Preparation method, product and application of cyclophosphamide sodium alginate nanospheres

Abstract

The present invention relates to the technical field of anti-cancer drugs, and particularly to a preparation method, product and application of cyclophosphamide sodium alginate nanospheres. The preparation method of the cyclophosphamide sodium alginate nanospheres of the present invention comprises the following steps: dissolving cyclophosphamide in an ε-polylysine aqueous solution to obtain a cyclophosphamide solution; dissolving an amphiphilic alginate in water to obtain an amphiphilic alginate aqueous solution; mixing the cyclophosphamide solution with the amphiphilic alginate aqueous solution to obtain a self-assembly system, and performing self-assembly to obtain the cyclophosphamide sodium alginate nanospheres. The present invention provides a preparation method of cyclophosphamide sodium alginate nanospheres. By encapsulating cyclophosphamide with an amphiphilic alginate and preparing it into a nanosphere drug, the sustained release of the cyclophosphamide drug can be achieved, the decomposition half-life of the drug can be prolonged, the drug utilization rate can be improved, and the drug dosage can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-cancer drugs, and particularly to a preparation method, product and application of cyclophosphamide sodium alginate nanospheres. Background Art

[0002] Cyclophosphamide (CTX) is a nitrogen mustard derivative that is hydrolyzed in the human body by excessive phosphoramidase or phosphatase present in the liver or tumor to become the active phosphoramide mustard. It has a broad anti-tumor spectrum and is the first so-called "latent" broad-spectrum anti-tumor drug, effective against both leukemia and solid tumors.

[0003] Cyclophosphamide is inactive in vitro and mainly exerts its effect by being hydrolyzed by liver P450 enzymes into aldophosphamide and then transported to tissues to form phosphoramide mustard. Cyclophosphamide can be inactivated by being converted into carboxyphosphamide by dehydrogenase or excreted in the form of acrolein, resulting in urinary tract toxicity. It belongs to the cell cycle non-specific drug, and its mechanism of action is the same as that of nitrogen mustard.

[0004] On October 27, 2017, the International Agency for Research on Cancer of the World Health Organization released a preliminary list of carcinogens for reference, and cyclophosphamide is included in the list of Group 1 carcinogens.

[0005] As an anti-tumor drug, it is used for malignant lymphoma, multiple myeloma, breast cancer, small cell lung cancer, ovarian cancer, neuroblastoma, retinoblastoma, Ewing sarcoma, soft tissue sarcoma, as well as acute leukemia and chronic lymphocytic leukemia, etc. It also has a certain therapeutic effect on testicular tumors, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, rhabdomyoma, and osteosarcoma. Currently, it is mostly combined with other anti-cancer drugs to form a combined chemotherapy regimen.

[0006] Cyclophosphamide has high toxicity, a short half-life, and low bioavailability. Currently, there are only tablets and no sustained-release preparations. Summary of the Invention

[0007] Based on the above, the present invention provides a preparation method, product and application of cyclophosphamide sodium alginate nanospheres, which solves the problem of cyclophosphamide sustained release.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] One of the technical solutions of the present invention is a preparation method of cyclophosphamide sodium alginate nanospheres, including the following steps:

[0010] Dissolve cyclophosphamide in an aqueous solution of ε-polylysine to obtain a cyclophosphamide solution;

[0011] Dissolve amphiphilic alginate in water to obtain an amphiphilic alginate aqueous solution;

[0012] Mix the cyclophosphamide solution with the amphiphilic alginate aqueous solution to obtain a self-assembly system, and perform self-assembly to obtain the cyclophosphamide sodium alginate nanospheres.

[0013] The second technical solution of the present invention is the cyclophosphamide sodium alginate nanospheres prepared according to the above preparation method.

[0014] The third technical solution of the present invention is the application of the above cyclophosphamide sodium alginate nanospheres in the preparation of anti-tumor drugs.

[0015] The fourth technical solution of the present invention is an anti-tumor drug, and the active ingredient includes the above cyclophosphamide sodium alginate nanospheres.

