Preparation method and application of folic acid modified beta-acid loaded chitosan nanoparticles

Abstract

The invention discloses a preparation method and application of folic acid modified beta-acid loaded chitosan nanoparticles. The preparation method comprises the following steps: (1) dissolving FA, NHS and EDC in DMSO according to a molar ratio of 1: 1.5: 1.5; (2) adding chitosan into a 1% acetic acid solution, and stirring for dissolving; (3) adding the activated folic acid active ester in the step (1) into the chitosan aqueous solution in the step (2), and mixing and stirring to obtain a mixed solution 1; (4) adding a beta-acid ethanol solution into the mixed solution 1 prepared in the step (3), and stirring to obtain a mixed solution 2; and (5) mixing and stirring a sodium tripolyphosphate solution and the mixed solution 2 prepared in the step (4), and centrifuging to obtain the folic acid modified beta-acid loaded chitosan nanoparticles. The nanoparticles prepared in the invention not only have a certain bacteriostatic effect on staphylococcus aureus, but also can be successfully ingested by HCT116 cells, and in-vivo safety experiments of mice find that the drug-loaded nanoparticles are safe.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and cancer treatment, and particularly relates to a preparation method and application of folic acid-modified chitosan nanoparticles loaded with β-acid. Background Art

[0002] Colorectal cancer (CRC) is the most common cancer in the world, with nearly 1.4 million new cases and approximately 774,000 deaths globally each year. Due to late diagnosis and poor treatment, CRC treatment has become a major economic and health burden for humanity. Currently, the main clinical treatments for CRC are surgery, radiotherapy, and chemotherapy, among which chemotherapy is one of the most promising cancer treatment methods. Conventional CRC treatments have some limitations, such as insufficient efficacy, severe adverse reactions, and innate and acquired drug resistance. It is worth noting that CRC patients with unresectable tumors are usually resistant to conventional chemotherapy. 5-Fluorouracil (5-FU) is a first-line drug for treating CRC, but resistance to 5-FU is a major clinical challenge. Therefore, exploring targeted therapy, neoadjuvant chemotherapy, and immunotherapy for CRC has become a breakthrough in cancer treatment. Despite the heterogeneity of CRC leading to certain differences in treatment, researchers are still seeking to develop more alternative and effective drugs to ensure human safety and health. Interestingly, targeted drugs can identify patients with overexpressed cancer cell surface receptors, enabling appropriate selection of patients for personalized treatment and achieving ideal targeted therapy. Nanoparticle drugs can improve the drug delivery system, greatly enhancing the efficacy of the drug while reducing the occurrence of side effects. In addition, nanoparticles can be modified to further improve the selectivity and specificity of treatment. Therefore, finding new anticancer drugs targeting the key pathogenic factors of CRC has become a major breakthrough. Importantly, the abuse of traditional antibiotics has led to the emergence of drug-resistant pathogens, so it is necessary to avoid infection during cancer treatment. It has been reported that after long-term chemotherapy for cancer, the patient's body immunity severely declines, making them vulnerable to bacterial attacks, and inflammatory cells can also affect cancer metastasis.

[0003] To enhance the targeting of drug carriers, nanoparticles are modified to achieve better anti-cancer effects. Folic acid (FA) is an essential micronutrient involved in DNA, RNA, and protein metabolism. Generally, low or even deficient FA levels are associated with many diseases, such as neurological diseases, cancer, and cardiovascular diseases. In addition, FA receptors are overexpressed on the surface of human tumor cells (breast, ovary, uterus, kidney, colon, and lung), making folic acid an ideal tumor marker for targeted drug delivery. Compared with normal cells, the expression of FA receptors on tumor cells is about 100 - 300 times higher. Moreover, compared with other ligands, FA has advantages such as receptor specificity, good coupling property, accessibility, and non-immunogenicity, enabling site-specific delivery of FA-modified nanoparticles. When FA-conjugated nanoparticles bind to FA receptors on tumor cells, they are internalized through receptor-mediated endocytosis, thus enhancing the retention of drugs in the tumor environment.

