RADIOPROTECTIVE NANODRUG FOR SMALL INTESTINE AND PREPARATION METHOD THEREOF

Abstract

The present invention relates to a method for constructing a nanodrug with adhesion in small intestine, including activating a basic amino acid with a small molecular catalyst, adding a polysaccharide solution and reacting to obtain an amphiphilic polymer; then adding a drug solution and mixing uniformly, to obtain drug-loaded nanoparticles including a hydrophilic portion that is the basic amino acid and a hydrophobic portion that is the polysaccharide and drug, wherein the drug has radioprotective effect or can inhibit ionizing radiation-induced cell death; and adding the drug-loaded nanoparticles to a dopamine solution to obtain a nanodrug including the basic amino acid and polydopamine on the surface after the reaction. The present invention provides an oral nanodrug with adhesion in intestinal tract, and the nanodrug has good biocompatibility, adhesion in small intestine and mucus barrier penetration ability, and can withstand the acid and alkali environment in the gastrointestinal tract.

Claims

1. A method for preparing a nanodrug for small intestine, comprising the following steps: (1) activating a hydrophilic basic amino acid with a small-molecule catalyst in an acidic buffer solution, then adding a polysaccharide solution, mixing uniformly and reacting at pH 4.5-5.5 and 20-30° C. to obtain an amphiphilic high-molecular polymer; then adding a drug solution and mixing uniformly, to obtain drug-loaded nanoparticles comprising a hydrophilic portion that is the basic amino acid and a hydrophobic portion that is the polysaccharide and drug, wherein the drug has radiation protection effect or has the effect of inhibiting ionizing radiation-induced cell death, and the drug is positively charged in agastricacid environment; and (2) adding the drug-loaded nanoparticles obtained in Step (1) to a dopamine solution, and reacting at pH 8.0-10.0 and 25-50° C., to obtain a nanodrug having a surface modified with polydopamine after the reaction is complete.

2. The preparation method according to claim 1, wherein in Step (1), the small-molecular catalyst is N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

3. The preparation method according to claim 2, wherein in Step (1), the molar ratio of the basic amino acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:4:4.

4. The preparation method according to claim 1, wherein in Step (1), the basic amino acid is selected from arginine, lysine, histidine and any combination thereof.

5. The preparation method according to claim 1, wherein in Step (1), the polysaccharide is selected from the group consisting of chitosan, dextran, alginic acid, cellulose and any combination thereof; and the molar ratio of the carboxyl group of the basic amino acid to the amino group of the polysaccharide is 1:1.

6. The preparation method according to claim 1, wherein in Step (1), the drug is selected from the group consisting of thalidomide, cysteamine thiosulfate, amifostine, genistein, resveratrol, 3,3-diindolylmethane, Entolimod, and Ex-RAD and any combination thereof; the concentration of the drug solution is 1.0 mg/mL; and the weight ratio of the drug to the polysaccharide encapsulated by the amino acid is 1:100.

7. The preparation method according to claim 1, wherein in Step (2), the concentration of the dopamine solution is 2.0 mg/mL; and the weight ratio of the nanoparticles to dopamine is 1:4.

8. A nanodrug for small intestine prepared by the preparation method according to claim 1, comprising nanoparticles and polydopamine modified on the surface of the nanoparticles, wherein the nanoparticles comprise a hydrophobic polysaccharide, a hydrophilic basic amino acid and a hydrophobic drug, wherein the polysaccharide and the basic amino acid are covalently bonded, the drug is located inside the nanoparticles and is positively charged in agastricacid environment, and the nanodrug has a particle size of 100-500 nm.

