Preparation method of oral radiation protection nano-drug

Abstract

The invention discloses a preparation method of an oral radiation protection nano-drug, which comprises the following steps: grafting amifostine onto carboxymethyl chitosan under the condition of normal temperature through a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (EDC / NHS) catalytic system in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the pH response type radiation protection nano-drug is synthesized by a one-step method. Intestinal radiation protection can be achieved by orally taking the pH response type radiation protection nano-drug, the nano-drug has pH sensitivity and can achieve targeted intestinal radiation protection, the bioavailability of the small molecule drug amifostine in the gastrointestinal tract can be effectively improved, the biotoxicity of the small molecule drug amifostine can be effectively reduced, and the nano-drug has important significance in the field of intestinal radiation protection.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to a preparation method of an oral nano-drug for radiation protection. Background Art

[0002] With the rapid development and wide application of nuclear energy and nuclear technologies in multiple fields such as industry and medicine, the health problems brought about by ionizing radiation have become increasingly prominent and have become one of the focuses of current social concern. Ionizing radiation can cause varying degrees of damage to multiple organ systems in the human body. Among them, the small intestine tissue has become a common site of radiation damage due to its high sensitivity and relatively large organ volume. The clinical manifestations of radiation-induced intestinal injury are diverse, including but not limited to diarrhea, hematochezia, electrolyte disorders, and may even be life-threatening in extreme cases. However, currently, the preventive and therapeutic means for radiation-induced intestinal injury in clinical practice are still relatively scarce, and there is no widely recognized effective drug or treatment strategy. Therefore, developing a radiation protection agent for radiation-induced intestinal injury is of extremely important practical significance and clinical value for reducing accidental irradiation or the radiation hazards that may be suffered during planned radiotherapy.

[0003] Currently, the clinical intervention measures for radiation-induced intestinal injury mainly rely on drug treatment and surgical treatment. In terms of drug treatment, traditional antidiarrheal drugs, antiemetic drugs, mucosal protectants, and antioxidant nutrients (such as vitamins A, B, C, and E) are widely used to relieve the clinical symptoms of gastrointestinal injury. However, these drugs can only relieve the symptoms to a certain extent. In cases of severe symptoms or ineffective drug treatment, intestinal transplantation may be required through surgery, but the surgical risk is high and the recovery is slow. In addition to the above traditional drugs, radiation protection drugs such as free radical scavengers (DOI: 10.1016 / j.freeradbiomed.2018.10.) and thiol preparations (DOI: 10.1634 / theoncologist.12-6-738) have also shown certain application potential in the field of intestinal radiation protection. In particular, amifostine, as the only selective normal tissue radioprotectant approved by the US Food and Drug Administration (FDA) currently, its clinical use provides a new idea for the prevention and treatment of radiation-induced intestinal injury.

[0004] Nevertheless, the existing radiation protection drugs still have many deficiencies. Most drugs are small molecule compounds, with the disadvantages of short biological half-life and obvious toxic and side effects (such as nausea, vomiting, hypotension, etc.). In addition, the targeting of these drugs in the body is poor, and it is difficult to effectively accumulate in the small intestine, thus limiting their radiation protection effect on the intestine. Moreover, the harsh physiological environment in the gastrointestinal tract, especially the strong action of gastric acid, will further reduce the bioavailability of such drugs.

[0005] In summary, developing a radioprotective agent for radiation-induced intestinal injury with high efficiency, low toxicity, good targeting and bioavailability is a key technical problem that urgently needs to be solved in this field currently. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a preparation method of an oral radioprotective nanomedicine, which realizes intestinal radiation protection through oral administration. This nanomedicine has pH sensitivity and can achieve targeted intestinal radiation protection, effectively improving the bioavailability of the small molecule drug amifostine in the gastrointestinal tract and reducing its biological toxicity, and is of great significance in the field of intestinal radiation protection.