[0004] The selection of carrier materials is a key factor in the preparation of nano-delivery systems, and their physicochemical properties and the formation of nanoparticles have a great impact on the controlled release and physiological activity of drugs. Some research reports that different biopolymers, such as chitosan, sodium alginate, gelatin, etc., when provided with FA modification, can significantly improve the effective delivery of active ingredients. Chitosan (CS) is an ideal and most widely used biocarrier for food and nutritional health care. As a naturally sourced biodegradable bio-polyamino sugar, CS has excellent biocompatibility, low toxicity, low price, etc., and has been widely used in the food and pharmaceutical industries. Different forms of drug delivery systems based on CS have been found in many fields, and CS nanoparticles are one of the most advanced drug delivery systems for improving the efficacy of anti-cancer drugs. CS nanoparticles can be used for the controlled release of different types of drugs to improve the tumor drug delivery effect. FA-conjugated CS derivative nanoparticles can improve the solubility and stability of drugs, control drug release, enhance drug efficacy and bioavailability, and reduce drug adverse reactions. At the same time, FA-modified nanoparticles can not only be used for oral administration but also for mucosal (nasal cavity, lungs, etc.) administration. Such nanoparticles have good application potential in tumor-specific anti-cancer drug delivery, gene therapy, and imaging diagnosis.

[0005] In recent years, anticancer drugs / active ingredients such as curcumin, doxorubicin, thymoquinone, and vincristine have been loaded into nanoparticles modified with FA to improve tumor-targeted drug delivery. When a natural active product has excellent properties such as antibacterial and anticancer effects, the active ingredient can play a great role if it is used as a potential active ingredient for treating cancer. Humulus is a perennial plant of the Cannabaceae family and is widely cultivated worldwide. Due to its unique flavor, hops are mainly used in the beer brewing industry. Different bioactive ingredients have been found in hops and have been proven to have positive health benefits and biological activities, including antioxidant, anticancer, anti-allergic, and antibacterial effects. Interestingly, β-acid is an active ingredient extracted from the soft resin of hops, and it has been found to have excellent antibacterial, antioxidant, and anticancer properties. Therefore, β-acid has potential application prospects in the pharmaceutical industry.

[0006] This technology ensures the safety of β-acid-loaded nanoparticles modified with folic acid in vivo, and the above nanoparticles not only have targeting properties for colorectal cancer but also have good antibacterial effects. Summary of the Invention

[0007] The object of the present invention is to provide a preparation method and application of chitosan nanoparticles loaded with β-acid modified with folic acid.

[0008] A preparation method of chitosan nanoparticles loaded with β-acid modified with folic acid specifically includes the following steps:

[0009] (1) Dissolve folic acid, N-hydroxysuccinimide, and 1-ethyl-3-(dimethylaminopropyl)carbodiimide in DMSO and stir to obtain folic acid active ester;

[0010] (2) Add chitosan to an acetic acid solution, stir to dissolve, and prepare an aqueous chitosan solution;

[0011] (3) Add the activated folic acid active ester in step (1) to the aqueous chitosan solution in step (2), mix and stir to obtain a mixed solution 1. After the reaction, adjust the pH value to 9 with NaOH, centrifuge to obtain a yellow precipitate, wash the yellow precipitate with saturated NaHCO3 solution, then wash with deionized water, and freeze-dry to obtain FA / CS powder. Dissolve the FA / CS powder in an acetic acid solution to obtain an FA / CS solution, and adjust the pH to 5.0 with NaOH;

[0012] (4) Dropwise add the β-acid ethanol solution to the solution prepared in step (3) and stir to obtain a mixed solution 2;

[0013] (5) Dropwise add the sodium tripolyphosphate solution to the mixed solution 2 prepared in step (4) and stir for 1 h, centrifuge and then freeze-dry to obtain the modified chitosan nanoparticles loaded with β-acid modified with folic acid.

[0014] Further, in step 1, the stirring is carried out at 30 °C in the dark for 3 h.

[0015] Further, in step 1, the molar ratio of folic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:1.5:1.5.

[0016] Further, in step 2, chitosan is dissolved in a 1% acetic acid solution to form an aqueous chitosan solution with a final concentration of 0.5%.

[0017] Further, the mixing and stirring in step (3) is carried out at 25 °C for 24 h.

[0018] Further, in step (3), centrifugation is carried out at 4 °C at 12000 rpm for 10 min.

[0019] Further, the yellow precipitate is washed 3 times with a saturated NaHCO3 solution, the precipitate is washed 3 times with deionized water, and freeze-dried for 24 h to obtain FA / CS powder.

[0020] Further, the concentration of the sodium tripolyphosphate solution in step (5) is 2 mg / mL.

[0021] Further, in step (5), the centrifugation parameter is centrifugation at 12000 rpm for 20 min.

[0022] Further, the antibacterial activity includes Staphylococcus aureus and Escherichia coli.