9. Use of the nanodrug according to claim 8 in the production of a radioprotective agent for small intestine.

10. The use according to claim 9, wherein the radioprotective agent is an oral drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is an SEM image of the prepared radioprotective nanodrug;

[0037] FIG. 2 shows the result of the hydrodynamic particle size of the radioprotective nanodrug;

[0038] FIG. 3 shows the standard ultraviolet absorption curve of the loaded drug thalidomide and the test result of the drug loading rate of the nanodrug;

[0039] FIG. 4 is a schematic diagram of the radioprotective nanodrug;

[0040] FIG. 5 shows the test result of in-vitro radiation protection effect of the radioprotective nanodrug;

[0041] FIG. 6 shows the test result of adhesion in intestinal tract of the radioprotective nanodrug;

[0042] FIG. 7 is the mode of action of the radioprotective nanodrug delivered by oral administration; and

[0043] FIG. 8 shows the effect of the radioprotective nanodrug in attenuation of radiation-induced intestinal damage.

REFERENCE NUMERALS

[0044] 1—chitosan; 2—arginine; 3—thalidomide; 4—polydopamine; 5—radioprotective nanodrug; 6—small intestinal mucus barrier; 7—intestinal villi; 8—intestinal crypt.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present invention, instead of limiting the scope of the present invention.

Example 1: Synthesis of Nanodrug

[0046] Arginine (0.867 g, 4.977 mmol) was dissolved in morpholinoethanesulfonic acid (40 mL, 25 mM, pH 5.0). Then, N-hydroxy succinimide (2.291 g, 19.908 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.816 g, 19.908 mmol) were added in sequence for activation for 2 h. Subsequently, a solution of chitosan (1.0 g, 4.977 mmol) dissolved in morpholinoethanesulfonic acid was added to the mixture, and reacted for 24 h with continuous stirring at room temperature. Sodium hydroxide (0.1 M) was then added to terminate the reaction. Subsequently, thalidomide (1.0 mg/mL, 10 mL) dissolved in a mixed solution of water and acetonitrile (v/v=1/1) was slowly added dropwise to the polymer solution (10 mg/mL, 100 mL). The resulting solution was continuously stirred and nitrogen was introduced overnight. Acetonitrile was removed, and the supernatant was collected and lyophilized after centrifugation. The lyophilized sample (20.0 mg) was transferred to a dopamine solution (2 mg/mL, 40 mL, pH 8.5), stirred at room temperature for 3 h, washed with deionized water, and centrifuged. The supernatant was collected to obtain a nanodrug.

[0047] FIG. 1 is an SEM image of the prepared radioprotective nanodrug. The result shows that the nanodrug has a small particle size and good dispersion. The nano-drug has a nearly spherical structure with a relatively smooth surface. It indicates that polydopamine is evenly coated on the surface of the nanoparticles by forming a coating.

[0048] FIG. 2 shows the test result of the hydrodynamic diameter of the radioprotective nanodrug. The result shows that the nanodrug has a small hydrodynamic diameter of about 214 nm and a PDI of 0.584. The particle size is suitable for penetrating the small intestinal mucus barrier, and facilitates the nanodrug to exert a radiation protection effect in the small intestinal crypt site.

[0049] FIG. 3 shows the standard ultraviolet absorption curve of the loaded thalidomide and the test result of the drug loading rate of the radioprotective nanodrug. By substituting the UV absorbency (FIG. 3B) of the ultrasonicated nanodrug solution into the calculation formulation (FIG. 3A, y=0.2245x+0.051, R.sup.2=0.9975) from the standard curve, the drug loading rate of the nanodrug is calculated to be about 22.98%.

[0050] FIG. 4 is a schematic diagram showing the structure of the prepared radioprotective nanodrug, including chitosan 1, arginine 2, thalidomide 3, and polydopamine 4. Chitosan 1 forms a network structure having a surface to which arginine 2 is linked, thalidomide 3 is enveloped in the network structure, and polydopamine 4 is located on the surface of the nanodrug.

