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

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

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a preparation method for exosome-loaded polymer for use in oral colon-targeting drug delivery, and belongs to the field of biomedicine. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic and nonspecific intestinal inflammatory disease. UC lesions mainly involve the mucosa and submucosa of the sigmoid colon and rectum, and may also extend to the descending colon or even the entire colon. The clinical manifestations of UC mainly include diarrhea, mucopurulent feces, abdominal pain and other symptoms. UC generally occurs in late adolescence and early adulthood, with an average age of onset of 17 to 40 years. UC still has unclear etiology, and is prone to recurrent attacks. Moreover, the UC has a long course of disease, is protracted and unhealed, shows a carcinogenic tendency, and is generally accompanied by a variety of extraintestinal symptoms. Therefore, the World Health Organization (WHO) has identified UC as one of the modem incurable diseases.

[0003] At present, there is no standardized operation for the surgical treatment of UC. Drug treatment in traditional Chinese medicine generally takes the form of retention enema administration. In Western medicine, the treatment is mainly conducted with drugs such as aminosalicylic acids, corticosteroids, and immunosuppressants. Existing traditional drugs for treating UC generally face short duration of drug effect and non-sustainable anti-inflammatory effects. These traditional drugs are also prone to disease recurrence and poor targeting, which are easy to cause some adverse reactions.

[0004] Therefore, there is an urgent need to develop an exosome (EXO)-loaded polymer for col on-targeted oral administration, which has high safety, desirable stability, durable antiinflammatory properties, and strong targeting. This polymer may show a broad market prospect for the treatment of UC. SUMMARY

[0005] To achieve the above purpose, the present disclosure provides a preparation method of exosome (EXO)-loaded polymer for colon-targeted oral administration. In the present disclosure, N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride (HTCC) and oxidized konjac glucomannan (OKGM) are used as an outer shell, and an EXO-loaded polymer for colon-targeted oral administration is wrapped through layer-by-layer (LBL) self-assembly. In this way, an LBL-self-assembled and EXO-loaded polymer system for colon-targeted oral administration may be obtained, which has high safety, desirable stability, durable anti-inflammatory properties, and strong targeting. The polymer systems are useful in the treatment of UC.

[0006] The present disclosure provides a preparation method of an EXO-loaded polymer for colon-targeted oral administration, including the following steps:

[0007] (1) collecting a culture supernatant of mesenchymal stem cells (MSCs), and extracting an EXO by differential centrifugation;

[0008] (2) adding periodate as an oxidant into an aqueous solution of konjac glucomannan (KGM), stirring to conduct a reaction, adding ethylene glycol, collecting a supernatant after conducting dialysis and solid-liquid separation, followed by freeze-drying to obtain oxidized KGM (OKGM);

[0009] (3) dissolving the OKGM obtained in step (2) and N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride (HTCC) in a phosphate-buffered saline (PBS) separately to obtain an OKGM solution and an HTCC solution;

[0010] (4) subjecting the HTCC solution obtained in step (3) and the EXO obtained in step (1) to constant rotation for 20 min to 30 min, washing with the PBS, conducting centrifugation, and collecting a precipitate to obtain an (MSCs-EXO)-HTCC polymer; and

[0011] (5) subjecting the (MSCs-EXO)-HTCC polymer obtained in step (4) and the OKGM solution obtained in step (3) to constant rotation for 20 min to 30 min, washing with the PBS, and conducting centrifugation to obtain an (MSCs-EXO)-(HTCC / OKGM) polymer; where

[0012] steps (4) and (5) are repeated to prepare a layer-by-layer self-assembled polymer loaded with the MSCs for colon-targeted oral administration, namely an (MSCs-EXO)-(HTCC / OKGM)n polymer, and n is a number of wrapping layers.

[0013] In one embodiment, in step (1), the supernatant of the MSCs is a cell supernatant collected when third-generation MSCs are fused to 70% to 80%, and then cultured for 48 h with a serum-free medium.

[0014] In one embodiment, the MSC is a kind of pluripotent stem cell with self-renewal and multi-lineage differentiation abilities.

[0015] In one embodiment, in step (1), the differential centrifugation specifically includes: conducting centrifugation on the supernatant of the MSCs at 300 g to 500 g for 10 min to 15 min, and collecting a supernatant I; conducting centrifugation on the supernatant I at 1800 g to 2000 g for 10 min to 15 min, and collecting a supernatant II; conducting centrifugation on the supernatant II at 10000 g to 11000 g for 60 min to 70 min, and collecting a supernatant III; conducting centrifugation on the supernatant III at 100000 g to 110000 g for 60 min to 70 min, and collecting an EXO precipitate; and resuspending the EXO precipitate in an appropriate amount of PBS, conducting centrifugation at 100000 g to 110000 g for 60 min to 70 min, and collecting an obtained precipitate to obtain a purified EXO.