[0023] The present invention has the following beneficial effects: The nanoparticles prepared in the present invention not only have a certain antibacterial effect on Staphylococcus aureus, but can also be successfully taken up by HCT116 cells. The safety experiment in mice found that the drug-loaded nanoparticles are safe. Description of the Drawings

[0024] Figure 1 Characterized the characteristic absorption peaks of CS, FA, TPP, β-acid, FA / CS, FA / CS / TPP, and FA / CS / TPP / β-acid;

[0025] Figure 2 Is the XRD diffraction pattern of the nanoparticles;

[0026] Figure 3 Is the scanning electron micrograph of the prepared nanoparticles;

[0027] Figure 4 Is the transmission electron micrograph of the morphology of the blank and drug-loaded nanoparticles;

[0028] Figure 5Graph for particle size detection and determination of the encapsulation efficiency (EE) and drug loading content (LC) of β-acid in nanoparticles by ultraviolet spectroscopy;

[0029] Figure 6 Graph for the cumulative percentage of β-acid released from nanoparticles at different simulated in vivo pH values;

[0030] Figure 7 Graph for the minimum bactericidal concentration (MBC) of β-acid and drug-loaded nanoparticles against Staphylococcus aureus and Escherichia coli;

[0031] Figure 8 Scanning electron microscopy images of β-acid and drug-loaded nanoparticles against Staphylococcus aureus and Escherichia coli;

[0032] Figure 9 Showing the effects of six materials on the viability of HCT116 cells;

[0033] Figure 10 Graph for the effects of six materials on the migration ability of HCT116 cells, and the effect is better than 5-FU, similar to the CCK-8 results;

[0034] Figure 11 The drug-loaded nanoparticles have good cell safety;

[0035] Figure 12 Confocal laser scanning microscopy (CLSM) images of the uptake of nanoparticles in HCT116 cells;

[0036] Figure 13 There is no significant change in the body weight of mice with different doses of nanoparticles;

[0037] Figure 14 Showing that there is no significant change in the liver and kidney function indexes of mice with different doses of nanoparticles;

[0038] Figure 15 Graph for the results of tissue sections of the heart, liver, spleen, lung and kidney of mice with different doses of nanoparticles. Detailed implementation mode

[0039] The following combines the attached Figures 1-15 and the implementation mode to further elaborate on the present invention:

[0040] Example 1

[0041] 1. Materials

[0042] Chitosan (CS) and folic acid were purchased from China Bailing Micro-Technology Co., Ltd. Sodium tripolyphosphate (TPP) and 5-fluorouracil (5-FU) were purchased from Shanghai Macklin Biochemical Co., Ltd. Hops extract was provided by Xinjiang Sapporo Agricultural Science and Technology Development Co., Ltd., and β-acid was extracted by our laboratory. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), pepsin, trypsin, and pancreatin were purchased from Aladdin Biochemical Technology Co., Ltd. Agar powder, yeast extract, pancreatin, and sodium chloride were provided by Beijing Aoboxing Biotechnology Co., Ltd. All chemical reagents were used without further purification.

[0043] 2. Preparation of folic acid FA-modified chitosan nanoparticles loaded with β-acid

[0044] (1) Folic acid FA, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with a molar ratio of 1:1.5:1.5 were dissolved in 40 ml of DMSO and stirred at 30 °C in the dark for 3 h.

[0045] (2) Chitosan CS was dissolved in 1% acetic acid solution to form a chitosan aqueous solution with a final concentration of 0.5%.

[0046] (3) The active ester of folic acid FA was slowly added to the chitosan CS solution and reacted at 25 °C for 24 h. At the end of the reaction, the pH was adjusted to 9.0 with NaOH solution, and centrifuged at 12,000 rpm for 10 min at 4 °C to obtain a yellow precipitate. The yellow precipitate was washed 3 times with saturated NaHCO3 solution, the precipitate was washed 3 times with deionized water, and freeze-dried for 24 h to obtain folic acid / chitosan FA / CS powder.

[0047] The folic acid / chitosan FA / CS was dissolved in 1% acetic acid solution, and the pH was adjusted to 5.0 with 3M NaOH.

[0048] (4) Different volumes of β-acid / ethanol solutions (7.5, 15, 22.5 mL) were slowly added dropwise to the FA / CS solution and stirred for 30 min.

[0049] STPP (2 mg / mL) solution was added dropwise and stirred for 1 h. The suspension was centrifuged at 12,000 rpm for 20 min and freeze-dried to obtain FA / CS / TPP / β-acid nanoparticles.