[0016] The present invention discloses the following technical effects:

[0017] The present invention provides a preparation method of cyclophosphamide sodium alginate nanospheres. Through the encapsulation of cyclophosphamide by amphiphilic alginate, it is prepared into a nanosphere drug, which can achieve the slow release of cyclophosphamide drug, extend the drug decomposition half-life, improve the drug utilization rate, and reduce the drug usage amount. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is an electron microscope photograph of the cyclophosphamide sodium alginate nanospheres prepared in Example 1 of the present invention.

[0020] Figure 2 It is the particle size distribution of the cyclophosphamide sodium alginate nanospheres prepared in Example 1 of the present invention.

[0021] Figure 3 It is the encapsulation efficiency and drug loading of the nanospheres at different cyclophosphamide concentrations in the present invention.

[0022] Figure 4 It is the drug slow release curve of the cyclophosphamide sodium alginate nanospheres prepared in Example 1 of the present invention.

[0023] Figure 5 It is the comparative analysis of cell viability between the cyclophosphamide sodium alginate nanospheres prepared in Example 1 of the present invention and cyclophosphamide tablets. Detailed Embodiments

[0024] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0025] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0028] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0029] One technical solution of the present invention, a preparation method of cyclophosphamide sodium alginate nanospheres, comprises the following steps:

[0030] Dissolve cyclophosphamide in an ε-polylysine aqueous solution to obtain a cyclophosphamide solution;

[0031] Dissolve amphiphilic alginate in water to obtain an amphiphilic alginate aqueous solution;

[0032] Mix the cyclophosphamide solution with the amphiphilic alginate aqueous solution to obtain a self-assembly system, and perform self-assembly to obtain the cyclophosphamide sodium alginate nanospheres.

[0033] In some embodiments of the present invention, the concentration of cyclophosphamide in the self-assembly system is 2.5 - 20 mg / mL; the concentration of ε-polylysine in the self-assembly system is 0.5 - 1.0 mg / mL. After dissolving cyclophosphamide in the ε-polylysine aqueous solution, it further includes the step of adjusting the pH to 7.2 - 7.4 with NaOH solution.

[0034] Too high or too low concentration of ε-polylysine in the self-assembly system will affect the drug encapsulation effect and the sustained-release performance of the drug. ε-Polylysine has an impact on the size of the nanospheres. Too high or too low concentration of ε-polylysine in the self-assembly system will affect the preparation of the nanospheres, and even nanoscale microspheres cannot be prepared. Therefore, the present invention limits the concentration of ε-polylysine in the self-assembly system to the above parameter range.

[0035] In some embodiments of the present invention, the concentration of amphiphilic alginate in the self-assembly system is 20 - 80 mg / mL; the self-assembly is carried out by ultrasonic method; the power of the ultrasonic wave is 500 - 1000 W. The time of ultrasonic self-assembly is 4 - 6 h. After the self-assembly is completed, it further includes the step of centrifuging to obtain the supernatant. The rotation speed of the centrifugation is 6000 - 8000 rpm, and the time is 10 - 20 min.

[0036] Too high or too low concentration of amphiphilic alginate in the self-assembly system will affect the preparation of the nanospheres, and even nanoscale microspheres cannot be prepared. Therefore, the present invention limits the concentration of amphiphilic alginate in the self-assembly system to the above parameter range.

[0037] The present invention does not particularly limit the concentrations of the cyclophosphamide solution and the amphiphilic alginate aqueous solution, as long as the concentrations of cyclophosphamide, ε-polylysine, and amphiphilic alginate in the self-assembly system obtained by mixing the cyclophosphamide solution and the amphiphilic alginate aqueous solution are within the above parameter ranges.