[0016] In one embodiment, in step (1), the differential centrifugation is conducted at 4°C.

[0017] In one embodiment, a preparation method of the OKGM specifically includes: dissolving 5 g of a KGM powder into 500 mL of deionized water by stirring, adding 10 mL of a 0.5 mol / L sodium periodate aqueous solution dropwise, and stirring in the dark at 40°C for 4 h; adding 10 mL of the ethylene glycol to a resulting reaction mixture and stirring for 2 h to neutralize unreacted periodate; conducting dialysis on a resulting solution using a dialysis membrane with a molecular weight cut-off (MWCO) of 12000 to 14000 for 3 d until an obtained dialysate does not have an iodate; subjecting a resulting reaction product to centrifugation at 2,500 r / min for 20 min, collecting a supernatant, and conducting vacuum freeze-drying to obtain the OKGM; and storing the obtained OKGM in a desiccator for later use.

[0018] In one embodiment, in step (3), the HTCC and the OKGM each are dissolved in the PBS at a final concentration of 0.01 mg / mL to 1.0 mg / mL.

[0019] In one embodiment, in steps (4) and (5), the washing with the PBS is conducted 2 to 3 times at a pH value of 7.2 to 7.4.

[0020] In one embodiment, the EXO and the HTCC solution are at a mass-to-volume ratio of (200-500) pg: (1-3) mL; and the EXO is dissolved in the PBS.

[0021] In one embodiment, the OKGM solution and the HTCC solution are at a volume ratio of (1-3):(1-3).

[0022] In one embodiment, steps (4) and (5) are repeated preferably 1 to 3 times, most preferably 2 times, to prepare an (MSCs-EXO)-(HTCC / OKGM)2 polymer.

[0023] The present disclosure further provides an LBL-self-assembled and MSCs-loaded polymer for colon-targeted oral administration prepared by the preparation method.

[0024] The present disclosure further provides a drug or a food including the LBL-self-assembled and MSCs-loaded polymer for colon-targeted oral administration.

[0025] The present disclosure further provides use of the LBL-self-assembled and MSCs-loaded polymer for colon-targeted oral administration in preparation of a drug for treating UC.

[0026] The present disclosure has the following beneficial effects:

[0027] (1) In the present disclosure, the self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)n prepared by LBL self-assembly has better biocompatibility, stability, safety, anti-inflammation properties, and sustained-release performances.

[0028] (2) In the present disclosure, the HTCC and the OKGM may release MSCs-EXO in a slow-controlled manner. Through homing properties of the MSCs-EXO, the regeneration and repair of ulcer tissues may be effectively promoted. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The present disclosure is further described in detail through the following examples, but it should be understood that the present disclosure is not limited by the following contents.

[0030] Stability test: uncoated EXO and LBL-coated EXO are placed in a simulated bile solution and a simulated gastric juice, and then treated in a water bath at 37°C for 2 h. After 2 h, the EXOs are collected by centrifugation, washed twice and then tested for structural integrity. It is mainly observed whether a membrane structure of the EXO is complete.

[0031] Mucoadhesive ability assay: freshly isolated porcine small intestines are washed and sectioned. The LBL-coated or uncoated EXOs are labeled with specific fluorescence, pre-fixated on an inner wall of the small intestine, incubated at 37°C for I h, and then analyzed using IVIS imaging.

[0032] Example 1

[0033] (1) Extraction of MSCs-derived EXO: when third-generation MSCs were fused to 70% to 80%, the MSCs were cultured in a serum-free medium for 48 h, and a cell supernatant was collected. The EXO was extracted by differential centrifugation, specifically: at 4°C, centrifugation was conducted on the supernatant of the MSCs at 300 g for 10 min, and a supernatant I was collected; centrifugation was conducted on the supernatant I at 2000 g for 10 min, and a supernatant II was collected; centrifugation was conducted on the supernatant II at 10000 g for 70 min, and a supernatant III was collected; centrifugation was conducted on the supernatant III at 100000 g for 70 min, and an EXO precipitate was collected; and the EXO precipitate was resuspended in an appropriate amount of PBS, centrifugation was conducted at 100000 g for 70 min, and an obtained precipitate was collected to obtain a purified EXO.

[0034] (2) Synthesis of OKGM: 5 g of a KGM powder was dissolved into 500 mL of deionized water by stirring, 10 mL of a 0.5 mol / L sodium periodate aqueous solution was added dropwise, and stirred in the dark at 40°C for 4 h. 10 mL of the ethylene glycol was added to a resulting reaction mixture and stirred for 2 h to neutralize unreacted periodate. Dialysis was conducted on a resulting solution using a dialysis membrane with a molecular weight cut-off (MWCO) of 12000 to 14000 for 3 d until an obtained dialysate did not have an iodate. A resulting reaction product was subjected to centrifugation at 2,500 r / min for 20 min, a supernatant was collected, and vacuum freeze-drying was conducted to obtain the OKGM; and dried OKGM was stored in a desiccator for later use.