[0050] 3. Characterization of FA / CS / TPP / β-acid

[0051] The FTIR of CS, β-acids, TPP, FA / CS / TPP, FA / CS / TPP / 7.5β-acids, FA / CS / TPP / 15β-acids, FA / CS / TPP / 22.5β-acids powders was measured in the range of 4000 - 500 cm-1 using a Vertex 70 FTIR spectrometer from Bruker in Beijing, China. The powders of FA / CS / TPP, FA / CS / TPP / 7.5β-acids, FA / CS / TPP / 15β-acids, FA / CS / TPP / 22.5β-acids were analyzed using a Bruker AXS D8 advanced diffractometer from Germany. The microstructures of FA / CS / TPP, FA / CS / TPP / 7.5β-acids, FA / CS / TPP / 15β-acids, and FA / CS / TPP / 22.5β-acids powders were observed using SEM (Regulus 8100, Hitachi, Tokyo, Japan) and TEM (Tecnai G220, FET, USA). The particle size, polydispersity index, and zeta potential of FA / CS / TPP, FA / CS / TPP / 7.5β-acids, FA / CS / TPP / 15β-acids, and FA / CS / TPP / 22.5β-acids were measured and analyzed using a nano Zetasizer.

[0052] (I) Infrared spectrum

[0053] First, the characteristic absorption peaks of CS, FA, TPP, β-acid, FA / CS, FA / CS / TPP, and FA / CS / TPP / β-acid were characterized by FTIR spectrometer Figure 1)。A strong and broad absorption peak appeared at 3365 cm-1 for CS, representing the -OH functional group. The absorption peaks observed at 2876 cm-1 were for the asymmetric and symmetric stretching vibrations of CH. Subsequently, the absorption peaks observed at 1655 cm-1, 1598 cm-1, and 1381 cm-1 were for the vibrations of the amide I, amide II, and amide III groups of CS. The absorption peak near 1194 cm-1 represented the C-O stretching vibration in CS. FA characterization found that the absorption peaks at 3544 cm-1, 3324 cm-1, and 3116 cm-1 were related to the stretching vibrations of the -NH, -OH, and -CH functional groups. The characteristic peaks of FA were mainly located at 1696 cm-1, 1605 cm-1, and 1485 cm-1, related to carboxylic acid vibrations, amino groups, C=C or C=N. By comparing the FTIR of FA / CS, FA, and CS, it was found that the absorption peaks of FA / CS disappeared at 1655 cm-1 and 1598 cm-1, while the new absorption peaks that appeared at 1512 cm-1 and 1609 cm-1 were attributed to the stretching vibration of the C=C in the aromatic ring backbone. These changes were caused by the connection of the carboxyl group of FA with the amino group of CS through a chemical reaction. In addition, the absorption peak at 1512 cm-1 was related to the bending vibration of NH (amine II) in FA / CS. This indicated that the carboxyl group in FA reacted with the amino group in CS to form an amide bond, demonstrating the successful synthesis of FA / CS coupling. The absorption peaks at 1169 cm-1 and 1213 cm-1 were related to the stretching vibration of the O-P-O group and the P=O vibration of TPP. In addition, the peak at 900 cm-1 was related to the stretching vibration of the P-O-P bridge in TPP. The peak at 1541 cm-1 for FA / CS / TPP came from the stretching vibration of CO-NH. As an active substance, the peak of β-acid was located at 3224 cm-1. In addition, the peak at 2971 cm-1 belonged to the C-H vibration, and the peak at 1665 cm-1 belonged to the C=O vibration of β-acid, which was consistent with our previous research. The addition of β-acid changed the infrared absorption spectrum. The -OH in β-acid interacted with the blank nanoparticles (FA / CS / TPP), shifting the absorption peak of -OH to 3403 cm-1 and forming a specific absorption peak of β-acid at 2971 cm-1. The FTIR experimental results indicated the successful preparation of folic acid-targeted chitosan nanoparticles loaded with β-acid.

[0054] (II) XRD Results

[0055] XRD technology is usually used to characterize the crystal structure of drug-loaded nanoparticles, and this study can provide information for subsequent dissolution of drugs from nanoparticles ( Figure 2)。It has been reported that when drugs are loaded in nanoparticles, some drugs are dispersed in amorphous or solid form. The XRD diffraction pattern of CS shows that there is an obvious diffraction peak at 2θ = 21°, but the diffraction peak is not sharp, indicating that CS mainly exists in the amorphous state. β-acid has obvious diffraction peaks at 2θ = 8.8°, 9.2° and 30.1°, indicating that β-acid is crystalline in nature. The XRD diffraction peaks of the cross-linking agent TPP are mainly located at 2θ = 19.7°, 20.3°, 20.8°, 26.0°, 35.0°, 35.7° and 36.2°. When TPP cross-links FA and CS, the diffraction peak of the formed blank nanoparticles is 2θ = 22.2°, and the diffraction peak is a broad peak, indicating that the nanoparticles formed by TPP cross-linking FA modified CS exist in amorphous form. After adding β-acid to FA / CS / TPP, compared with the blank nanoparticles (FA / CS / TPP), a diffraction peak appears again near 2θ = 8.7° in the drug-loaded nanoparticles, indicating that β-acid is successfully loaded in the nanoparticles but exists in amorphous form.