Example 2. In-Vitro Radiation Protection Effect Test

[0051] An appropriate amount of nanodrug (11.237 μg/mL) prepared in Example 1 were dispersed in a medium for culturing small intestinal crypt organoid, and C57BL/6J mouse small intestinal crypt organoids were cultured ex vivo, and irradiated with X ray at a dose of 14 Gy after 12 h. After radiation damage, the disintegrated small intestinal crypts (as shown in FIG. 5A) and the intact crypts with sharp edge (as shown in FIG. 5B) were calculated. As shown in FIG. 5C, the survival rate of the crypts treated with the nanodrug after irradiation is about 42.67%, which is significantly higher than that of the control group (*p<0.05). The result shows that the nanodrug has good radiation protection effect.

Example 3. Test of Adhesion in Intestinal Tract

[0052] The radioprotective nanodrug prepared in Example 1 was labeled with Cy5.5 fluorescent dye, and then resuspended in phosphate buffer solution. C57BL/6J mice were fasted for 12 h, and the dye-labeled nanodrug solution (4 mg/mL, 0.5 mL) was administered to mice in each group by intragastric administration. The mice were euthanized 6 h and 24 h after administration, and the small intestine tissues were taken for in-vitro fluorescence imaging. The instrument used was Kodak FX Pro in-vivo fluorescence imaging system. The wavelength of the excitation light was 630 nm and the wavelength of the emission light was 700 nm.

[0053] As shown in FIG. 6, 6 h after administration (FIG. 6A), the small intestine of mice shows a strong fluorescent signal, indicating that the drug has mostly been accumulated in the small intestine. 24 h after administration (FIG. 6B), the fluorescent signal in the small intestine tissue of the mice remains at a strong level, indicating that the nanodrug has good adhesion in small intestine.

[0054] FIG. 7 is a schematic diagram showing the mode of action of the radioprotective nanodrug delivered by oral administration. The radioprotective nanodrug 5 can withstand the acid and alkali environment in the gastrointestinal tract and thus can be avoided from decomposition and absorption into the blood under the action of gastric acid. Due to its adhesion ability in the intestinal fluid environment, the nanodrug can penetrate the small intestinal mucus barrier 6 to reach the intestinal villi 7, and further reach the small intestinal crypt 8.

Example 4. Test of Ability to Relieve Radiation-Induced Intestinal Damage

[0055] The radioprotective nanodrug (22.98 wt. %, containing 100 mg/kg thalidomide in 500 μL phosphate buffer) was administered to C57BL/6J mice (male, 8 weeks old) by intragastric administration 12 h before irradiation, and the simple irradiation group was given the same dose of phosphate buffer. The X-RAD 320i X-ray machine was used to irradiate the abdomen of the mice at a dose of 14 Gy and a dose rate of 1 Gy/min. 5 days after irradiation, the small intestine tissues of the mice were taken to make paraffin sections which were stained with hematoxylin-eosin. The main evaluation criterion of radiation-induced intestinal damage includes the number of pathologically detected crypts in the intestinal sample. The regeneration and repair of the intestinal tract after irradiation mainly rely on the stem cells in the crypt site, so the survival and intactness of the small intestinal crypts after irradiation can reflect the severity of radiation damage in the small intestine.

[0056] As shown in FIG. 8, the normal small intestinal crypt structure is shown in FIG. 8A, and the crypt structures almost disappear in the simple irradiation group 5 days after irradiation (as shown in FIG. 8B), indicating that ionizing radiation causes severe intestinal damage and the highly reduced crypts cannot exert the regeneration and repair function, which leads to the death of the mice. In the group treated with the radioprotective nanodrug (FIG. 8C), some small intestinal crypts still retain the original contour and regenerate 5 days after the irradiation (as shown in FIG. 8C, the arrow indicates the viable crypts), indicating that the small intestine tissue still has the repair and regeneration ability after irradiation, and thus the radioprotective nanodrug can greatly relieve radiation-induced intestinal injury.

[0057] While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. It should be appreciated that some improvements and variations can be made by those skilled in the art without departing from the technical principles of the present invention, which are also contemplated to be within the scope of the present invention.