Preparation method of oral radiation protection nanomedicine

Abstract

The application discloses a preparation method of an oral radiation protection nano drug, and the preparation method is characterized in that 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (EDC / NHS) catalytic systems are used to graft amifostine to carboxymethyl chitosan under normal temperature conditions, and a pH-responsive radiation protection nano drug is synthesized by one-step method. The oral pH-responsive radiation protection nano drug can realize intestinal radiation protection, the nano drug has pH sensitivity and can realize targeted intestinal radiation protection, can effectively improve the bioavailability of a small-molecule drug amifostine in a gastrointestinal tract and reduce the biological toxicity of the small-molecule drug, and has important significance in the field of intestinal radiation protection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a preparation method of an oral radiation protection nano-drug. BACKGROUND

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

[0003] Currently, the clinical interventions for radiation-induced intestinal damage mainly rely on drug therapy and surgical treatment. In terms of drug therapy, traditional antidiarrheal drugs, antiemetic drugs, mucosa protective agents, and antioxidant nutrients (such as vitamins A, B, C, and E) are widely used to alleviate the clinical symptoms of gastrointestinal damage. However, these drugs can only alleviate symptoms to a certain extent, and in cases of severe symptoms or ineffective drug therapy, intestinal transplantation through surgery may be required, but surgery is high-risk and slow to recover. 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 intestinal radiation protection. In particular, Amifostine, as the only selective normal tissue radiation protection agent approved by the US Food and Drug Administration (FDA), its use in clinical practice provides a new idea for the prevention and treatment of radiation-induced intestinal damage.

[0004] Despite this, the existing radiation protection drugs still have many shortcomings. Most of the drugs are small molecule compounds, which have the disadvantages of short biological half-life and obvious toxic side effects (such as nausea, vomiting, hypotension, etc.). In addition, these drugs have poor targeting in the body and are difficult to effectively enrich in the small intestine, thereby limiting their radiation protection effect on the intestine. In addition, 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 radiotherapy radiation protection agent with high efficiency, low toxicity, good targeting and bioavailability is a key technical problem to be solved in the current field. SUMMARY

[0006] To solve the above technical problems, the application provides a preparation method of an oral radiation protection nano drug, which realizes intestinal radiation protection through oral administration. The nano drug has pH sensitivity and can realize targeted intestinal radiation protection, effectively improves the bioavailability of the small molecule drug amifostine in the gastrointestinal tract and reduces its biological toxicity, and has important significance in the field of intestinal radiation protection.

[0007] The application is implemented by the following technical solutions:

[0008] The application provides a preparation method of a pH-responsive radiation protection nano drug, including the following steps:

[0009] Carboxymethyl chitosan (CS) is dissolved in a buffer solution, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCl) and N-hydroxysuccinimide (NHS) are added to the obtained carboxymethyl chitosan solution under light-proof conditions for activation, the pH value of the solution is maintained at 6.7-7.3 during the activation process, the mixed solution obtained after activation is added to an amifostine solution for reaction under light-proof conditions, the pH value is adjusted to 7.8-8.2 to terminate the reaction, and dialysis and drying treatment are performed to obtain the pH-responsive radiation protection nano drug.

[0010] The application grafts amifostine onto carboxymethyl chitosan through a 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride catalytic system (EDC / NHS) at room temperature, and synthesizes the Am@CS nano drug by one-step method. The modification of carboxymethyl chitosan makes the nano drug have excellent biocompatibility and pH response performance, can maintain the biological activity of amifostine in the gastrointestinal tract, improve the bioavailability of amifostine in intestinal radiation protection, effectively alleviate radiation-induced intestinal injury, and provide a new idea for preventing and treating radioactive intestinal injury.

[0011] Further, the buffer solution is a 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-ethyl carbodiimide hydrochloride is 1:(0.9-1.1).

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

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

[0016] Further, the pH value can be adjusted by using a sodium hydroxide solution in the activation process.

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

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

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

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

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

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

[0023] Further, dialysis is performed by using deionized water, and the molecular weight cut-off of the dialysis bag is 3000-4000Da.