[0056] The drug-loaded nanoparticles are amorphous. Analysis of the preparation process reveals that CS dissolved in the acidic solution may carry a positive charge, and the stable polymer nanoparticles formed by FA modification and cross-linking of CS with TPP also carry a positive charge. FA / CS / TPP interacts with β-acid to form FA / CS / TPP / β-acid, and the positive charge decreases after adding β-acid. All these ionic interactions play an important role in cross-linking and the subsequent formation of drug-loaded nanoparticles, enabling cross-linking to occur rapidly in solution and form stable polymers. β-acid is a small molecule that rapidly binds to the blank nanoparticles during the formation of drug-loaded nanoparticles, so β-acid exists in the polymer in amorphous form.

[0057] (III) Scanning Electron Microscope

[0058] As Figure 3 shown, FA / CS / TPP, FA / CS / TPP / 7.5β-acids, FA / CS / TPP / 15β-acids, FA / CS / TPP / 22.5β-acids are spherical or quasi-spherical, but serious aggregation and adhesion occur. Although the sizes of the nanoparticles are uneven, they are all slightly smaller than 300 nm. As the content of β-acid in the nanoparticles increases, the surface of the nanoparticles becomes smooth, which may be related to the fact that the presence of β-acid improves the adhesion of the nanoparticles to a certain extent and increases the aggregation of the nanoparticles. At the same time, the drug-loaded nanoparticles are prone to attracting each other and forming stable aggregates due to their high surface energy and surface activity. Thus, they are sticky. Compared with the nanoparticles without added β-acid, the adhesion of FA / CS / TPP nanoparticles decreases.

[0059] (IV) Transmission Electron Microscope, Charge and Particle Size Analysis

[0060] In addition, transmission electron microscopy was used to further analyze the morphology of blank and drug-loaded nanoparticles. As Figure 4 shown, similar to the SEM results, due to the presence of β-acid, the dispersibility of nanoparticles among the drug-loaded nanoparticles was severely weakened, resulting in no clear connection boundary between the nanoparticles. The reason for this phenomenon may be that intermolecular hydrogen bonds are formed between β-acid and FA / CS / TPP particles after incorporation.

[0061] When preparing drug-loaded nanoparticles by the ion gel method, the cations in FA / CS are crosslinked with the anions of TPP. In the detection of nanoparticle size, we found that although the content of β-acid gradually increased, there was no significant difference compared with FA / CS / TPP, and its particle size distribution was as follows: (286.67 ± 4.63), (266 ± 16.06), (286 ± 12.03), (265 ± 8.22) nm( Figure 5 ). The nanoparticle sizes observed by the particle size analysis method were larger than those observed by TEM and SEM, which may be due to the swelling of nanoparticles in water. On the contrary, in the TEM and SEM techniques, the nanoparticles were prepared by the direct drying method. The particle sizes observed by transmission electron microscopy and scanning electron microscopy under dry conditions cannot accurately reflect their sizes in body fluids. The aggregation of particles in fluids increases the effective volume of the particles. In addition, complex particles such as proteins and lipids in body fluids can attach to the particle surface, thus increasing the size of the nanoparticles. Evaluating the particle size in solution, especially in serum-containing medium as a body fluid model, is crucial. Therefore, the nanoparticle sizes calculated by particle size analysis are within an acceptable range, reflecting the success of the ion gel technology. In the zeta potential test, we found that the FA / CS / TPP nanoparticles carried +12.73 mV( Figure 5 ). As a negatively charged molecule, β-acid showed a decreasing trend with the addition of β-acid. In addition, the high positive charge on the nanoparticle surface is beneficial for the binding of the particles to the negatively charged cell membrane, making it an ideal platform for drug delivery. Using ion gel to encapsulate drugs is a commonly used strategy.