[0038] In some specific cases of the present invention, the concentration of cyclophosphamide in the cyclophosphamide solution can be limited to 2.5 - 100 mg / mL, the concentration of ε-polylysine in the cyclophosphamide solution can be 0.5 - 5.0 mg / mL, the concentration of amphiphilic alginate in the amphiphilic alginate aqueous solution can be 25 - 100 mg / mL, and the volume ratio of the cyclophosphamide solution to the amphiphilic alginate aqueous solution can be 1:(1 - 10) to regulate the concentrations of cyclophosphamide, ε-polylysine, and amphiphilic alginate in the self-assembly system to the above parameter ranges.

[0039] In some embodiments of the present invention, the amphiphilic alginate is sodium alginate modified with dodecyl glycidyl ether.

[0040] In some embodiments of the present invention, the preparation method of the amphiphilic alginate includes the following steps:

[0041] After adjusting the pH of the sodium alginate aqueous solution to be alkaline (pH = 8.8 - 9.2, more preferably pH = 9.0), sodium dodecyl sulfate and dodecyl glycidyl ether are added to obtain a reaction solution;

[0042] After heating the reaction solution, adjust the pH to acidic (pH = 3.8 - 4.2, more preferably pH = 4.0), add acetone (to remove unreacted dodecyl glycidyl ether), filter the obtained solid phase and dry it to obtain the amphiphilic alginate.

[0043] The purpose of adjusting the pH of the sodium alginate aqueous solution to 8.8 - 9.2 and adjusting the pH of the reaction solution to 3.8 - 4.2 after heating the reaction solution is to prepare nano - scale microspheres. Increasing or decreasing the above pH will affect the preparation of nano - microspheres, and even nano - scale microspheres cannot be made.

[0044] In some embodiments of the present invention, the mass concentration of the sodium alginate aqueous solution is 3% - 10%; the mass - to - volume ratio of sodium alginate to sodium dodecyl sulfate and dodecyl glycidyl ether is 1 g:(0.4 - 1.5) mL:(0.4 - 1) mL.

[0045] Too much or too little addition amount of dodecyl glycidyl ether will affect the yield of the amphiphilic alginate and also affect the drug - loading performance. Therefore, the present invention limits the addition amount of dodecyl glycidyl ether to the above - mentioned ratio range.

[0046] In some embodiments of the present invention, the heating temperature is 65 - 80 °C and the time is 7 - 9 h.

[0047] Too high or too low heating temperature, too long or too short heating time will affect the yield of the amphiphilic alginate and also affect the drug - loading performance.

[0048] The drying is specifically vacuum drying at 50 - 60 °C for 48 - 72 h. Before drying, it also includes the step of washing with acetone 2 - 3 times.

[0049] The present invention provides a method for preparing cyclophosphamide - amphiphilic sodium alginate nano - microspheres, including preparing amphiphilic alginate (SA - DGE), dissolving cyclophosphamide in an ε - polylysine aqueous solution, mixing it with an amphiphilic alginate (SA - DGE) solution, and forming a core - shell structure micelle in water to encapsulate the hydrophobic drug cyclophosphamide, achieving the effect of drug sustained release. Through the encapsulation of cyclophosphamide by amphiphilic alginate, drug sustained release can be realized, the drug decomposition half - life can be prolonged, the drug utilization rate can be improved, and the drug usage amount can be reduced.

[0050] The second technical solution of the present invention is the cyclophosphamide sodium alginate nanospheres prepared according to the above preparation method.

[0051] In the present invention, high performance liquid chromatography can be used to analyze the drug loading and encapsulation efficiency of the cyclophosphamide sodium alginate nanospheres.

[0052] The third technical solution of the present invention is the application of the above-mentioned cyclophosphamide sodium alginate nanospheres in the preparation of anti-tumor drugs.

[0053] The fourth technical solution of the present invention is an anti-tumor drug, the active ingredient of which includes the above-mentioned cyclophosphamide sodium alginate nanospheres.

[0054] The present invention will be further described below through examples.