[0024] The application protects the pH-responsive radiation protection nanodrug prepared by the above preparation method.

[0025] The application also protects the application of the above pH-responsive radiation protection nanodrug in the preparation of a radiation protection agent for radio-induced intestinal injury.

[0026] The application can achieve intestinal radiation protection by orally taking the pH-responsive radiation protection nanodrug, which has pH sensitivity and can achieve targeted intestinal radiation protection, effectively improve the bioavailability of the small-molecule drug amifostine in the gastrointestinal tract and reduce its biological toxicity, and improve the effect of intestinal radiation protection.

[0027] Under the condition of pH=2.0, the pH-responsive radiation protection nanodrug is aggregated together through electrostatic interaction, and the structure is very stable, which can hinder the contact between the internal amifostine and the external highly acidic gastric juice, and effectively protect the biological activity of amifostine.

[0028] Under the condition of pH=6.8, the electrostatic interaction between the pH-responsive radiation protection nanodrug is weakened, and the aggregated structure is gradually dispersed, which is expected to quickly pass through the gap between the reticular fibers of the small intestinal mucus layer and play a radiation protection role.

[0029] The beneficial effects of the present application are as follows:

[0030] 1. The pH-responsive radiation protection nanodrug provided by the present application 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 radiation protection nanodrug provided by the present application has good biocompatibility, and the targeted enrichment thereof in the intestinal tract is conducive to enhancing the precise protection effect of the pH-responsive radiation protection nanodrug on the intestinal tissue and also can reduce the toxic and side effects on other tissues and organs to a certain extent.

[0032] 3. The pH-responsive radiation protection nanodrug provided by the present application can achieve effective intestinal radiation protection, and the intestinal radiation protection capacity thereof is superior to that of free amifostine. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The application principle schematic diagram for the pH-responsive radiation protection nanodrug provided by the present application for preparing a radiation protection agent for radioactive intestinal injury.

[0034] Figure 2 The pH-responsive capability test result diagram of the Am@CS nanodrug prepared in Example 1; wherein, A is the particle size distribution diagram of the Am@CS nanodrug dissolved in a phosphate buffer, B is the particle size distribution diagram of the Am@CS nanodrug under simulated gastric juice conditions, C is the actual object diagram and TEM diagram of the Am@CS nanodrug under simulated gastric juice conditions, D is the particle size distribution diagram of the Am@CS nanodrug under simulated intestinal juice conditions, and E is the actual object diagram and TEM diagram of the Am@CS nanodrug under simulated intestinal juice conditions.

[0035] Figure 3 The hydrolysis efficiency diagram of the Am@CS nanodrug and free amifostine in simulated gastrointestinal environment, wherein A is the Am@CS nanodrug, and B is free amifostine.

[0036] Figure 4 The intestinal targeting capability test result diagram of the Am@CS nanodrug prepared in Example 1; wherein, A is the typical in vitro fluorescence distribution image of the peripheral blood and main tissue organs of C57BL / 6 mice after oral gavage administration of the fluorescently labeled Am@CS nanodrug for 6, 24 and 48 hours, and B is the fluorescence intensity quantitative detection result diagram.

[0037] Figure 5 The representative image of the H&E staining pathological tissue section and the data diagram of the crypt survival rate of the small intestinal tissue of the mice after different treatments; wherein, A is the representative image of the H&E staining pathological tissue section, and B is the data diagram of the crypt survival rate. DETAILED DESCRIPTION

[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 art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0039] The application provides a preparation method of a pH-responsive radiation protection nanodrug, comprising the following steps:

[0040] Carboxymethyl chitosan (CS) is dissolved in a buffer solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) and N-hydroxysuccinimide (NHS) are added to the obtained carboxymethyl chitosan solution to activate under light-proof conditions, the pH value of the solution is maintained at 6.7-7.3 during the activation process, and the mixed solution obtained after the activation is added to an amifostine (Am) solution to react under light-proof conditions, and the reaction equation is as follows:

[0041]

[0042] The reaction is terminated by adjusting the pH value to 7.8-8.2, dialysis and drying treatment are performed, and the pH-responsive radiation protection nanodrug is obtained.