[0062] (V) Drug loading and encapsulation efficiency

[0063] The ultraviolet spectroscopy method was used to determine the encapsulation efficiency (EE) and drug loading (LC) of β-acid in the nanoparticles. β-acid is a hydrophobic drug, which is stabilized by electrostatic interaction and hydrogen bond. The LCs of different drug-loaded nanoparticles were (20.97 ± 0.89%), (20.79 ± 1.04%) and (32.19 ± 1.11%) respectively, and the EEs were (44.61 ± 1.80%), (45.31 ± 1.49%) and (43.41 ± 1.31%) respectively( Figure 5)。The encapsulation efficiency and drug loading rate are parameters related to the encapsulation ability of different raw materials for drugs and the quality of the preparation. These parameters mainly depend on the raw material type, drug polarity, and nanoparticle preparation method.

[0064] (VI) Release

[0065] The release of β-acid from the nanoparticles was studied in simulated gastric fluid, small intestinal fluid, and colonic fluid at pH = 1.2, 6.8, and 7.8. The cumulative percentage of β-acid released from the nanoparticles is as Figure 6 shown. Similar release curves were found under the three different pH conditions. Biphasic kinetics of β-acid release was observed: a sudden release followed by a slow release. β-acid was rapidly released during the initial rapid release phase and continuously degraded slowly by diffusion during the later stable phase ( Figure 6 A - C).

[0066] The effect of different digestive enzymes on the release of β-acid was studied by adding different digestive enzymes to the simulated solution ( Figure 6(D - F). The results showed that at pH = 1.2, the release rate and release efficiency of the nanoparticles were the highest, indicating that trypsin had an impact on the release of the nanoparticles. During the rapid release phase at pH = 1.2, the release rates of the artificial small intestinal juice of different drug - loaded nanoparticles were 22.5% (FA / CS / TPP / 7.5β - acids), 34% (FA / CS / TPP / 15β - acids), and 31% (FA / CS / TPP / 22.5β - acids), respectively. The subsequent steady release phases were 26% (FA / CS / TPP / 7.5β - acids), 35% (FA / CS / TPP / 15β - acids), and 37.5% (FA / CS / TPP / 22.5β - acids), respectively. At pH = 6.8, the total β - acid release amounts were close to 26.9% (FA / CS / TPP / 7.5β - acids), 36.4% (FA / CS / TPP / 15β - acids), and 38.3% (FA / CS / TPP / 22.5β - acids), respectively. At pH = 7.8, the release rates of the active ingredient β - acids were 12.4% (FA / CS / TPP / 7.5β - acids), 12.7% (FA / CS / TPP / 15β - acids), and 4.9% (FA / CS / TPP / 22.5β - acids), respectively. This indicated that in the presence of enzymes, the total drug release amount was the highest at pH = 6.8, followed by pH = 7.8, suggesting that trypsin and pancreatin could accelerate the decomposition of the nanoparticles, resulting in the release of β - acids. Similar to the results of drug release without enzymes, the release amount was the least at pH = 1.2, indicating that the possibility of the drug carrier releasing β - acids under acidic conditions was small. Therefore, reducing the direct exposure of the drug to intestinal epithelial cells during actual drug administration would help overcome the side effects of the drug, and it was expected that the in - vivo release would be different compared to the in - vitro situation.

[0067] Example 2

[0068] This test example aims to verify the antibacterial, anti - cancer effects and safety of the folic - acid - modified chitosan nanoparticles loaded with β - acids prepared in the above - mentioned example, as follows:

[0069] (I) Inhibitory effects on Staphylococcus aureus and Escherichia coli

[0070] The specific verification steps include: taking the strains of Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC25923), adding nutrient broth under sterile conditions, expanding the culture, and culturing at 37 °C for 24 h. Selecting typical colonies and dissolving them in 0.9% (w / v) physiological saline to make the reading of the bacterial turbidimeter 0.50 McFarland turbidity units, with a concentration of 1×10 8CFU / mL. Disperse β - acids and drug - loaded nanoparticles in ethanol to obtain solutions with different concentrations (8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg / mL). Take 50 μL of bacterial solution (concentration is 1×10 7 CFU / mL) and 100 μL of the drug solution with different concentrations (8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg / mL), incubate at 37 °C for 24 h, spread 100 μL of the bacterial solution cultured the next day on the LB solid medium and incubate for 24 h, observe and record the bacterial growth situation.