[0007] The present invention is realized through the following technical solutions:

[0008] The present invention provides a preparation method of a pH-responsive radioprotective nanomedicine, including the following steps:

[0009] Dissolve carboxymethyl chitosan (CS) in a buffer solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) and N-hydroxysuccinimide (NHS) to the obtained carboxymethyl chitosan solution for activation under dark conditions, keep the pH value of the solution at 6.7 - 7.3 during the activation process, add the activated mixed solution to the amifostine solution for reaction under dark conditions, adjust the pH value to 7.8 - 8.2 to terminate the reaction, and perform dialysis and drying treatments to obtain the pH-responsive radioprotective nanomedicine.

[0010] In the present invention, amifostine is grafted onto carboxymethyl chitosan by the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (EDC / NHS) catalytic system in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride under normal temperature conditions to synthesize Am@CS nanomedicine by a one-step method. The modification of carboxymethyl chitosan endows the nanomedicine with excellent biocompatibility and pH-responsive performance, can maintain the biological activity of amifostine in the gastrointestinal tract, improve the bioavailability of amifostine in intestinal radiation protection, effectively relieve radiation-induced intestinal injury, and provide new ideas for the prevention and treatment of radiation-induced intestinal injury.

[0011] Further, the buffer solution is phosphate buffer solution.

[0012] Further, the concentration of carboxymethyl chitosan in the carboxymethyl chitosan solution is 0.001 - 0.05 g / mL.

[0013] Further, the mass ratio of carboxymethyl chitosan to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(0.9 - 1.1).

[0014] Furthermore, the mass ratio of the carboxymethyl chitosan to N-hydroxysuccinimide is 1:(0.5 - 0.7).

[0015] Furthermore, the activation time is 0.2 - 1 h.

[0016] Furthermore, during the activation process, the pH value can be adjusted with a sodium hydroxide solution.

[0017] Furthermore, the volume ratio of the mixed solution to the amifostine solution is (2 - 5):1.

[0018] Furthermore, the amifostine solution is prepared by dissolving amifostine in a phosphate buffer solution.

[0019] Furthermore, the concentration of amifostine in the amifostine solution is 0.005 - 0.02 g / mL.

[0020] Furthermore, the reaction time is 24 - 48 h.

[0021] Furthermore, the reaction is terminated by adjusting the pH value to 7.8 - 8.2 with a sodium hydroxide solution.

[0022] Furthermore, the concentration of the sodium hydroxide solution is 0.1 - 0.3 M.

[0023] Furthermore, dialysis is carried out with deionized water, and the cut-off molecular weight of the dialysis bag is 3000 - 4000 Da.

[0024] The present invention protects the pH-responsive radiation protection nano-drug prepared by the above preparation method.

[0025] The present invention also protects the application of the above pH-responsive radiation protection nano-drug in the preparation of a radiation protection agent for radioactive intestinal injury.

[0026] The present invention can achieve intestinal radiation protection by orally administering the pH-responsive radiation protection nano-drug. This nano-drug has pH sensitivity and can achieve targeted intestinal radiation protection, effectively improving the bioavailability of the small molecule drug amifostine in the gastrointestinal tract and reducing its biological toxicity, and enhancing the intestinal radiation protection effect.

[0027] Under the condition of pH = 2.0, the pH-responsive radiation protection nano-drugs aggregate together through electrostatic interaction, and the structure is very stable, which can prevent the internal amifostine from contacting with the highly acidic gastric juice outside, effectively protecting the biological activity of amifostine.

[0028] Under the condition of pH = 6.8, the electrostatic interaction between the pH-responsive radiation protection nano-drugs weakens, and the aggregated structure gradually disperses, and is expected to quickly pass through the gaps between the reticular fibers of the small intestinal mucus layer to play a radiation protection role.

[0029] Advantages of the present invention:

[0030] 1. The pH-responsive radioprotective nano-drug provided by the present invention has pH-responsive performance and acid resistance in the stomach, can effectively protect the biological activity of amifostine, and improve the oral bioavailability of the small molecule drug amifostine.

[0031] 2. The pH-responsive radioprotective nano-drug provided by the present invention has good biocompatibility. Its targeted enrichment in the intestine is beneficial to enhancing its precise protective effect on intestinal tissues, and can also reduce the toxic and side effects on other tissues and organs to a certain extent.

