Oral microcapsules containing exosomes, their preparation and application in radiation-induced intestinal injury

Abstract

The present invention discloses oral microcapsules containing exosomes, a preparation method thereof, and an application thereof in radiation-induced intestinal injury, belonging to the field of treating radiation-induced intestinal injury. The present invention aims to solve the technical problem that gastric acid can corrode proteins, resulting in the inactivation of exosomes. The method of the present invention: dissolving sodium alginate in PBS to obtain a sodium alginate solution, adding exosomes thereto, and stirring until uniform to obtain a mixed solution; then transferring it to a capillary tube, placing it in an electric field, generating droplets under the action of the electric field, and dropping them into a CaCl2 solution below for curing to obtain the microcapsules; and its application as a preparation for treating radiation-induced intestinal injury drugs. The microcapsules of the present invention promote the repair of radiation-induced intestinal injury by protecting intestinal epithelial cells. It has been proven that the SA microcapsules encapsulating and delivering MSC-exos have excellent therapeutic effects on RIII.

Description

Technical Field

[0001] The present invention belongs to the field of treating radiation-induced intestinal injury, and specifically relates to an orally administered microcapsule containing exosomes and a preparation method thereof; and the use of an orally administered microcapsule containing exosomes for the preparation of a medicament for treating radiation-induced intestinal injury. Background Art

[0002] Ionizing radiation is widely used in various fields such as industry, military, and medical treatment, and the risk of accidental radiation exposure is gradually increasing. When ionizing radiation acts on the body, it electrolytically separates water to generate free radicals, causing oxidative damage to biological macromolecules such as DNA, RNA, and proteins, and causing damage to the body. At the same time, due to the rapid renewal of intestinal cells, they are highly sensitive to radiation. When the entire intestine receives ionizing radiation with a dose exceeding 5 Gy, radiation-induced intestinal injury (RIII) will occur. Currently, for RIII, clinical treatment is basically mainly symptomatic treatment such as antidiarrheal, anti-inflammatory, and nutritional support. For example, loperamide or diphenoxylate compound is used to treat diarrhea, anticonvulsants are given to reduce colic, and antibiotics are used to inhibit the overgrowth of intestinal bacteria, lacking effective treatment measures.

[0003] Exosomes (exo) are extracellular vesicles secreted by cells with a diameter of about 40 - 100 nm, which contain various biological macromolecules such as RNA, DNA, and proteins. It has been found that exosomes are widely present in body fluids, carry and transmit biological macromolecules, form intercellular communication, and thus affect the occurrence and progression of some diseases. Mesenchymal stem cells are a type of pluripotent stem cells originating from mesenchymal tissues. Exosomes derived from mesenchymal stem cells have been widely studied due to their characteristics of regulating immunity and promoting tissue regeneration and repair. Generally, the administration method of exosomes is mostly intravenous injection or intraperitoneal injection. The exosomes after intravenous injection are quickly transferred to the liver for metabolism, difficult to concentrate in the damaged intestinal site, and have poor targeting; the exosomes after intraperitoneal injection are dispersed in the abdominal cavity and cannot act on the damaged intestinal epithelial cells. Therefore, oral administration shows great advantages. Oral administration is convenient in form and is carried out under non-invasive conditions, facilitating self-administration by patients. However, due to oral administration passing through gastric acid, gastric acid will corrode proteins, resulting in the inactivation of exosomes. Therefore, there is an urgent need for an oral carrier for exosome delivery. Summary of the Invention

[0004] The present invention aims to solve the technical problem that gastric acid will corrode proteins, resulting in the inactivation of exosomes, and provides an orally administered microcapsule containing exosomes, a preparation method thereof, and its application in radiation-induced intestinal injury.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The object of the present invention is to provide a method for preparing oral microcapsules containing exosomes, comprising the following steps:

[0007] Step 1: Dissolve sodium alginate in PBS to obtain a sodium alginate solution, then add exosomes and stir until uniform to obtain a mixed solution;

[0008] Step 2: Transfer the mixed solution of Step 1 into a capillary tube, place it in an electric field, and under the action of the electric field, generate droplets, which are dropped into the lower CaCl2 solution for solidification to obtain the microcapsules.

[0009] According to the method described in claim 1, characterized in that, in Step 1, the protein quantification of the exosomes is 10 mg / ml, the mass concentration of the sodium alginate solution is 1%, and the exosomes are added according to a volume ratio of 2:1 of the exosomes to the sodium alginate solution.

[0010] Further limited, the inner diameter of the capillary tube is 60 mm - 100 mm.

[0011] Further limited, in Step 2, the flow rate of the mixed solution is 40 μl / min - 60 μl / min.

[0012] Further limited, in Step 2, the collection distance is 4 cm - 6 cm.

[0013] Further limited, in Step 2, the voltage is 8 kv - 10 kv.

[0014] Further limited, in Step 2, the mass concentration of the CaCl2 solution is 2% - 4%.

[0015] Prepare oral microcapsules containing exosomes by any of the above methods.

[0016] Further limited, the average diameter of the microcapsules is 80 μm - 140 μm.

[0017] Use of the microcapsules prepared by any of the above methods or the above microcapsules as a preparation for a drug for treating radiation-induced intestinal injury.

[0018] Further limited, when treating radiation-induced intestinal injury, dissolve the microcapsules in PBS.

