Fluorouracil sustained-release nano-microsphere as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses fluorouracil sustained-release nano-microspheres as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, dissolving fluorouracil in a polyarginine aqueous solution, uniformly stirring, and adjusting the pH value to 7.2-7.4 to obtain a fluorouracil solution; s2, dissolving amphiphilic alginate in water, sequentially adding carboxymethyl chitosan and quaternized gelatin, and uniformly stirring to obtain a composite carrier solution; and S3, mixing the fluorouracil solution obtained in the step S1 with the composite carrier solution obtained in the step S2, performing ultrasonic treatment, performing self-assembly, performing centrifugation, and collecting precipitates to obtain the fluorouracil nano-microspheres. The invention discloses a fluorouracil sustained-release nano-microsphere as well as a preparation method and application thereof. The fluorouracil sustained-release nano-microsphere can be used for remarkably improving the encapsulation efficiency and the drug loading capacity of fluorouracil, realizing long-acting sustained release of drugs, enhancing the tumor targeting property and the cell uptake efficiency and improving the treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fluorouracil sustained-release nanosphere, its preparation method, and its application. Background Technology

[0002] 5-Fluorouracil (5-FU) is a commonly used antimetabolite antitumor drug in clinical practice. It interferes with the DNA synthesis and cell division of tumor cells by inhibiting thymidylate synthase, and has a definite therapeutic effect on various solid tumors such as colorectal cancer, gastric cancer, and breast cancer. It is widely used in the clinical treatment of malignant tumors.

[0003] However, fluorouracil's inherent molecular characteristics have significant drawbacks that limit its clinical efficacy. This drug has a small molecular weight, good water solubility, wide distribution in the body, and a rapid metabolic rate, with a plasma half-life typically less than 20 minutes. This results in low bioavailability, making it difficult to achieve effective drug concentrations at tumor sites and leading to a short duration of action. Furthermore, to maintain the necessary effective blood drug concentrations for treatment, frequent administration or continuous intravenous infusion is required, increasing the patient's treatment burden and medication adherence issues. It also easily triggers severe systemic toxic side effects, such as leukopenia and thrombocytopenia due to bone marrow suppression, nausea, vomiting, and diarrhea caused by gastrointestinal mucosal damage, and stomatitis, significantly impacting patient treatment tolerance and quality of life.

[0004] Existing technologies have attempted to develop drug delivery systems to improve their pharmacokinetic performance and targeting. For example, microspheres or nanoparticle carriers are constructed using natural polymers such as alginate and chitosan. However, existing delivery systems still have significant shortcomings. Some carriers rely solely on single or two-component polymers for construction, making it difficult to form a stable three-dimensional network structure. This results in low drug encapsulation efficiency, insufficient drug loading, and a tendency for premature degradation or burst release in vivo, failing to achieve long-term sustained release. Furthermore, some existing delivery systems have limited efficiency in targeting and uptake of tumor cells, making it difficult to effectively improve drug accumulation at the tumor site. Therefore, they still cannot fully resolve the contradiction between the therapeutic efficacy and toxic side effects of fluorouracil. Summary of the Invention

[0005] The present invention aims to provide a fluorouracil sustained-release nanosphere, its preparation method and application. The fluorouracil sustained-release nanosphere can significantly improve the encapsulation efficiency and drug loading of fluorouracil, achieve long-term sustained drug release, enhance tumor targeting and cellular uptake efficiency, and improve therapeutic effect.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for preparing fluorouracil sustained-release nanospheres includes the following steps: S1. Dissolve fluorouracil in an aqueous solution of polyarginine, stir until homogeneous, and adjust the pH to 7.2-7.4 to obtain a fluorouracil solution; S2. Dissolve amphiphilic alginate in water, then add carboxymethyl chitosan and quaternized gelatin in sequence, stir until homogeneous, and obtain a composite carrier solution; S3. The fluorouracil solution obtained in S1 is mixed with the composite carrier solution obtained in S2, and the mixture is subjected to ultrasonic treatment for self-assembly. After centrifugation, the precipitate is collected to obtain fluorouracil nanospheres.