[0043] The pH-responsive radiation protection nanodrug provided by the application can be used for preparing a radiation protection agent for radio-induced intestinal injury, and the application principle schematic diagram is as shown in the figure. Figure 1 The pH-responsive radiation protection nanodrug realizes intestinal radiation protection by oral administration, has pH-responsive performance and acid resistance under the condition of gastric juice pH=2.0, is aggregated together through electrostatic interaction, has a very stable structure, can hinder the contact between the internal amifostine and the external highly acidic gastric juice, and effectively protects the biological activity of the amifostine; under the condition of intestinal juice pH=6.8, the electrostatic interaction between the pH-responsive radiation protection nanodrugs is weakened, the aggregated structure is gradually dispersed, and it is expected to quickly pass through the gaps between the reticular fibers of the small intestinal mucus layer and play a radiation protection role.

[0044] The application will be further described in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting to the application.

[0045] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0046] Example 1

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

[0048] Amifostine (0.1 g) was dissolved in a phosphate buffer (10 mL) to obtain an amifostine solution. Carboxymethyl chitosan (0.1 g) was dissolved in a phosphate buffer (30 mL) and continuously stirred until the solution became clear and transparent. Subsequently, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (95.9 mg) and N-hydroxysuccinimide (57.6 mg) were added to the solution and activated in the dark for 30 minutes. The pH of the solution was maintained at about 7.0. The resulting solution was gradually added to the amifostine solution. The mixture was stirred at room temperature in the dark for 24 hours. The pH of the reaction solution was adjusted to 8.0 using a sodium hydroxide solution (0.15 M) to terminate the reaction. The Am@CS polymer was dialyzed against deionized water for 3 days (the molecular weight cut-off of the dialysis bag was 3500 Da). Finally, the product was freeze-dried by a vacuum freeze dryer to obtain the Am@CS nanodrug.

[0049] The Am@CS nanodrug prepared in Example 1 was tested for pH responsiveness, and the test results are shown in FIG. 1. Figure 2 As shown in FIG. 1, A is a particle size distribution diagram of the Am@CS nanodrug dissolved in a phosphate buffer. The hydrated particle size of the nanodrug dissolved in the phosphate buffer is about 209.1 nm, and the dispersity is 0.108. B is a particle size distribution diagram of the Am@CS nanodrug under simulated gastric fluid conditions. C is a real object diagram (left) and a transmission electron microscope (TEM) diagram (right, scale bar: 1 μm) of the Am@CS nanodrug under simulated gastric fluid conditions. Under the simulated gastric fluid pH = 2.0 conditions, the Am@CS nanodrug has a pH-responsive performance that causes it to aggregate together by electrostatic interaction, the particle size significantly increases, and white flocculent precipitate is separated out. This structure is very stable and can hinder the contact between the internal amifostine and the external highly acidic gastric juice, effectively protecting the biological activity of amifostine. D is a particle size distribution diagram of the Am@CS nanodrug under simulated intestinal fluid conditions. E is a real object diagram (left) and a TEM diagram (right, scale bar: 500 nm, inset scale bar: 200 nm) of the Am@CS nanodrug under simulated intestinal fluid conditions. Under the simulated intestinal fluid pH = 6.8 conditions, the electrostatic interaction between the Am@CS nanodrug is weakened, the aggregation structure gradually disperses, and the original particle size (275.8 nm) can be basically restored. The TEM image shows that the Am@CS nanodrug is a spherical particle with uniform size, which is expected to quickly pass through the gaps between the reticular fibers of the small intestinal mucus layer and play a radioprotective role.