[0055] Example 1

[0056] Step 1: Dissolve 3 g of sodium alginate in 100 mL of deionized water (the reaction is carried out in a 250 ml three-necked flask equipped with a magnetic bar, a reflux condenser through a spherical condenser, an inlet tube for reactants, and the flask is placed in a constant temperature water bath with a temperature of 30 °C), stir for 3 h until evenly dissolved to obtain solution A.

[0057] Step 2: Adjust the pH of the above solution A to 9.0 with 0.2 M aqueous NaOH solution, then add 3 mL of sodium dodecyl sulfate (SDS), and dropwise add 3 mL of dodecyl glycidyl ether (DGE) through a dropper to obtain a reaction solution; heat the reaction solution to 70 °C, react for 8 h and then cool to room temperature to obtain solution B.

[0058] Step 3: Adjust the pH of the above solution B to 4.0 with acetic acid, add 300 mL of acetone to remove unreacted DEG, then filter to obtain a solid phase, wash the solid phase 3 times with acetone and then dry it in vacuum at 55 °C for 48 h to obtain dodecyl glycidyl ether modified sodium alginate (i.e., amphiphilic alginate, labeled as SA-DGE).

[0059] Step 4: Dissolve 0.1 g of ε-polylysine in 20 mL of aqueous solution, stir until evenly dissolved to obtain an ε-polylysine aqueous solution, then dissolve 1.0 g of cyclophosphamide evenly in 20 mL of the ε-polylysine aqueous solution, add a small amount of 1 M NaOH solution to adjust the pH to 7.2, and dissolve evenly to obtain solution C.

[0060] Step 5: Measure 4 mL of an aqueous solution of amphiphilic alginate (SA-DGE) with a concentration of 0.025 g / mL (the amphiphilic alginate aqueous solution is prepared by dissolving amphiphilic alginate in water), mix it evenly with 1 mL of Solution C, and perform ultrasonic self-assembly for about 4 h to obtain a mixed solution. Then, centrifuge the mixed solution at a rotation speed of 8000 rpm for 20 min to obtain a supernatant drug-loaded nanomicroparticle product (i.e., cyclophosphamide sodium alginate nanomicroparticles), and analyze its drug loading and encapsulation efficiency by high-performance liquid chromatography. The encapsulation efficiency and drug loading are 9.89% and 18.24% respectively.

[0061] Figure 1 and Figure 2 are the electron microscope photograph and particle size distribution diagram of the cyclophosphamide sodium alginate nanomicroparticles prepared in Example 1; from Figure 1 and Figure 2 it can be seen that the microsphere particles are complete and uniform, and the average particle size is 58 nm.

[0062] Figure 3 are the encapsulation efficiency and drug loading of the nanomicroparticles at different cyclophosphamide concentrations (i.e., on the basis of Example 1, only the cyclophosphamide concentration in the self-assembly system is adjusted); from Figure 3 it can be seen that as the cyclophosphamide concentration increases, the encapsulation efficiency of the prepared cyclophosphamide sodium alginate nanomicroparticles increases, but the drug loading decreases.

[0063] Figure 4 is the sustained-release curve of the cyclophosphamide drug (the absolute content of cyclophosphamide in the tablets and cyclophosphamide sodium alginate nanomicroparticles is the same during the experiment). In the figure, a is the cyclophosphamide tablet (abbreviation: tablet; purchased; 50 mg per tablet), and b is the cyclophosphamide sodium alginate nanomicroparticles prepared in Example 1 (abbreviation: nanomicroparticles); from Figure 4 it can be seen that for the tablet, the drug release rate is 80% at 0.5 h and reaches 100% at 1 h. However, for the nanomicroparticles, the drug release rate is only 33% at 1 h. The drug release rate reaches 89% at 50 h and 95% at 75 h. Compared with the cyclophosphamide tablet, the nanomicroparticles have fully achieved the purpose of slow release.