[0007] Preferably, in S1, the concentration of fluorouracil in the fluorouracil solution is 2.5-20.0 mg / mL, and the concentration of polyarginine is 0.5-1.0 mg / mL.

[0008] Preferably, in S2, the amphiphilic alginate is hexadecyl isocyanate-modified sodium alginate.

[0009] Preferably, the method for preparing the hexadecyl isocyanate-modified sodium alginate includes the following steps: T1. Adjust the pH of the sodium alginate aqueous solution to 8.8-9.2, add sodium dodecyl sulfate and hexadecyl isocyanate to obtain the reaction solution; T2. Heat the reaction solution obtained in T1 to 65-80℃ and react for 7-9 hours. Adjust the pH to 3.8-4.2, add acetone to precipitate, filter and dry to obtain hexadecyl isocyanate modified sodium alginate.

[0010] Preferably, the sodium alginate aqueous solution has a mass concentration of 3%-10%, and the mass ratio of sodium alginate to sodium dodecyl sulfate and hexadecyl isocyanate is 1:0.4-1.5:0.4-1.0.

[0011] Preferably, in S2, the concentration of amphiphilic alginate in the composite carrier solution is 20-80 mg / mL, the concentration of carboxymethyl chitosan is 1.0-4.0 mg / mL, and the concentration of quaternized gelatin is 0.5-3.0 mg / mL.

[0012] Preferably, in S3, the ultrasonic treatment power is 500-1000W, the time is 4-6h, the centrifugation speed is 6000-8000rpm, and the centrifugation time is 10-20min.

[0013] The present invention also provides fluorouracil nanospheres prepared by the preparation method described above.

[0014] Preferably, the diameter of the fluorouracil nanospheres is 50-300 nm.

[0015] The present invention also provides the application of the fluorouracil nanospheres in the preparation of antitumor drugs.

[0016] The present invention also provides an antitumor drug, the active ingredient of which includes the aforementioned fluorouracil sustained-release nanospheres.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a fluorouracil sustained-release nanosphere, its preparation method, and its applications. By constructing a ternary polymeric synergistic system of amphiphilic alginate, carboxymethyl chitosan, and quaternized gelatin, a stable three-dimensional cross-linked network is formed through multiple electrostatic interactions and hydrogen bonding among the three components, significantly improving the encapsulation efficiency and drug loading of fluorouracil. This ternary network structure is dense and homogeneous, effectively immobilizing drug molecules. The network structure forms a multi-level diffusion barrier, effectively slowing the release rate of fluorouracil and significantly prolonging the drug's in vivo half-life.

[0018] The preparation method disclosed in this invention is simple to operate, has mild conditions, and leaves no organic solvent residue, making it suitable for the industrial production of green chemical drug delivery.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 Scanning electron microscope image of the fluorouracil nanospheres prepared in Example 2; Figure 2 The encapsulation efficiency and drug loading of nanospheres prepared at different fluorouracil concentrations in Examples 2-7 are compared. Figure 3 This is a comparison of the in vitro sustained-release curves of the fluorouracil nanospheres prepared in Example 2 and commercially available fluorouracil tablets; Figure 4 The graph shows a comparative analysis of the effects of fluorouracil nanospheres prepared in Example 2 and commercially available fluorouracil tablets at a concentration of 25 μg / mL on the viability of mouse colon cancer cells CT26. Figure 5 The figure shows a comparative analysis of the effects of fluorouracil nanospheres prepared in Example 2 and commercially available fluorouracil tablets at a concentration of 25 μg / mL on the viability of mouse breast cancer 4T1 cells. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] Test material source: Fluorouracil was purchased from Sigma-Aldrich.