[0032] 3. The pH-responsive radioprotective nano-drug provided by the present invention can achieve effective intestinal radiation protection, and its intestinal radiation protection ability is superior to that of free amifostine. Description of the drawings

[0033] Figure 1 Schematic diagram of the application principle of the pH-responsive radioprotective nano-drug provided by the present invention for preparing a radioprotective agent for radioactive intestinal injury.

[0034] Figure 2 Test result chart of the pH-responsive ability of the Am@CS nano-drug prepared in Example 1; among them, A is the particle size distribution chart of the Am@CS nano-drug dissolved in phosphate buffer, B is the particle size distribution chart of the Am@CS nano-drug under simulated gastric juice conditions, C is the physical map and TEM map of the Am@CS nano-drug under simulated gastric juice conditions, D is the particle size distribution chart of the Am@CS nano-drug under simulated intestinal juice conditions, and E is the physical map and TEM map of the Am@CS nano-drug under simulated intestinal juice conditions.

[0035] Figure 3 Hydrolysis efficiency chart of the Am@CS nano-drug and free amifostine in a simulated gastrointestinal environment; among them, A is the Am@CS nano-drug and B is free amifostine.

[0036] Figure 4 Test result chart of the intestinal targeting ability of the Am@CS nano-drug prepared in Example 1; among them, A is the typical in vitro fluorescence distribution images of peripheral blood and main tissues and organs at 6, 24, and 48 hours after C57BL / 6 mice were orally gavaged with fluorescently labeled Am@CS nano-drug, and B is the quantitative detection result chart of fluorescence intensity.

[0037] Figure 5 Representative images of H&E stained pathological tissue sections of mouse small intestine tissues and crypt survival rate data charts after different treatments; among them, A is the representative image of the H&E stained pathological tissue section, and B is the crypt survival rate data chart. Detailed implementation manners

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0039] The present invention provides a method for preparing a pH-responsive radiation protection nanomedicine, comprising the following steps:

[0040] Dissolve carboxymethyl chitosan (CS) in a buffer solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) and N-hydroxysuccinimide (NHS) to the obtained carboxymethyl chitosan solution for activation under light-shielded conditions, and maintain the pH value of the solution at 6.7 - 7.3 during the activation process. Add the activated mixed solution to an amifostine (Am) solution for reaction under light-shielded conditions. The reaction equation is:

[0041]

[0042] Adjust the pH value to 7.8 - 8.2 to terminate the reaction, and perform dialysis and drying treatments to obtain the pH-responsive radiation protection nanomedicine.

[0043] The pH-responsive radiation protection nanomedicine provided by the present invention can be used to prepare a radiation protection agent for radioactive intestinal injury. The schematic diagram of the application principle is as Figure 1 shown. The present invention realizes intestinal radiation protection by orally administering the pH-responsive radiation protection nanomedicine. Under the condition of gastric juice pH = 2.0, the pH-responsive radiation protection nanomedicine has pH-responsive performance and acid resistance, aggregates together through electrostatic interaction, and has a very stable structure, which can prevent the internal amifostine from contacting with the external highly acidic gastric juice and effectively protect the biological activity of amifostine; under the condition of intestinal juice pH = 6.8, the electrostatic interaction between the pH-responsive radiation protection nanomedicines weakens, and the aggregated structure gradually disperses, and is expected to quickly pass through the gaps between the reticular fibers of the small intestinal mucus layer to play a radiation protection role.

[0044] The following further illustrates the present invention with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0045] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0046] Example 1

[0047] A preparation method of a pH-responsive radioprotective nano-drug (Am@CS) comprises the following steps:

[0048] Dissolve amifostine (0.1 g) in phosphate buffer solution (10 mL), and stir to obtain an amifostine solution. Dissolve carboxymethyl chitosan (0.1 g) in phosphate buffer solution (30 mL), and continuously stir until the solution becomes clear and transparent. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (95.9 mg) and N-hydroxysuccinimide (57.6 mg), and activate them in the dark for 30 minutes. Keep the pH of the solution at about 7.0. Gradually add the obtained solution to the amifostine solution, and stir the mixture in the dark at room temperature for 24 hours. Then adjust the pH value of the reaction solution to 8.0 with sodium hydroxide solution (0.15 M) to terminate the reaction. Dialyze the Am@CS polymer with deionized water for 3 days (the cut-off molecular weight of the dialysis bag is 3500 Da). Finally, freeze-dry the product with a vacuum freeze dryer to obtain the Am@CS nano-drug.