[0064] Figure 5Cell viability analysis of cyclophosphamide sodium alginate nanospheres prepared in Example 1 of the present invention and cyclophosphamide tablets acting on A549 cells simultaneously. The drug concentrations of both cyclophosphamide tablets and cyclophosphamide sodium alginate nanospheres are 20 μg / mL. The control group (Control) does not add drugs (when performing the cell viability experiment, each sample has a culture medium. For the tablet sample, it is culture medium + tablet; for the cyclophosphamide sodium alginate nanosphere sample, it is culture medium + cyclophosphamide sodium alginate nanosphere; for the control sample (Control), only the culture medium is used without adding drugs). The tablets with the same absolute concentration of cyclophosphamide and cyclophosphamide sodium alginate nanospheres are simultaneously applied to the lung cancer cell A549 and cultured for 24 h, 48 h, and 72 h respectively. Subsequently, the effect of the drugs on the viability of A549 cells is calculated. The results show that when the cyclophosphamide tablets act for 24 h, the viability of A549 cells is 67.82%. Continuing to act for 48 h and 72 h, it is found that the cell viability subsequently increases and reaches 89.84% of the control group at 72 h. However, when the cyclophosphamide sodium alginate nanospheres act for 24 h, the viability of A549 cells is 72.16%, showing no difference from the cyclophosphamide tablet group. Continuing to act for 48 h and 72 h, it is found that the cell viability subsequently continues to decrease, dropping to 42.73% of the control group at 48 h and 20.74% of the control group at 72 h, showing a significant difference from the cyclophosphamide tablet group. This indicates that for the same concentration of cyclophosphamide, cyclophosphamide sodium alginate nanospheres have a better effect on inhibiting the growth of lung cancer cell A549 compared to tablets.

[0065] The embodiments described above are only used to describe the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing cyclophosphamide sodium alginate nanoparticles, characterized in that: The following steps are involved: dissolving cyclophosphamide in an ε-polylysine aqueous solution to obtain a cyclophosphamide solution; dissolving amphiphilic alginate in water to obtain an amphiphilic alginate aqueous solution; The cyclophosphamide solution is mixed with the amphiphilic alginate aqueous solution to obtain a self-assembly system, and self-assembly is performed to obtain the cyclophosphamide sodium alginate nanoparticles; The amphiphilic alginate is sodium alginate modified by dodecyl glycidyl ether; The concentration of cyclophosphamide in the self-assembly system is 2.5-20 mg / mL; The concentration of the amphiphilic alginate in the self-assembly system is 20-80 mg / mL.

2. The method for preparing cyclophosphamide sodium alginate nanoparticles according to claim 1, characterized in that: The concentration of ε-polylysine in the self-assembly system is 0.5-1.0 mg / mL.

3. The method for preparing cyclophosphamide sodium alginate nanoparticles according to claim 1, characterized in that: The self-assembly is carried out in an ultrasonic manner.

4. The method for preparing cyclophosphamide sodium alginate nanoparticles according to claim 1, characterized in that: The preparation method of the amphiphilic alginate comprises the following steps: After adjusting the pH of the sodium alginate aqueous solution to 8.8-9.2, sodium lauryl sulfate and lauryl glycidyl ether were added to obtain a reaction solution; After heating the reaction solution, the pH is adjusted to 3.8-4.2, acetone is added, and the solution is filtered. The obtained solid phase is dried to obtain the amphiphilic alginate.

5. The method for preparing cyclophosphamide sodium alginate nanoparticles according to claim 4, characterized in that: The mass concentration of the sodium alginate aqueous solution is 3%-10%; the mass volume ratio of sodium alginate to the sodium dodecyl sulfate and dodecyl glycidyl ether is 1g:(0.4-1.5)mL:(0.4-1)mL.

6. The method for preparing cyclophosphamide sodium alginate nanoparticles according to claim 4, characterized in that: The heating temperature is 65-80°C and the heating time is 7-9h.

7. Cyclophosphamide sodium alginate nanoparticles prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the cyclophosphamide sodium alginate nanoparticles according to claim 7 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: The effective ingredients include the cyclophosphamide sodium alginate nanoparticles according to claim 7.

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