[0071] Use the two - fold dilution method to determine the MIC and MBC of β - acids and drug - loaded nanoparticles against Escherichia coli and Staphylococcus aureus, and use SEM to measure the morphological changes of bacteria (the specific results are as Figure 7 , 8 shown). The FA / CS / TPP / 15β - acids nanoparticles still have an inhibitory effect on Staphylococcus aureus at a concentration of 0.03125 mg / ml. For Escherichia coli, pure β - acids have no inhibitory effect at 8 mg / mL, while FA / CS / TPP / 15β - acids have an inhibitory effect on Escherichia coli ( Figure 7 ). Therefore, we subsequently selected FA / CS / TPP / 15β - acids for research. Treat Escherichia coli and Staphylococcus aureus with FA / CS / TPP / 15β - acids and observe the bacterial morphology by scanning electron microscopy. After treatment with the drug - loaded nanoparticles, the morphological structure of the bacteria changes significantly. Normal Escherichia coli and Staphylococcus aureus have complete structures. After treatment, the surfaces of Escherichia coli and Staphylococcus aureus shrink, and some bacteria even rupture ( Figure 8 ). β - acids have antibacterial activity, the nanoparticles are positively charged in aqueous solution, while the membranes of bacteria and cancer cells are negatively charged. Electrostatic attraction enables the bacteria or cell membranes to interact more effectively.

[0072] (II) Inhibitory effect on colorectal cancer cells

[0073] The specific verification steps include: Take colorectal cancer cells HCT116 and human normal intestinal epithelial cells NCM460, and seed the cells in 96 - well plates respectively (1×10 per well 4Cell density). Allow the cells to grow for 6 hours to allow cell attachment. Replace the medium in the 96-well plate with the drug-containing medium (McCoy's 5A for HCT116 cells; RPMI 1640 medium for NCM460 cells) (80, 40, 20, 10, 5 μg / mL) and incubate for 24 h. The next day, aspirate the drug-containing medium and replace it with fresh medium. Add 10 μL of CCK-8 solution to each well and incubate at 37 °C for 1 h. Measure the optical density (OD) of each well at 450 nm using an enzyme-linked immunosorbent assay.

[0074] The CCK-8 method was used to evaluate the in vitro cytotoxicity of pure β-acid, 5-FU (5-fluorouracil), FA / CS / TPP, FA / CS / TPP / 7.5β-acid, FA / CS / TPP / 15β-acid, and FA / CS / TPP / 22.5β-acid nanoparticles against HCT116 and NCM460 cell lines. Figure 9 The effects of the six materials on the activity of HCT116 cells are shown. The experimental results indicate that the drug-loaded nanoparticles at concentrations greater than 40 μg / mL have a significantly better inhibitory effect on HCT116 cells than β-acid and 5-FU. For HCT116 cells, the reason for the inhibitory effect of the drug-loaded nanoparticles on their proliferation may be that the nanoparticles target FA, increasing the entry of β-acid into cancer cells to exert its effect. Folate-modified nanoparticles improve the bioavailability of the drug through an active targeting strategy and the EPR effect. In addition, the sustained release of the drug from the nanoparticles allows for continuous exposure to the drug, which helps to enhance the cytotoxic effect.

[0075] This study also analyzed the effects of different concentrations (0 - 40 μg / mL) of drug-loaded nanoparticles on the proliferation of normal cells to show that they are non-toxic to normal cells and can be used as potential therapeutic agents. We found that even at a concentration of 40 μg / mL, co-incubation of the nanoparticles with NCM460 cells for 24 h had no effect on cell proliferation, indicating that the drug-loaded nanoparticles have good cell safety ( Figure 11 ). Studies have shown that nanocarriers are of great significance for delivering sufficient amounts of anticancer drugs to cancer cells, which helps to minimize the side effects of anticancer drugs.

[0076] In addition, the present invention used a scratch assay to further study the effect of drug-loaded nanoparticles (FA / CS / TPP / 22.5β-acids) on cell migration. The experimental results showed that the presence of the drug-loaded nanoparticles could effectively inhibit the proliferation of HCT116 cells, and the effect was better than that of 5-FU, similar to the CCK-8 results ( Figure 10 ). In summary, the toxic effect of folate-modified β-acid-loaded nanoparticles on colorectal cancer cells was effectively demonstrated in HCT116 cells.

[0077] To study the targeting of the prepared nanoparticles to folate receptors, the applicant selected nanoparticles loaded with coumarin-6 for co-culture with HCT116 cells and analyzed them using a confocal microscope. It has been reported that folate receptors are well-known tumor markers that are highly expressed on the membrane surface of colorectal cancer cells. Both free folic acid and folate-functionalized nanomaterials can recognize and tightly couple with folate receptors. It can be clearly seen from the CLSM images ( Figure 12 ) that after co-incubation of the nanoparticles loaded with coumarin-6 with HCT116 cells, strong red fluorescence of DID and green fluorescence of C6 appeared inside the cells. This indicates that the nanoparticles loaded with coumarin-6 can be significantly internalized into HCT116 cells through folate receptor-mediated endocytosis. In addition, we also co-incubated the coumarin-6-loaded nanoparticles pretreated with free FA with HCT116 cells to block folate receptors and further confirm their recognition ability. As shown in Figure 12 , the fluorescence intensity inside the cells decreased significantly, indicating that the folate receptors on the surface of HCT116 cells were coupled with free folic acid, largely blocking the uptake of nanoparticles by the cells. The above results show that folic acid-modified nanoparticles promote folate receptor-mediated endocytosis and are expected to become targeted nanocarriers for folate receptor-positive tumors.