[0035] (3) Synthesis of a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)n: the HTCC and the OKGM were dissolved in PBS at a final concentration of 0.1 mg / mL. A cationic polymer HTCC and the EXO were mixed at a constant slow speed and a room temperature for 30 min, and washed 2 to 3 times to obtain an (MSCs-EXO)-HTCC polymer. An anionic polymer OK GM and the MSCs-HTCC were mixed at a constant slow speed and a room temperature for 30 min to obtain a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM).

[0036] Step (3) was repeated 1 to 2 times to obtain a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)2 and a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)3, respectively.

[0037] The (MSCs-EXO)-(HTCC / OKGM), (MSCs-EXO)-(HTCC / OKGM)2, and (MSCs-EXO)-(HTCC / OKGM)3 were tested for stability and mucoadhesive properties, respectively.

[0038] Stability test: the membrane structure of (MSCs-EXO)-(HTCC / OKGM) is destroyed in simulated gastric juice after two hours, which is not enough to protect EXOs from bile salts or gastric acid. However, the (MSCs-EXO)-(HTCC / OKGM)2 can still prevent acid and bile salt erosion for 2 h when being exposed to simulated gastric juice or simulated bile salt solution at 37°C. It is seen that the polymer had desirable stability. The (MSCs-EXO)-(HTCC / OKGM)3 have better stability.

[0039] The experimental results of mucoadhesive ability assay shows that for the (MSCs-EXO)-(HTCC / OKGM)2: after 1 h, the level of EXO wrapped in LBL can be detected is nearly three times higher than that of bare EXO. After 2 h, the EXO level can still be more than two times higher. Within 6 h, the level of EXO wrapped in LBL is significantly higher than that of the bare EXO. After 12 h, the difference gradually decreases due to the saturation of EXO. It is seen that LBL-encapsulated (MSCs-EXO)-(HTCC / OKGM)2 have a stronger mucoadhesive ability and can stay in the intestinal tract for a longer time. However, the time required for the release of (MSCs-EXO)-(HTCC / OKGM)3-encapsulated EXOs is prolonged, with a release delay of more than 4 h.

[0040] Example 2

[0041] (1) Extraction of MSCs-derived EXO: when third-generation MSCs were fused to 70% to 80%, the MSCs were cultured in a serum-free medium for 48 h, and a cell supernatant was collected. The EXO was extracted by differential centrifugation, specifically: at 4°C, centrifugation was conducted on the supernatant of the MSCs at 300 g for 15 min, and a supernatant I was collected; centrifugation was conducted on the supernatant I at 1,800 g for 15 min, and a supernatant II was collected; centrifugation was conducted on the supernatant II at 11000 g for 60 min, and a supernatant III was collected; centrifugation was conducted on the supernatant III at 110000 g for 60 min, and an EXO precipitate was collected; and the EXO precipitate was resuspended in an appropriate amount of PBS, centrifugation was conducted at 110000 g for 60 min, and an obtained precipitate was collected to obtain a purified EXO.

[0042] (2) Synthesis of OKGM: 5 g of a KGM powder was dissolved into 500 mL of deionized water by stirring, 10 mL of a 0.5 mol / L sodium periodate aqueous solution was added dropwise, and stirred in the dark at 40°C for 4 h. 10 mL of the ethylene glycol was added to a resulting reaction mixture and stirred for 2 h to neutralize unreacted periodate. Dialysis was conducted on a resulting solution using a dialysis membrane with a molecular weight cut-off (MWCO) of 12000 to 14000 for 3 d until an obtained dialysate did not have an iodate. A resulting reaction product was subjected to centrifugation at 2,500 r / min for 20 min, a supernatant was collected, and vacuum freeze-drying was conducted to obtain the OKGM; and dried OKGM was stored in a desiccator for later use.

[0043] (3) Synthesis of a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)n: the HTCC and the OKGM were dissolved in PBS at a final concentration of 0.5mg / mL. A cationic polymer HTCC and the EXO were mixed at a constant slow speed and a room temperature for 30 min, and washed 3 times to obtain an (MSCs-EXO)-HTCC polymer. An anionic polymer OKGM and the MSCs-HTCC were mixed at a constant slow speed and a room temperature for 30 min to obtain a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM).

[0044] Step (3) was repeated 1 time to obtain a self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)2.