[0024] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0025] Example 1 A cetyl isocyanate-modified sodium alginate, the preparation method of which includes the following steps: T1. Dissolve 3g of sodium alginate in 100mL of deionized water. The reaction is carried out in a 250mL three-necked flask equipped with a magnetic stir bar, connected to a spherical condenser reflux condenser and a dropping device. The flask is placed in a 30℃ constant temperature water bath and stirred for 3 hours until completely dissolved. The pH is adjusted to 9, 0.3g of sodium dodecyl sulfate is added, and 3g of hexadecyl isocyanate is added dropwise to obtain the reaction solution. T2. Heat the reaction solution obtained in T1 to 70°C and react for 8 hours. Cool to room temperature, adjust the pH to 4, add 300 mL of acetone to precipitate the reaction product, remove unreacted hexadecyl isocyanate, filter the product, wash the product three times with acetone, and then dry it under vacuum at 55°C for 48 hours to obtain hexadecyl isocyanate modified sodium alginate.

[0026] Example 2 A method for preparing fluorouracil sustained-release nanospheres includes the following steps: S1. Dissolve 0.1g of polyarginine in 10mL of deionized water and stir until homogeneous to obtain a polyarginine aqueous solution. Dissolve 1.0g of fluorouracil in the polyarginine aqueous solution and stir until homogeneous. Adjust the pH to 7.2 using a 5% NaOH solution to obtain a fluorouracil solution. S2. Dissolve 0.05g of hexadecyl isocyanate-modified sodium alginate prepared in Example 1 in 10mL of deionized water, add 0.025g of carboxymethyl chitosan and 0.025g of quaternized gelatin in sequence, stir evenly to obtain a composite carrier solution. S3. Mix 1 mL of the fluorouracil solution obtained in S1 with 4 mL of the composite carrier solution obtained in S2, and sonicate at 800 W for 4 h to perform self-assembly. Centrifuge at 8000 rpm for 20 min, collect the precipitate, wash with deionized water, and obtain fluorouracil nanospheres.

[0027] Example 3 The preparation method is the same as in Example 2, except that the volume of the fluorouracil solution in S3 is 0.050 mL.

[0028] Example 4 The preparation method is the same as in Example 2, except that the volume of the fluorouracil solution in S3 is 0.125 mL.

[0029] Example 5 The preparation method is the same as in Example 2, except that the volume of the fluorouracil solution in S3 is 0.250 mL.

[0030] Example 6 The preparation method is the same as in Example 2, except that the volume of the fluorouracil solution in S3 is 0.500 mL.

[0031] Example 7 The preparation method is the same as in Example 2, except that the volume of the fluorouracil solution in S3 is 0.750 mL.

[0032] The effectiveness of the fluorouracil nanospheres provided in the above embodiments was verified through the following experiments.

[0033] 1. Scanning electron microscopy analysis was performed on the fluorouracil nanospheres provided in Example 2, and the results are as follows: Figure 1 As shown.

[0034] Depend on Figure 1 It can be seen that the fluorouracil nanospheres have a uniform particle size distribution and an intact structure, with an average particle size of 134 nm.

[0035] 2. The encapsulation efficiency and drug loading of the nanospheres at different fluorouracil concentrations provided in Examples 2-7 were determined, and the results are as follows: Figure 2 As shown.

[0036] Depend on Figure 2 It can be seen that as the concentration of fluorouracil increases, the encapsulation efficiency of the prepared fluorouracil nanospheres increases, but the drug loading decreases.

[0037] 3. The sustained-release curves of commercially available fluorouracil tablets and the fluorouracil nanospheres prepared in Example 2 were determined using existing experimental protocols. The drug dosage for both was 50 mg. The results are as follows: Figure 3 As shown.

[0038] Depend on Figure 3 It can be seen that the release rate of fluorouracil tablets is 78% at 0.5h and 98% at 1h; while the release rate of fluorouracil nanospheres is only 35% at 1h, 90% at 48h, and 96% at 72h, showing superior sustained-release performance.

[0039] 4. The effects of commercially available fluorouracil tablets and the fluorouracil nanospheres prepared in Example 2 on the viability of mouse colon cancer CT26 cells were determined. Three groups were set up: control group (no drug), tablet group (25 μg / mL), and fluorouracil nanosphere group (25 μg / mL). Fluorouracil nanospheres and commercially available fluorouracil tablets were applied to mouse colon cancer CT26 cells, respectively. After 24 h of culture, the effects of the drugs on CT26 cell viability were calculated. The results are as follows: Figure 4 As shown.

