Fluorouracil sustained-release nanomicrospheres, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly discloses a fluorouracil sustained-release nano-microsphere 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, adjusting the pH to 7.2-7.4, and obtaining a fluorouracil solution; S2, dissolving an amphiphilic alginate in water, sequentially adding carboxymethyl chitosan and quaternized gelatin, uniformly stirring, and obtaining a composite carrier solution; and S3, mixing the fluorouracil solution obtained in S1 with the composite carrier solution obtained in S2, performing self-assembly through ultrasonic treatment, centrifuging, and collecting the precipitate to obtain fluorouracil nano-microsphere. The fluorouracil sustained-release nano-microsphere can significantly improve the encapsulation efficiency and drug loading of fluorouracil, realize long-acting sustained release of the drug, enhance the tumor targeting and cell uptake efficiency, and improve the treatment effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a fluorouracil sustained-release nanomicrosphere and a preparation method and application thereof. BACKGROUND

[0002] As a clinically common anti-metabolite antitumor drug, fluorouracil (5-Fluorouracil, 5-FU) can interfere with 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, and is widely used in the field of clinical treatment of malignant tumors.

[0003] However, fluorouracil has significant defects in its molecular characteristics, which restrict its clinical application effect. The drug has a small molecular weight, good water solubility, wide distribution in the body and fast metabolism rate, and the plasma half-life is usually less than 20 min, resulting in low drug bioavailability, difficulty in forming an effective drug concentration at the tumor site, and short duration of action. In order to maintain the effective blood drug concentration required for treatment, the patient needs to be given drugs frequently or continuously intravenously, which not only increases the patient's treatment burden and drug compliance, but also easily causes serious systemic toxic and side effects, such as leukopenia and thrombocytopenia caused by bone marrow suppression, nausea, vomiting, diarrhea caused by gastrointestinal mucosa damage, and oral mucositis, which greatly affects the patient's treatment tolerance and quality of life.

[0004] In the prior art, drug delivery systems have been developed to improve their pharmacokinetic properties and targeting. For example, natural polymer materials such as alginate and chitosan are used to construct microsphere or nanoparticle carriers. However, the existing delivery systems still have obvious deficiencies. Some carriers are only constructed by single or double components of high polymer, which is difficult to form a stable three-dimensional network structure, resulting in low drug encapsulation efficiency, insufficient drug loading capacity, and easy premature degradation or drug burst in the body, which cannot achieve long-term sustained release. In addition, some existing delivery systems have limited targeting recognition and uptake efficiency for tumor cells, which cannot effectively improve the enrichment of drugs in tumor sites, and still cannot fully solve the contradiction between the therapeutic effect and the toxic and side effects of fluorouracil. SUMMARY

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

[0006] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] A preparation method of a fluorouracil sustained-release nanomicrosphere, comprising the following steps:

[0008] S1, dissolving fluorouracil in polyarginine aqueous solution, stirring uniformly, adjusting pH to 7.2-7.4, obtaining fluorouracil solution;

[0009] S2, dissolving amphiphilic alginate in water, adding carboxymethyl chitosan and quaternized gelatin in sequence, stirring uniformly, obtaining composite carrier solution;

[0010] S3, mixing fluorouracil solution obtained in S1 with composite carrier solution obtained in S2, self-assembling through ultrasonic treatment, centrifuging, collecting precipitate, obtaining fluorouracil nanomicrosphere.

[0011] 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.

[0012] Preferably, in S2, the amphiphilic alginate is hexadecyl isocyanate modified sodium alginate.

[0013] Preferably, the preparation method of the hexadecyl isocyanate modified sodium alginate comprises the following steps:

[0014] T1, adjusting the pH of sodium alginate aqueous solution to 8.8-9.2, adding sodium dodecyl sulfate and hexadecyl isocyanate, obtaining reaction liquid;

[0015] T2, heating the reaction liquid obtained in T1 to 65-80℃, reacting for 7-9 h, adjusting pH to 3.8-4.2, adding acetone for precipitation, filtering and drying, obtaining hexadecyl isocyanate modified sodium alginate.

[0016] Preferably, the mass concentration of the sodium alginate aqueous solution is 3%-10%, and the mass ratio of sodium alginate, sodium dodecyl sulfate and hexadecyl isocyanate is 1:0.4-1.5:0.4-1.0.

[0017] 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.

[0018] Preferably, in S3, the power of the ultrasonic treatment is 500-1000 W, the time is 4-6 h, the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 10-20 min.

