WO2022142448A1

WO2022142448A1 - Preparation method for exosome-loaded polymer for use in oral colon-targeting drug delivery

Info

Publication number: WO2022142448A1
Application number: PCT/CN2021/116897
Authority: WO
Inventors: 邓超; 陈敬华; 胡祎炜; 康明珠; 李梦雨; 段亦都; 潘子豪; 张夏鼎
Original assignee: 江南大学
Priority date: 2020-12-29
Filing date: 2021-09-07
Publication date: 2022-07-07
Also published as: GB202307931D0; CN112618515B; CN112618515A; GB2616153A

Abstract

A preparation method for an exosome-loaded self-assembled polymer for use in oral colon-targeting drug delivery. The polymer is formed by compounding chitosan quaternary ammonium salt (HTCC), oxidized konjac glucomannan (OKGM), and a mesenchymal stem cell-derived exosome (MSCs-EXO) by means of layer-by-layer (LBL) wrapping and LBL encapsulation. The polymer has certain biocompatibility, stability, safety, anti-inflammatory, and sustained release performance, and can be used as a drug-carrying system to carry the exosome so as to repair an ulcerous part.

Description

A kind of preparation method of exosome-loaded oral colon-targeted drug delivery polymer

technical field

The invention relates to a preparation method of a polymer for oral colon-targeted drug delivery loaded with exosomes, and belongs to the field of biomedicine.

Background technique

Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease of the intestinal tract, which mainly involves the mucosa and submucosa of the sigmoid colon and rectum, and can also extend to the descending colon or even the entire colon. The main clinical manifestations are diarrhea, mucus, pus and blood in the stool and abdominal pain. Ulcerative colitis usually develops in late adolescence and early adulthood, with an average age of 17 to 40 years. Because the etiology of ulcerative colitis is still unclear, the disease is prone to repeated attacks, the course of the disease is long, the disease is prolonged, and it has a tendency to become cancerous and is accompanied by a variety of extraintestinal symptoms. Therefore, WHO has identified it as one of the modern incurable diseases. one.

At present, there is no standardized standard surgical procedure for the surgical treatment of ulcerative colitis. Drug treatment in traditional Chinese medicine often adopts the method of retention enema administration, and western medicine mainly chooses aminosalicylic acid, adrenocortical hormones, immunosuppressive drugs and other drugs for treatment. The existing traditional medicines for the treatment of ulcerative colitis often face the problems of short duration of efficacy and unsustainable anti-inflammatory effect, and are prone to a series of problems such as easy relapse of the disease, poor targeting, and easy to cause some adverse reactions.

Therefore, a highly targeted exosome-loaded oral colon-targeted polymer with high safety, stability and long-lasting anti-inflammatory properties was developed, which has a good effect on the treatment of ulcerative colitis. market prospects.

SUMMARY OF THE INVENTION

In order to achieve the above object, the present invention provides a method for preparing a polymer for oral colon targeted drug delivery loaded with exosomes. The quaternary ammonium salt of chitosan and oxidized konjac glucomannan are used as shells. The exosome-loaded oral colon-targeted drug delivery method was coated to obtain a safe, stable, durable anti-inflammatory and highly targeted drug for the treatment of ulcerative colitis. Layer-by-layer self-assembled polymer system for oral colon-targeted drug delivery loaded with exosomes.

The present invention first provides a preparation method of an exosome-loaded polymer for oral colon-targeted administration, the method steps comprising:

(1) Collect the mesenchymal stem cell culture supernatant, and extract exosomes by differential centrifugation;

(2) adding oxidant periodate in the aqueous solution of konjac glucomannan and stirring reaction, then adding ethylene glycol, through dialysis, solid-liquid separation to get supernatant, and freeze-drying to prepare oxidized konjac glucomannan;

(3) dissolving oxidized konjac glucomannan and chitosan quaternary ammonium salt prepared in step (2) in PBS buffer to obtain oxidized konjac glucomannan solution and chitosan quaternary ammonium salt solution;

(4) The chitosan quaternary ammonium salt solution obtained in step (3) and the exosomes obtained in step (1) were constantly rotated for 20-30 minutes, washed with PBS, centrifuged, and the precipitate was collected to obtain (MSCs-EXO)- HTCC polymers;

(5) The (MSCs-EXO)-HTCC polymer obtained in step (4) and the oxidized konjac glucomannan solution obtained in step (3) were constantly rotated for 20-30 minutes, washed with PBS, and centrifuged to obtain (MSCs- EXO)-(HTCC/OKGM) polymer;

Repeat steps (4) and (5) to prepare a layer-by-layer self-assembled polymer loaded with stem cells for oral colon targeted drug delivery, namely (MSCs-EXO)-(HTCC/OKGM) _n polymer, where n is Number of wrapping layers.