[0049] Perform a pH-responsive ability test on the Am@CS nano-drug prepared in Example 1. The test results are as Figure 2 shown. Among them, A is the particle size distribution diagram of the Am@CS nano-drug dissolved in phosphate buffer solution. When the nano-drug is dissolved in phosphate buffer solution, the hydrated particle size is about 209.1 nm, and the dispersity is 0.108. B is the particle size distribution diagram of the Am@CS nano-drug under simulated gastric juice conditions. C is the physical picture (left picture) and transmission electron microscope (TEM) picture (right picture, scale bar is 1 μm) of the Am@CS nano-drug under simulated gastric juice conditions. Under the condition of simulated gastric juice with pH = 2.0, the pH-responsive performance of the Am@CS nano-drug enables it to aggregate together through electrostatic interaction, the particle size increases significantly, and a white flocculent precipitate precipitates. This structure is very stable, which can prevent the internal amifostine from contacting the external highly acidic gastric juice and effectively protect the biological activity of amifostine. D is the particle size distribution diagram of the Am@CS nano-drug under simulated intestinal juice conditions. E is the physical picture (left picture) and TEM picture (right picture, scale bar is 500 nm, inset scale bar is 200 nm) of the Am@CS nano-drug under simulated intestinal juice conditions. Under the condition of simulated intestinal juice with pH = 6.8, the electrostatic interaction between the Am@CS nano-drugs weakens, and the aggregated structure gradually disperses, and can basically restore the original particle size (275.8 nm) and presents as spherical particles with uniform size under the TEM image, and is expected to quickly pass through the gaps between the reticular fibers of the small intestinal mucus layer to play a radioprotective role.

[0050] Figure 3Hydrolysis efficiency diagrams of Am@CS nanomedicine and free amifostine in simulated gastric and intestinal environments. Here, A represents Am@CS nanomedicine and B represents free amifostine. By comparing the process of hydrolysis of free amifostine and Am@CS nanomedicine to produce free sulfhydryl groups in simulated digestive fluids, it can be found that free amifostine rapidly hydrolyzes and releases almost 100% of free sulfhydryl groups within the first 20 minutes in simulated gastric fluid, while Am@CS nanomedicine only releases approximately 26% of free sulfhydryl groups in simulated gastric fluid within 2 hours, indicating that Am@CS nanomedicine has good gastric acid stability and thus maintains its biological activity before reaching the small intestine. In the simulated intestinal fluid environment, the hydrolysis process of Am@CS nanomedicine shows obvious sustained-release characteristics, with approximately 30% hydrolysis within 1 hour and more than 6 hours required for complete hydrolysis. However, the content of terminal active sulfhydryl groups of free amifostine continuously decreases after complete hydrolysis, indicating that the sulfhydryl groups produced by hydrolysis gradually react with each other to form disulfides and lose their biological activity.

[0051] The intestinal targeting ability of the Am@CS nanomedicine prepared in Example 1 was tested. The test method was as follows: The Am@CS nanomedicine prepared in Example 1 was labeled with Cy5.5 fluorescent dye and then resuspended in phosphate buffer. After fasting C57BL / 6J mice for 12 hours, the dye-labeled nanomedicine solution (8 mg / mL, 0.3 mL) was administered to the mice by gavage, and the peripheral blood and main tissue organs of the mice were subjected to in vitro fluorescence imaging and quantitative analysis of the average fluorescence intensity at 6 hours, 24 hours, and 48 hours respectively.