[0019] The application further provides fluorouracil nanomicrosphere prepared by the preparation method.

[0020] Preferably, the fluorouracil nanospheres have a diameter of 50-300 nm.

[0021] The application also provides the fluorouracil nanospheres for use in the preparation of an antitumor drug.

[0022] The application also provides an antitumor drug, the effective component of which comprises the fluorouracil sustained-release nanospheres.

[0023] Compared with the prior art, the application has the following advantages and technical effects:

[0024] The application discloses fluorouracil sustained-release nanospheres, a preparation method and application thereof.

[0025] The preparation method disclosed by the application is simple in operation, mild in conditions and free of organic solvent residues, and is suitable for industrialized production of green chemical drug delivery.

[0026] The technical solutions of the application are further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A scanning electron microscope image of fluorouracil nanospheres prepared in Example 2;

[0028] Figure 2 A comparison curve of the encapsulation efficiency and drug loading of nanospheres prepared in Examples 2-7 at different fluorouracil concentrations;

[0029] Figure 3 A comparison diagram of in-vitro sustained-release curves of fluorouracil nanospheres prepared in Example 2 and commercially available fluorouracil tablets;

[0030] Figure 4 A comparison analysis diagram of the influence of fluorouracil nanospheres prepared in Example 2 and commercially available fluorouracil tablets on the viability of mouse colon cancer cells CT26 at a concentration of 25 mu g / mL;

[0031] Figure 5 A comparison analysis diagram of the influence of fluorouracil nanospheres prepared in Example 2 and commercially available fluorouracil tablets on the viability of mouse breast cancer cells 4T1 at a concentration of 25 mu g / mL. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further described below with reference to the drawings and examples.

[0033] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by a person with ordinary skills in the art to which the present application belongs.

[0034] The source of the test material: fluorouracil is purchased from Sigma-Aldrich Company.

[0035] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art, which can be purchased through commercial channels.

[0036] Example 1

[0037] A kind of hexadecyl isocyanate modified sodium alginate, the preparation method includes the following steps:

[0038] T1, 3g sodium alginate is dissolved in 100mL deionized water, the reaction is carried out in 250mL three-necked flask, the flask is equipped with a magnetic stirring rod, connected with a spherical condenser reflux condensing device and dropwise addition device, placed in a 30℃ constant temperature water bath, stirred for 3h to completely dissolve, pH is adjusted to 9, 0.3g sodium dodecyl sulfate is added, and 3g hexadecyl isocyanate is added dropwise to obtain a reaction solution;

[0039] T2, the reaction solution obtained in T1 is heated to 70℃, and the reaction is carried out for 8h, and then cooled to room temperature, and the pH is adjusted to 4, 300mL of acetone is added to precipitate the reaction product, the unreacted hexadecyl isocyanate is removed, the product is filtered, washed with acetone 3 times, and then dried at 55℃ under vacuum for 48h to obtain hexadecyl isocyanate modified sodium alginate.

[0040] Example 2

[0041] A preparation method of fluorouracil sustained-release nanomicrospheres, comprising the following steps:

[0042] S1, 0.1g polyarginine is dissolved in 10mL deionized water, stirred uniformly, to obtain a polyarginine aqueous solution, 1.0g fluorouracil is dissolved in the polyarginine aqueous solution, stirred uniformly, and the pH is adjusted to 7.2 using a 5% NaOH solution to obtain a fluorouracil solution;

[0043] S2, 0.05g hexadecyl isocyanate modified sodium alginate prepared in example 1 is dissolved in 10mL deionized water, 0.025g carboxymethyl chitosan and 0.025g quaternary ammonium gelatin are added in turn, and stirred uniformly to obtain a composite carrier solution;

[0044] S3, 1 mL of the fluorouracil solution obtained in S1 was mixed with 4 mL of the composite carrier solution obtained in S2, self-assembled by ultrasonic treatment at a power of 800 W for 4 h, centrifuged at 8000 rpm for 20 min, and the precipitate was collected and washed with deionized water to obtain fluorouracil nanomicrospheres.

[0045] Example 3

[0046] The preparation method was the same as that in Example 2, except that the volume of the fluorouracil solution in S3 was 0.050 mL.

[0047] Example 4

[0048] The preparation method was the same as that in Example 2, except that the volume of the fluorouracil solution in S3 was 0.125 mL.