Further, when the mesenchymal stem cell supernatant collected in step (1) is the third-generation mesenchymal stem cell fusion to 70-80%, the serum-free medium is replaced, and the cell supernatant collected after culturing for 48 hours.

Further, the mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multi-directional differentiation capabilities.

Further, the specific operation of the differential centrifugation method described in step (1) is as follows: centrifuge the collected mesenchymal stem cell supernatant at 300-500g for 10-15min, and collect the supernatant; centrifuge at 1800-2000g for 10-15min, collect Supernatant; centrifuge at 10000-11000g for 60-70min, collect the supernatant; centrifuge at 100000-110000min for 60-70min, collect the precipitate; finally resuspend the exosome pellet in an appropriate amount of PBS, centrifuge at 100000-110000g for 60-70min, collect the precipitate, Obtain purified exosomes.

Further, the differential centrifugation in step (1) is all performed at 4°C.

Further, the preparation of the oxidized konjac glucomannan specifically includes: adding 5g KGM (oxidized konjac glucomannan, Konjac Glucomannan, KGM for short) powder in 500mL deionized water, stirring and dissolving, adding 10mL 0.5mol/L high Aqueous sodium iodate solution was stirred at 40°C in the dark for 4 hours; then 10 mL of ethylene glycol was added to the reaction mixture and stirred for 2 hours to neutralize the unreacted periodate; the solution was dialyzed with a dialysis membrane (MWCO: 12,000-14,000) for 3 hours until the dialysate does not contain iodate; the reaction product is centrifuged at 2500r/min for 20min, the supernatant is taken, vacuum freeze-dried to obtain OKGM (oxidized konjac glucomannan), and the dried sample is stored in a desiccator for the next step.

Further, the chitosan quaternary ammonium salt (N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride, HTCC) and oxidized konjac glucomannan (OKGM) used in the step (3) were in PBS. The final concentrations of dissolution were all 0.01-1.0 mg/mL.

Further, in the steps (4) and (5), the PBS is a phosphate buffered saline solution, the pH is 7.2-7.4, and the washing is performed 2-3 times.

Further, the mass-volume ratio of the exosomes to the chitosan quaternary ammonium salt solution is 200-500 μg: 1-3 mL; wherein, the exosomes are dissolved in PBS.

Further, the volume ratio of the oxidized konjac glucomannan solution to the chitosan quaternary ammonium salt solution is 1-3:1-3.

Further, steps (4) and (5) are preferably repeated 1 to 3 times, most preferably twice, to prepare (MSCs-EXO)-(HTCC/OKGM) ₂ polymer.

The present invention provides the layer-by-layer self-assembled polymer prepared by the above preparation method and loaded with stem cells for oral colon-targeted drug delivery.

The present invention provides a medicine or food comprising the layer-by-layer self-assembled polymer loaded with stem cells for oral colon-targeted drug delivery.

The present invention provides the application of the layer-by-layer self-assembled polymer loaded with stem cells for oral colon-targeted drug delivery in the preparation of a medicament for treating ulcerative colitis.

The beneficial effects obtained by the present invention:

(1) The present invention adopts (MSCs-EXO)-(HTCC/OKGM) _n self-assembled polymer prepared by layer-by-layer self-assembly (LBL) technology, which has good biocompatibility , stability, safety, anti-inflammatory and sustained release properties.

(2) In the present invention, HTCC and OKGM can release MSCs-EXO in a slow-controlled manner, and utilize the homing properties of MSCs-EXO, which can effectively promote the regeneration and repair of ulcer site tissue.

Detailed ways

The present invention will be further described in detail below through specific examples and comparative examples, but it should be understood that the present invention is not limited by these contents.

Stability test: Uncoated exosomes and LBL-coated exosomes were placed in simulated bile solution and simulated gastric juice, and were water bathed at 37 °C for 2 h. After 2 h, exosomes were collected by centrifugation, washed twice, and tested for structural integrity, mainly whether the membrane structure was complete.

Mucoadhesion assay: Freshly isolated porcine small intestine was washed and sectioned. LBL-coated or uncoated exosomes were labeled with specific fluorescence, pre-fixed to the lining of the small intestine, then incubated at 37°C for 1 h and visualized and analyzed using IVIS imaging.

Example 1

(1) Extraction of mesenchymal stem cell-derived exosomes: When the third-generation mesenchymal stem cells are confluent to 70-80%, the serum-free medium is replaced, cultured for 48 hours, and the cell supernatant is collected. Use differential centrifugation to extract exosomes. The specific operations are as follows: at 4 °C, centrifuge the collected mesenchymal stem cell supernatant at 300 g for 10 min to collect the supernatant; centrifuge at 2000 g for 10 min to collect the supernatant; The supernatant was centrifuged at 100,000g for 70min, and the precipitate was collected; finally, the exosome precipitate was resuspended in an appropriate amount of PBS, centrifuged at 100,000g for 70min, and the precipitate was collected to obtain purified exosomes.