[0040] Depend on Figure 4 It was found that after 24 hours of treatment, the cell viability of the tablet group was 67.02%, while that of the nanosphere group was 53.27%, indicating a better inhibitory effect on the proliferation of mouse colon cancer cells CT26.

[0041] 5. The effects of commercially available fluorouracil tablets and the fluorouracil nanospheres prepared in Example 2 on the viability of mouse breast cancer cells 4T1 were determined. Three groups were set up: control group (no drug), tablet group (25 μg / mL), and fluorouracil nanosphere group (25 μg / mL). Fluorouracil nanospheres and commercially available fluorouracil tablets were applied to mouse breast cancer cells 4T1, respectively. After 24 h of culture, the effects of the drugs on the viability of 4T1 cells were calculated. The results are as follows: Figure 5 As shown.

[0042] Depend on Figure 5 It was found that after 24 hours of treatment, the cell viability of the tablet group was 72.48%, while that of the nanosphere group was 45.75%, indicating that the tablet group had a better inhibitory effect on the proliferation of mouse breast cancer cells 4T1.

[0043] The above results indicate that the fluorouracil nanospheres prepared in this invention are superior to commercially available fluorouracil tablets in terms of drug sustained release, cellular uptake, and inhibition of tumor cell proliferation, and have good prospects for drug delivery and clinical application.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing fluorouracil sustained-release nanospheres, characterized in that, Includes the following steps: S1. Dissolve fluorouracil in an aqueous solution of polyarginine, stir until homogeneous, and adjust the pH to 7.2-7.4 to obtain a fluorouracil solution; S2. Dissolve amphiphilic alginate in water, then add carboxymethyl chitosan and quaternized gelatin in sequence, stir until homogeneous, and obtain a composite carrier solution; S3. The fluorouracil solution obtained in S1 is mixed with the composite carrier solution obtained in S2, and the mixture is subjected to ultrasonic treatment for self-assembly. After centrifugation, the precipitate is collected to obtain fluorouracil nanospheres.

2. The preparation method according to claim 1, characterized in that, In S1, the concentration of fluorouracil in the fluorouracil solution is 2.5-20.0 mg / mL, and the concentration of polyarginine is 0.5-1.0 mg / mL.

3. The preparation method according to claim 1, characterized in that, In S2, the amphiphilic alginate is hexadecyl isocyanate modified sodium alginate.

4. The preparation method according to claim 3, characterized in that, The method for preparing the hexadecyl isocyanate-modified sodium alginate includes the following steps: T1. Adjust the pH of the sodium alginate aqueous solution to 8.8-9.2, add sodium dodecyl sulfate and hexadecyl isocyanate to obtain the reaction solution; T2. Heat the reaction solution obtained in T1 to 65-80℃ and react for 7-9 hours. Adjust the pH to 3.8-4.2, add acetone to precipitate, filter and dry to obtain hexadecyl isocyanate modified sodium alginate.

5. The preparation method according to claim 4, characterized in that, The sodium alginate aqueous solution has a mass concentration of 3%-10%, and the mass ratio of sodium alginate to sodium dodecyl sulfate and hexadecyl isocyanate is 1:0.4-1.5:0.4-1.

0.

6. The preparation method according to claim 1, characterized in that, In S2, the concentration of amphiphilic alginate in the composite carrier solution is 20-80 mg / mL, the concentration of carboxymethyl chitosan is 1.0-4.0 mg / mL, and the concentration of quaternized gelatin is 0.5-3.0 mg / mL.

7. The preparation method according to claim 1, characterized in that, In S3, the ultrasonic treatment power is 500-1000W, the time is 4-6h, the centrifugation speed is 6000-8000rpm, and the centrifugation time is 10-20min.

8. Fluorouracil nanospheres prepared by the preparation method according to any one of claims 1-7.

9. The use of the fluorouracil nanospheres as described in claim 8 in the preparation of antitumor drugs.

10. An antitumor drug, characterized in that, The active ingredient comprises the fluorouracil sustained-release nanospheres as described in claim 8.

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