[0049] Example 5

[0050] The preparation method was the same as that in Example 2, except that the volume of the fluorouracil solution in S3 was 0.250 mL.

[0051] Example 6

[0052] The preparation method was the same as that in Example 2, except that the volume of the fluorouracil solution in S3 was 0.500 mL.

[0053] Example 7

[0054] The preparation method was the same as that in Example 2, except that the volume of the fluorouracil solution in S3 was 0.750 mL.

[0055] The fluorouracil nanomicrospheres provided in the above examples were verified for effectiveness by the following tests.

[0056] 1. The fluorouracil nanomicrospheres provided in Example 2 were subjected to scanning electron microscope analysis, and the results are shown in Figure 1 .

[0057] As can be seen from Figure 1 , the fluorouracil nanomicrospheres have uniform particle size distribution, complete structure, and an average particle size of 134 nm.

[0058] 2. The entrapment efficiency and drug loading of the nanomicrospheres at different fluorouracil concentrations provided in Examples 2-7 were determined, and the results are shown in Figure 2 .

[0059] As can be seen from Figure 2 , as the fluorouracil concentration increases, the entrapment efficiency of the prepared fluorouracil nanomicrospheres increases, but the drug loading decreases.

[0060] 3. The release curve of the commercially available fluorouracil tablets and the fluorouracil nanoscale microsphere drug prepared in Example 2 was determined using the existing test protocol, wherein the drug dosage was 50 mg, and the results are shown in Table 3. Figure 3

[0061] As shown in Table 3, the release rate of the fluorouracil tablets was 78% at 0.5 h and 98% at 1 h, while the release rate of the fluorouracil nanoscale microspheres was only 35% at 1 h, 90% at 48 h, and 96% at 72 h, which showed better release performance. Figure 3

[0062] 4. The effect of the commercially available fluorouracil tablets and the fluorouracil nanoscale microspheres prepared in Example 2 on the viability of mouse colon cancer cells CT26 was determined, and three groups were set: a control group Control (no drug), a tablet group (25 μg / mL), and a fluorouracil nanoscale microsphere group (25 μg / mL). The fluorouracil nanoscale microspheres and the commercially available fluorouracil tablets were respectively applied to the mouse colon cancer cells CT26, and after 24 h of culture, the effect of the drugs on the viability of the CT26 cells was calculated, and the results are shown in Table 4. Figure 4

[0063] As shown in Table 4, after 24 h of action, the cell viability of the tablet group was 67.02%, and the cell viability of the nanoscale microsphere group was 53.27%, which had a better inhibitory effect on the proliferation of the mouse colon cancer cells CT26. Figure 4

[0064] 5. The effect of the commercially available fluorouracil tablets and the fluorouracil nanoscale microspheres prepared in Example 2 on the viability of mouse breast cancer cells 4T1 was determined, and three groups were set: a control group Control (no drug), a tablet group (25 μg / mL), and a fluorouracil nanoscale microsphere group (25 μg / mL). The fluorouracil nanoscale microspheres and the commercially available fluorouracil tablets were respectively applied to the mouse breast cancer cells 4T1, and after 24 h of culture, the effect of the drugs on the viability of the 4T1 cells was calculated, and the results are shown in Table 5. Figure 5

[0065] As shown in Table 5, after 24 h of action, the cell viability of the tablet group was 72.48%, and the cell viability of the nanoscale microsphere group was 45.75%, which had a better inhibitory effect on the proliferation of the mouse breast cancer cells 4T1. Figure 5 The above results show that the fluorouracil nanoscale microspheres prepared in the present application are superior to the commercially available fluorouracil tablets in terms of drug release effect, cell uptake rate, and inhibition of tumor cell proliferation, and have good drug delivery and clinical application prospects.

[0066]

[0067] ​​​​​​It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

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. Mix the fluorouracil solution obtained in S1 with the composite carrier solution obtained in S2, and perform self-assembly by ultrasonic treatment. Centrifuge and collect the precipitate to obtain fluorouracil nanospheres. 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; In S2, the amphiphilic alginate is hexadecyl isocyanate modified sodium alginate; 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. 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. 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.

2. 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.

3. Fluorouracil nanospheres prepared by the preparation method according to any one of claims 1-2.

4. The application of the fluorouracil nanospheres as described in claim 3 in the preparation of antitumor drugs.

5. An antitumor drug, characterized in that, The active ingredient comprises the fluorouracil sustained-release nanospheres as described in claim 4.

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