[0045] After detection, it is found that the self-assembled polymer (MSCs-EXO)-(HTCC / OKGM)2 can still prevent acid and bile salt erosion for 2 h when being exposed to simulated gastric juice or simulated bile salt solution at 37°C. It is seen that the polymer have desirable stability and delayed the release of EXOs.

[0046] Comparative Example 1

[0047] Comparative Example 1 was the EXO directly extracted from step (1) of Example 1 without subsequent encapsulation.

[0048] The test results are as follows: when being exposed to the simulated gastric juice at 37°C, the common, unwrapped HTCC / OKGM EXO have a damaged structure.

[0049] Comparative Example 2

[0050] In this comparative example, other steps were the same as those in Example 1, but the KGM was not oxidized. According to the method of Example 1, the KGM and the HTCC were directly used for self-assembly.

[0051] The studies find that the above two components can not be self-assembled, because the KGM aqueous solution has viscosity and poor fluidity, and subsequent experiments are failed.

[0052] Comparative Example 3

[0053] When HTCC had a concentration of less than 0.01 mg / mL, the HTCC was not sufficiently positively-charged to support the binding to the EXO. When the HTCC had a concentration of greater than 1 mg / mL, the HTCC showed certain cytotoxicity to the EXO.

[0054] Although the present disclosure has been disclosed as above in preferred examples, it is not intended to limit the present disclosure. Those skilled in the art may make various variations and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to that defined by the claims.

Claims

21 11 241. A preparation method of an exosome (EXO)-loaded polymer for colon-targeted oral administration, comprising the following steps:(1) collecting a culture supernatant of mesenchymal stem cells (MSCs), and extracting an EXO by differential centrifugation;(2) adding periodate as an oxidant into an aqueous solution of konjac glucomannan (KGM), stirring to conduct a reaction, adding ethylene glycol, conducting dialysis and solid-liquid separation and collecting a supernatant, and conducting freeze-drying to obtain oxidized KGM (OKGM);(3) dissolving the OKGM obtained in step (2) and N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride (HTCC) in a phosphate-buffered saline (PBS) separately to obtain an OKGM solution and an HTCC solution;(4) subjecting the HTCC solution obtained in step (3) and the EXO obtained in step (1) to constant rotation for 20 min to 30 min, washing with the PBS, conducting centrifugation, and collecting a precipitate to obtain an (MSCs-EXO)-HTCC polymer; and(5) subjecting the (MSCs-EXO)-HTCC polymer obtained in step (4) and the OKGM solution obtained in step (3) to constant rotation for 20 min to 30 min, washing with the PBS, and conducting centrifugation to obtain an (MSCs-EXO)-(HTCC / OKGM) polymer; whereinsteps (4) and (5) are repeated to prepare a layer-by-layer self-assembled polymer loaded with the MSCs for colon-targeted oral administration, namely an (MSCs-EXO)-(HTCC / OKGM)n polymer, and n is a number of wrapping layers.

2. The preparation method according to claim 1, wherein a preparation method of the OKGM specifically comprises: dissolving 5 g of a KGM powder into 500 mL of deionized water by stirring, adding 10 mL of a 0.5 mol / L sodium periodate aqueous solution dropwise, and stirring in the dark at 40°C for 4 h; adding 10 mL of the ethylene glycol to a resulting reaction mixture and stirring for 2 h to neutralize unreacted periodate; conducting dialysis on a resulting solution using a dialysis membrane with a molecular weight cut-off (MWCO) of 12000 to 14000 for 3 d until an obtained dialysate does not have an iodate; subjecting a resulting reaction product to centrifugation at 2,500 r / min for 20 min, collecting a supernatant, and conducting vacuum freeze-drying to obtain the OKGM; and storing the obtained OKGM in a desiccator for later use.

3. The preparation method according to claim 1, wherein in step (3), the HTCC and the OKGM each are dissolved in the PBS at a final concentration of 0.01 mg / mL to 1.0 mg / mL.21 11 244. The preparation method according to claim 1, wherein in steps (4) and (5), the washing with the PBS is conducted 2 to 3 times at a pH value of 7.2 to 7.4.

5. The preparation method according to claim 1, wherein the EXO and the HTCC solution are at a mass-to-volume ratio of (200-500) pg: (1-3) mL.

6. The preparation method according to any one of claims 1 to 5, wherein the OKGM solution and the HTCC solution are at a volume ratio of (1-3):(1-3).

7. The preparation method according to claim 1, wherein steps (4) and (5) are repeated 1 to 3 times.

8. An EXO-loaded polymer for colon-targeted oral administration, having a layer-by-layer self-assembled structure of (MSCs-EXO)-(HTCC / OKGM)n, wherein MSCs represents mesenchymal stem cells, EXO represents exosome, HTCC represents N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride, OKGM represents oxidized konjac glucomannan, and n is a number of wrapping layers.