acid nanoparticles and 5-FU, the tumors located in the colon disappeared. When assessing the body weight of mice, the body weight of the orthotopic colon cancer model showed a trend of first increasing and then stabilizing, while the 5-FU group showed a trend of first increasing and then decreasing. Figure 7 (A) Compared with the control group, the tumor volume and number in other drug-treated groups were reduced, which undoubtedly indicates that drug-loaded nanoparticles have a significant ability to inhibit tumor growth. Figure 7 (C and D in section 7). H&E staining was used to assess the severity of colon cancer and tumor cell apoptosis. Figure 7 (H&E). As can be seen, the colonic muscle layer and mucosa of healthy mice (PBS group) are clearly layered and structurally intact, with no diseased cells observed in the magnified H&E images. In contrast, tumor cells in the control group are severely invasive, destroying the mucosal and muscular layers of the colonic tissue. Notably, after treatment with FA / CS / TPP / 22.5β-acid, the colonic sections were as healthy as those in the normal mouse group, without tumors. The tumor invasion of the anterior mucosal structure was well preserved, indicating a significant therapeutic effect.

[0058] In in vivo antitumor studies, drug-loaded nanoparticles (FA / CS / TPP / 22.5β-acid) were found to have good antitumor effects; therefore, they were chosen to be loaded into sodium alginate hydrogel for the treatment of colorectal cancer. Similar to the in vivo safety studies of the nanoparticles, oral administration of different doses of the hydrogel showed normal results within the normal range after 14 days. Figure 8 (A). Experimental results showed that the intake of hydrogel did not affect changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), creatinine (CREA), and urea (UREA). Therefore, the intake of hydrogel did not alter liver and kidney function. Furthermore, H&E staining was performed on various mouse organs. The corresponding results indicated that no significant toxicity was observed with different doses of hydrogel. Figure 8 (Middle B). Therefore, an orthotopic tumor-bearing mouse model was used with a dose of 400 mg / kg. After 14 days of treatment, the mice treated with hydrogel showed no significant change in body weight, while the control group mice showed a significant decrease in body weight. At the end of the experiment, imaging examination showed that the hydrogel did not reduce the length of the colon in the mice. The tumor implanted in the colon region shrank significantly, which confirmed that the drug-loaded hydrogel has excellent ability to inhibit tumor growth. Figure 8 C). H&E staining ( Figure 8(D) shows typical tumor features in the tissues of the control group, 5-FU group, and drug-loaded hydrogel-treated group, including damage to the mucosal structures on the colonic wall. After hydrogel treatment, tissue sections showed that the tumor area was confined, while the mucosal structures at the invasive front remained intact, indicating significant treatment efficacy.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a folic acid-modified chitosan nanoparticle-sodium alginate hydrogel loaded with β-acid, characterized in that, Includes the following steps: (1) Folic acid, 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide were dissolved in DMSO and stirred to obtain folic acid active ester; (2) Add folic acid active ester to CS solution to react and obtain FA / CS powder, dissolve it in acetic acid solution, and adjust the pH to obtain FA / CS solution; (3) Add the β-acid ethanol solution to the FA / CS solution, stir, then add the TPP solution and continue stirring to obtain FA / CS / TPP / β-acid; (4) Sodium alginate solution and CaCl2 solution were mixed to obtain Alg hydrogel; (5) FA / CS / TPP / β-acid and Alg hydrogel were mixed to obtain folic acid modified chitosan nanoparticles loaded with β-acid-sodium alginate hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of folic acid, 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide is 1:1~2:1~2; the stirring time is 2~4h.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the CS solution is 0.5~1g / mL; the volume ratio of the CS solution to the folic acid active ester is 4~6:

1.

4. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the β-acid ethanol solution is 0.04~0.2 mg / mL; the concentration of the FA / CS solution is 5~25 mg / mL; the concentration of the TPP solution is 1~3 mg / mL; and the volume ratio of the β-acid ethanol solution, FA / CS solution and TPP solution is 5~25:90~110:10~30.

5. The preparation method according to claim 1, characterized in that, In step (3), the stirring time is 20-40 min; the stirring time is 0.8-1.2 h.

6. The preparation method according to claim 1, characterized in that, In step (4), the concentration of sodium alginate solution is 1-2%; the concentration of CaCl2 solution is 1-2%; and the volume ratio of sodium alginate solution to CaCl2 solution is 1-3:

1.

7. The preparation method according to claim 1, characterized in that, In step (5), the volume ratio of the FA / CS / TPP / β-acid to the Alg hydrogel is 10:2~4.

8. A folic acid-modified chitosan nanoparticle-sodium alginate hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the folic acid-modified chitosan nanoparticles-sodium alginate hydrogel of claim 8 in the preparation of a colorectal cancer treatment drug.

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