(2) Synthesis of OKGM: Add 5 g of KGM powder to 500 mL of deionized water, stir to dissolve, add 10 mL of 0.5 mol/L sodium periodate aqueous solution dropwise, and stir at 40 °C for 4 h in the dark. Then 10 mL of ethylene glycol was added to the reaction mixture and stirred for 2 h to neutralize unreacted periodate. The solution was dialyzed against a dialysis membrane (MWCO: 12,000-14,000) for 3 days until the dialysate was free of iodate. The reaction product was centrifuged at 2500 r/min for 20 min, the supernatant was taken, vacuum freeze-dried to obtain OKGM, and the dried samples were stored in a desiccator for use in the next step.

(3) Synthesis of (MSCs-EXO)-(HTCC/OKGM) _n self-assembled polymers: Chitosan quaternary ammonium salt (HTCC) and oxidized konjac glucomannan (OKGM) were dissolved in PBS, and the final concentrations were all is 0.1 mg/mL. The cationic polymer chitosan quaternary ammonium salt and exosomes were mixed with constant slow rotation at room temperature for 30 min, and washed 2-3 times to obtain (MSCs-EXO)-HTCC polymer. Then, the anionic polymer oxidized konjac glucomannan was mixed with MSCS-HTCC under constant slow rotation at room temperature for 30 min to obtain (MSCs-EXO)-(HTCC/OKGM) self-assembled polymer.

Repeat the above step (3) 1-2 times to obtain (MSCs-EXO)-(HTCC/OKGM) ₂ self-assembled polymer and (MSCs-EXO)-(HTCC/OKGM) ₃ self-assembled polymer, respectively.

(MSCs-EXO)-(HTCC/OKGM), (MSCs-EXO)-(HTCC/OKGM) ₂ and (MSCs-EXO)-(HTCC/OKGM) ₃ prepared above were tested for stability, mucoadhesion, respectively. Performance testing.

Stability test: The membrane structure of (MSCs-EXO)-(HTCC/OKGM) was destroyed in simulated gastric fluid for two hours, which was not enough to protect exosomes completely from bile salts or gastric acid. However, (MSCs-EXO)-(HTCC/OKGM) ₂ exposed to simulated gastric juice at 37°C or a polymer that simulates bile salt solution can prevent the erosion of acid and bile salts for 2 h, which shows that it has good stability. The stability of (MSCs-EXO)-(HTCC/OKGM) ₃ was better.

The experimental results of the mucoadhesion assay showed that for (MSCs-EXO)-(HTCC/OKGM) ₂ , the level of LBL-encapsulated exosomes was nearly three times higher than that of naked exosomes after 1 h, and 2 h. The level of LBL-encapsulated exosomes was significantly higher than that of bare exosomes within 6 h, and the difference gradually decreased after 12 h due to the saturation of exosomes. It can be seen that the LBL-encapsulated (MSCs-E XO)-(HTCC/OKGM) ₂ had stronger mucoadhesion ability and stayed longer in the intestinal tract. However, the time required for the release of (MSCs-EXO)-(HTCC/OKGM) ₃ -encapsulated exosomes was prolonged, and its release was delayed by more than 4 h.

Example 2

(1) Extraction of mesenchymal stem cell-derived exosomes: When the third-generation mesenchymal stem cells are confluent to 70-80%, the serum-free medium is replaced, cultured for 48 hours, and the cell supernatant is collected. The exosomes were extracted by differential centrifugation. The specific operations were as follows: at 4°C, centrifuge the collected mesenchymal stem cell supernatant at 300 g for 15 min to collect the supernatant; centrifuge at 1800 g for 15 min to collect the supernatant; centrifuge at 11,000 g for 60 min to collect The supernatant was centrifuged at 110,000g for 60min to collect the precipitate; finally, the exosome precipitate was resuspended in an appropriate amount of PBS, centrifuged at 110,000g for 60min, and the precipitate was collected to obtain purified exosomes.

(3) Synthesis of (MSCs-EXO)-(HTCC/OKGM) _n self-assembled polymers: Chitosan quaternary ammonium salt (HTCC) and oxidized konjac glucomannan (OKGM) were dissolved in PBS, and the final concentrations were all is 0.5 mg/mL. The cationic polymer chitosan quaternary ammonium salt and exosomes were mixed with constant slow rotation at room temperature for 30 min and washed three times to obtain (MSCs-EXO)-HTCC polymer. Then, the anionic polymer oxidized konjac glucomannan was mixed with MSCS-HTCC under constant slow rotation at room temperature for 30 min to obtain (MSCs-EXO)-(HTCC/OKGM) self-assembled polymer.