[0052] The test results are as Figure 4 shown. Here, A is a typical in vitro fluorescence distribution image of the peripheral blood and main tissue organs of C57BL / 6 mice at 6, 24, and 48 hours after oral gavage with fluorescently labeled Am@CS nanomedicine, and B is a graph of the quantitative detection results of fluorescence intensity. As can be seen from Figure 4 it, the Am@CS nanomedicine was not significantly distributed in tissues and organs such as blood, liver, lung, and kidney, but was highly enriched in the digestive tract, especially the small intestine, and had a long retention time. The targeted enrichment of the Am@CS nanomedicine in the intestine is beneficial to enhancing its precise protective effect on intestinal tissues and can also reduce the toxic and side effects on other tissues and organs to a certain extent.

[0053] Two hours after C57BL / 6 mice were orally administered Am@CS nanomedicine, they received 14 Gy of whole abdominal X-ray irradiation. Five days after irradiation, histopathological analysis was performed on their intestinal tissue sections and compared with different treatment groups such as the simple irradiation group. The test results are as Figure 5 shown.Figure 5 Representative images of H&E (hematoxylin-eosin) stained pathological tissue sections and crypt survival rate data graphs of mouse small intestine tissues after different treatments; among them, A is a representative image of the H&E stained pathological tissue section (scale bar is 100 μm), and B is the crypt survival rate data graph. Compared with the normal group (Control, only gavaged with PBS) where the intestinal morphological structure of the mice is clear, the number of crypts is normal, and the villus structure is intact, a large number of crypt stem cells in the mice of the simple irradiation group (IR+PBS), the carboxymethyl chitosan treatment group (IR+CS), and the free amifostine treatment group (IR+Amifostine) died, the crypt structure was significantly lost, and the villus structure was also damaged. However, the mice orally gavaged with the Am@CS nanodrug (IR+Am@CS) could largely maintain the integrity of the crypt-villus structure. By observing and counting the intestinal crypt survival rates of mice in different treatment groups, it was found that the number of surviving crypts in the irradiated mice treated with the Am@CS nanodrug was significantly higher than that in the simple irradiation group, the carboxymethyl chitosan treatment group, and the free amifostine treatment group, indicating that compared with free amifostine, the Am@CS nanodrug could significantly improve the bioavailability of amifostine in the gastrointestinal tract, significantly enhance its radiation protection effect, maintain the structure and function of the intestinal crypt-villus in mice, and relieve the intestinal damage induced by radiation.

[0054] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly explaining the present invention, and are not intended to limit the implementation manners of the present invention. Those skilled in the art should understand that other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a pH-responsive radiation protection nano-drug, characterized in that, It includes the following steps: Dissolve carboxymethyl chitosan in a buffer solution. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the obtained carboxymethyl chitosan solution and carry out activation under light-shielded conditions. During the activation process, maintain the pH value of the solution at 6.7 - 7.

3. Add the activated mixed solution to the amifostine solution and carry out a reaction under light-shielded conditions. Adjust the pH value to 7.8 - 8.2 to terminate the reaction, and carry out dialysis and drying treatments to obtain the pH-responsive radiation protection nano-drug.

2. The preparation method according to claim 1, characterized in that, The concentration of carboxymethyl chitosan in the carboxymethyl chitosan solution is 0.001 - 0.05 g / mL.

3. The preparation method according to claim 1, characterized in that, The mass ratio of carboxymethyl chitosan to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(0.9 - 1.1).

4. The preparation method according to claim 1, characterized in that, The mass ratio of carboxymethyl chitosan to N-hydroxysuccinimide is 1:(0.5 - 0.7).

5. The preparation method according to claim 1, characterized in that, The activation time is 0.2 - 1 h.

6. The preparation method according to claim 1, wherein, The volume ratio of the mixed solution to the amifostine solution is (2 - 5):

1.

7. The preparation method according to claim 1, characterized in that, The concentration of amifostine in the amifostine solution is 0.005 - 0.02 g / mL.

8. The preparation method according to claim 1, wherein, The reaction time is 24 - 48 h.

9. A pH-responsive radiation protection nano-drug prepared by the preparation method according to any one of claims 1 - 8.

10. Use of the pH-responsive radiation protection nano-drug according to claim 9 in the preparation of a radiation protection agent for radioactive intestinal injury.

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