[0078] (III) Anticancer therapeutic effect and safety in vivo

[0079] Evaluate the safety of the drug-loaded nanoparticles in mice. Grouped according to different administration doses (Control, low-dose group of FA / CS / TPP, medium-dose group of FA / CS / TPP, high-dose group of FA / CS / TPP, low-dose group of FA / CS / TPP / β-acid, medium-dose group of FA / CS / TPP / β-acid, high-dose group of FA / CS / TPP / β-acid) for treatment. At different time points, corresponding blood specimens and body weights were collected. It was found that there were no significant changes in blood physiological indexes such as body weight (as shown in Figure 13 ), red blood cell level (Table 1), white blood cell level, and lymphocyte level compared with the blank control group, and there were no significant changes in liver and kidney function indexes (as shown in Figure 14 ). In addition, the main organs of the mice, including the heart, liver, spleen, lungs, and kidneys, were taken and tissue pathological sections were made. The results of the tissue sections showed that there was no obvious effect on the mouse tissues at this administration dose (as shown in Figure 15 ). Based on the above results, the nanoparticles prepared by the present invention obviously have good biosafety.

[0080] Table 1

[0081]

[0082] This specific embodiment is only an interpretation of the present invention and does not limit the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A method for preparing folic acid-modified chitosan nanoparticles loaded with β-acid, characterized in that: The following steps are involved: (1) dissolving folic acid, N-hydroxysuccinimide and 1-ethyl-3-dimethylaminopropylcarbodiimide in DMSO and stirring to obtain an active ester of folic acid; (2) adding chitosan to the acetic acid solution, stirring and dissolving, and preparing a chitosan aqueous solution; (3) adding the activated folic acid active ester in step (1) to the chitosan aqueous solution in step (2) and mixing to obtain a mixed solution 1; adjusting the pH value to 9 with NaOH after the reaction is completed, centrifuging to obtain a yellow precipitate; washing the yellow precipitate with a saturated NaHCO3 solution, then washing with deionized water, and freeze-drying to obtain FA / CS powder; dissolving the FA / CS powder in an acetic acid solution to obtain a FA / CS solution; and adjusting the pH value to 5.0 with NaOH; (4) adding the β-acid ethanol solution dropwise to the solution prepared in step (3), and stirring to obtain a mixed solution 2; (5) The sodium tripolyphosphate solution is added dropwise to the mixed solution 2 prepared in step (4) and stirred for 1 hour. After centrifugation, the mixture is freeze-dried to obtain the folic acid-modified chitosan nanoparticles loaded with β-acid.

2. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: In the step 1, stirring is carried out at 30° C. in the dark for 3 h.

3. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: In the step 1, the molar ratio of folic acid, N-hydroxysuccinimide and 1-ethyl-3-dimethylaminopropylcarbodiimide is 1:1.5:1.

5.

4. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: In the step 2, chitosan is dissolved in 1% acetic acid solution to form a chitosan aqueous solution with a final concentration of 0.5%.

5. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: The mixing and stirring in step (3) is reacted at 25° C. for 24 hours.

6. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: In step (3), the centrifugation is performed at 12000 rpm at 4°C for 10 min.

7. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: The yellow precipitate was washed three times with saturated NaHCO3 solution, and the precipitate was washed three times with deionized water. FA / CS powder was obtained after freeze-drying for 24 h.

8. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: The concentration of the sodium tripolyphosphate solution in step (5) is 2 mg / mL.

9. The method for preparing a folic acid-modified chitosan nanoparticle loaded with β-acid according to claim 1, characterized in that: In the step (5), the centrifugation parameter is 12000 rpm for 20 min.

10. A folic acid-modified chitosan nanoparticle loaded with β-acid prepared by the preparation method according to any one of claims 1 to 4.

11. Use of folic acid-modified chitosan nanoparticles loaded with β-acid prepared by the preparation method according to claim 5, characterized in that: The antibacterial agents include Staphylococcus aureus and Escherichia coli.

Images