Repeat the above step (3) once again to obtain (MSCs-EXO)-(HTCC/OKGM) ₂ self-assembled polymers.

After testing, it was found that the (MSCs-EXO)-(HTCC/OKGM) ₂ self-assembled polymer was exposed to simulated gastric juice at 37 °C or the polymer simulated bile salt solution could prevent the erosion of acid and bile salts for 2 h, with good performance. Stability; delayed exosome release.

Comparative Example 1

Comparative Example 1 is the exosomes directly extracted in step (1) of Example 1, without the subsequent encapsulation process.

The test results are as follows: when exposed to simulated gastric juice at 37 °C, the structure of ordinary, unencapsulated HTCC/OKGM exosomes is destroyed.

Comparative Example 2

The remaining steps are the same as in Example 1, without oxidizing konjac glucomannan, and directly using konjac glucomannan and chitosan quaternary ammonium salt for self-assembly according to the method in Example 1.

The study found that the combination of the two could not achieve the self-assembly process, because the aqueous solution of konjac glucomannan was viscous and had poor fluidity, and subsequent experiments could not be carried out.

Comparative Example 3

When the concentration of chitosan quaternary ammonium salt is less than 0.01mg/mL, the positive charge of chitosan quaternary ammonium salt is not enough to support the binding with exosomes; when the concentration of chitosan quaternary ammonium salt is greater than 1 mg/mL, the Glycan quaternary ammonium salts showed certain cytotoxicity to exosomes.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims

A preparation method of a polymer for oral colon-targeted drug delivery loaded with exosomes, characterized in that the method steps include:

(1) Collect the mesenchymal stem cell culture supernatant, and extract exosomes by differential centrifugation;

(2) adding oxidant periodate in the aqueous solution of konjac glucomannan and stirring reaction, then adding ethylene glycol, through dialysis, solid-liquid separation to get supernatant, and freeze-drying to prepare oxidized konjac glucomannan;

(3) dissolving oxidized konjac glucomannan and chitosan quaternary ammonium salt prepared in step (2) in PBS buffer to obtain oxidized konjac glucomannan solution and chitosan quaternary ammonium salt solution;

(4) The chitosan quaternary ammonium salt solution obtained in step (3) and the exosomes obtained in step (1) were constantly rotated for 20-30 minutes, washed with PBS, centrifuged, and the precipitate was collected to obtain (MSCs-EXO)- HTCC polymers;

(5) The (MSCs-EXO)-HTCC polymer obtained in step (4) and the oxidized konjac glucomannan solution obtained in step (3) were constantly rotated for 20-30 minutes, washed with PBS, and centrifuged to obtain (MSCs- EXO)-(HTCC/OKGM) polymer;

Repeat steps (4) and (5) to prepare a layer-by-layer self-assembled polymer loaded with stem cells for oral colon targeted drug delivery, namely (MSCs-EXO)-(HTCC/OKGM) n polymer, where n is Number of wrapping layers.
The preparation method according to claim 1, wherein the preparation of the oxidized konjac glucomannan specifically comprises: adding 5g KGM powder to 500mL deionized water, stirring and dissolving, adding dropwise 10mL 0.5mol/L periodic acid Aqueous sodium solution, stirred at 40°C in the dark for 4 hours; then, 10 mL of ethylene glycol was added to the reaction mixture and stirred for 2 hours to neutralize the unreacted periodate; the solution was dialyzed with a dialysis membrane (MWCO: 12,000-14,000) for 3 days , until the dialysate does not contain iodate; the reaction product was centrifuged at 2500 r/min for 20 min, the supernatant was taken, vacuum freeze-dried to obtain OKGM, and the dried sample was stored in a desiccator for use in the next step.
The preparation method according to claim 1, characterized in that, in the step (3), the final concentrations of chitosan quaternary ammonium salt and oxidized konjac glucomannan dissolved in PBS are both 0.01-1.0 mg/mL.
The preparation method according to claim 1, wherein in the steps (4) and (5), the PBS is a phosphate buffered saline solution, the pH is 7.2-7.4, and the washing is performed 2 to 3 times.
The preparation method according to claim 1, wherein the mass-volume ratio of the exosomes to the chitosan quaternary ammonium salt solution is 200-500 μg: 1-3 mL.
The preparation method according to any one of claims 1 to 5, wherein the volume ratio of the oxidized konjac glucomannan solution to the chitosan quaternary ammonium salt solution is 1-3:1-3.
The preparation method according to claim 1, wherein steps (4) and (5) are repeated 1 to 3 times.
The exosome-loaded oral colon-targeted administration polymer prepared by the preparation method according to any one of claims 1 to 7.
A medicament comprising the exosome-loaded oral colon-targeted polymer of claim 8.
The application of the exosome-loaded oral colon-targeted drug delivery polymer according to claim 8 in the preparation of a medicament for the treatment of ulcerative colitis.