[0050] Figure 3Figure of hydrolysis efficiency of Am@CS nanodrug and free amifostine in simulated gastric and intestinal environment, wherein A is Am@CS nanodrug, and B is free amifostine. By comparing the process of hydrolysis of free amifostine and Am@CS nanodrug to generate free thiol in simulated digestive juice, it can be found that free amifostine is rapidly hydrolyzed to release almost 100% free thiol within the first 20 minutes in simulated gastric juice, while Am@CS nanodrug only releases about 26% free thiol in simulated gastric juice for 2 hours, indicating that Am@CS nanodrug has good gastric acid stability, thereby maintaining its biological activity before reaching the small intestine. In the simulated intestinal environment, the hydrolysis process of Am@CS nanodrug shows obvious sustained-release characteristics, with about 30% hydrolysis within 1 hour, and complete hydrolysis requiring more than 6 hours. While the content of terminal active thiol of free amifostine continuously decreases after complete hydrolysis, indicating that the thiol generated by hydrolysis gradually reacts with each other to form disulfide, losing biological activity.

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

[0052] The test results are shown in Figure 4 , wherein A is a typical in vitro fluorescence distribution image of peripheral blood and main organs of C57BL / 6 mice at 6, 24 and 48 hours after oral gavage administration of fluorescently labeled Am@CS nanodrug, and B is a fluorescence intensity quantitative detection result graph. As can be seen from Figure 4 , Am@CS nanodrug does not show obvious distribution in blood, liver, lung and kidney, but is largely enriched in the digestive tract, especially the small intestine, and has a long residence time. The targeted enrichment of Am@CS nanodrug in the intestinal tract is conducive to enhancing its precise protection of intestinal tissue and to reducing the toxic and side effects on other tissue organs to some extent.

[0053] After C57BL / 6 mice were orally administered with Am@CS nanodrug for 2 hours, they received 14Gy of whole abdominal X-ray irradiation. After 5 days of irradiation, histopathological analysis was performed on the intestinal tissue sections, and compared with the simple irradiation group and other different treatment groups. The test results are shown in Figure 5 .Figure 5 Representative images of H&E staining (hematoxylin-eosin staining) pathological tissue sections and crypt survival rate data graph of small intestinal tissue of mice after different treatments; wherein, A is a representative image of H&E staining pathological tissue sections (scale bar is 100 μm), and B is a crypt survival rate data graph. Compared with the normal group (Control, only intragastric administration of PBS) mice, the intestinal morphology of the irradiation group (IR+PBS), the carboxymethyl chitosan treatment group (IR+CS) and the free amifostine treatment group (IR+Amifostine) mice was clear, the number of crypts was normal, and the villus structure was complete. However, the crypt stem cells of the mice in the irradiation group, the carboxymethyl chitosan treatment group and the free amifostine treatment group died in large numbers, the crypt structure was obviously lost, and the villus structure was also destroyed. However, the mice orally administered with Am@CS nanomedicines (IR+Am@CS) can greatly maintain the integrity of the crypt-villus structure. It was found through observation and statistics of the intestinal crypt survival rate of mice in different treatment groups that the number of surviving crypts of the irradiated mice treated with Am@CS nanomedicines was significantly higher than that of the irradiation group, the carboxymethyl chitosan treatment group and the free amifostine treatment group, indicating that compared with free amifostine, Am@CS nanomedicines can significantly improve the bioavailability of amifostine in the gastrointestinal tract, significantly enhance its radiation protection effect, maintain the structure and function of the crypt-villus of the mouse intestine, and alleviate the radiation-induced intestinal injury.

[0054] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Those skilled in the art should understand that on the basis of the above description, other different forms of changes or variations can also be made. Here, it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.

Claims

1. A method for preparing a pH-responsive radiation-protective nanomedicine, characterized in that, Includes the following steps: Carboxymethyl chitosan was dissolved in a buffer solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the resulting carboxymethyl chitosan solution and activated under light-protected conditions. During activation, the pH of the solution was maintained at 6.7-7.

3. The resulting mixed solution was added to amifostine solution and reacted under light-protected conditions. The pH was adjusted to 7.8-8.2 to terminate the reaction. The solution was then dialyzed and dried to obtain the pH-responsive radiation protection nanomedicine.

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 the 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, characterized in that, 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, characterized in that, The reaction time is 24-48 hours.

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

10. The application of the pH-responsive radiation protection nanomedicine of claim 9 in the preparation of radiation protection agents for radioactive intestinal injury.

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