[0019] In the present invention, the reaction of sodium alginate (SA) with calcium ions forms a colloidal product. The carboxyl groups in sodium alginate undergo a cross-linking reaction with calcium ions to form a three-dimensional network structure, thereby forming a colloidal gel. In an alkaline condition, the cross-linking between sodium alginate and calcium ions is disrupted, leading to gel depolymerization; while in an acidic condition, this cross-linking is enhanced and the gel becomes more stable. Using the gel formed by sodium alginate and calcium ions as an oral carrier for exosome delivery enables exosomes to be protected from gastric acid corrosion, and in alkaline intestinal fluid, the sodium alginate carrier dissolves and the exosomes are released, acting on damaged intestinal epithelial cells, thus achieving the effect of treating radiation-induced intestinal injury.

[0020] The present invention uses microfluidic technology to prepare sodium alginate microcapsules containing exosomes. First, the inner diameter of the needle tip of the gavage needle in mouse gavage is about 0.5 mm. If the capsule particle size exceeds this range, the capsule will rupture at the needle tip during gavage, and the particle size should not be too low either. According to previous exploration, since the quantitative administration of exosomes to each mouse is 1 mg, when the capsule diameter is less than 60 μm, the gavage liquid is too viscous and prone to clogging the needle. Therefore, the particle size of the capsules prepared in this experiment is controlled between 80 μm and 140 μm, which can not only enable the capsules to pass through the needle smoothly but also avoid needle clogging caused by too small capsule diameter.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] In the present invention, exo is encapsulated in SA for oral delivery of drugs for radiation-induced intestinal injury. Its SA shell has a stable structure under acidic conditions and dissolves under alkaline conditions. MSC-exo has a strong immunomodulatory effect on tissue injury repair. However, due to the action of a large amount of gastric acid and enzymes in the stomach, exo rarely reaches the intestinal site through different administration methods. By encapsulating with SA, exo can be protected from gastric acid corrosion and is concentrated and released in the small intestine. Also, since exo inhibits inflammation through immunomodulation and promotes tissue injury repair. The microcapsules of the present invention promote the repair of radiation-induced intestinal injury by protecting intestinal epithelial cells. The SA microcapsules encapsulating and delivering MSC-exos of the present invention have been proven to have excellent therapeutic effects on RIII. The oral SA microcapsules of the present invention can be widely applied in various fields.

[0023] In order to further understand the features and technical content of the present invention, please refer to the following detailed description of the present invention and the drawings. However, the attached drawings are only for reference and illustration purposes and are not used to limit the present invention. Brief Description of the Drawings

[0024] Figure 1 It is a schematic diagram of preparation by microfluidic technology;

[0025] Figure 2 It is an experimental schematic diagram of treating radiation-induced intestinal injury by orally administering vesicles containing exosomes;

[0026] Figure 3 It is the survival analysis of mice after 17 Gy abdominal irradiation;

[0027] Figure 4 It is the weight change of mice after 17 Gy abdominal irradiation;

[0028] Figure 5 It is the representative HE staining of small intestinal sections of mice 4 days after 14 Gy abdominal irradiation;

[0029] Figure 6 It is the number of crypts per millimeter of mice 4 days after 14 Gy abdominal irradiation;

[0030] Figure 7 It is the intestinal villus length of mice 4 days after 14 Gy abdominal irradiation. Detailed implementation manners

[0031] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0032] Example 1

[0033] Step 1: Weigh sodium alginate and dissolve it in PBS to prepare a 1% sodium alginate solution. Take 10 ml of exosomes with a protein quantification of 10 mg / ml and dissolve them in 5 ml of the sodium alginate solution, and stir evenly to obtain a mixed solution.

[0034] Step 2: Transfer the mixed solution of Step 1 to a capillary tube, place it in an electric field, and under the action of the electric field, generate droplets, which are dropped into the 3% CaCl2 solution below for solidification to obtain the microcapsules.

[0035] Among them, the flow rate of the sodium alginate and exosome mixed solution is 40 μl / min, the collection distance is 4 cm, the voltage is 8 kv, and the average diameter of the generated microcapsules is 80 μm - 140 μm.

[0036] Dissolve the microcapsules prepared in this example in 5 ml of PBS, and use the following tests to verify the invention effect.

[0037] I. Establishment and treatment of a radiation-induced intestinal injury mouse model

[0038] This experiment was divided into two dose rate groups of 17 Gy and 14 Gy. The 17 Gy group was used to observe the rescue effect of different administration groups on the survival of mice after high-dose irradiation, and the 14 Gy group was used to observe the therapeutic effect of different administration groups on the radiation-induced intestinal injury in mice. Each group was further divided into a con group, an IR group, an IR+exo group, an IR+SA group, and an IR+SA-exo group, with 5 mice in each group, for a total of 50 mice.

[0039] Each mouse in the non-con group was injected with 100 μl of 0.1% sodium pentobarbital for anesthesia. After anesthesia, the mouse was fixed on the irradiation plate and placed at a position 1.5 m away from the cobalt source. Other areas except the abdomen were blocked with a lead plate, and irradiation began.

[0040] 2 h after the irradiation ended, the con group and the IR group mice were each gavaged with 200 μl of PBS, the IR+exo group mice were each gavaged with 200 μl of exosomes (5 mg / ml), the IR+SA group mice were each administered 200 μl of SA solution, and the IR+SA-exo group mice were each given 200 μl of SA-exo (after 100 μl of 10 mg / ml exo was encapsulated with SA, PBS was added to adjust the volume to 200 μl).

[0041] Three administrations were given at 2 h, 24 h, and 48 h after the irradiation ended. The 17 Gy group was used to observe the survival of different administration groups; the 14 Gy group was used to observe the treatment of radiation-induced intestinal injury in mice with different administration groups, and the mice were sacrificed for sampling at 96 h.

[0042] 1. SA-exo improved the survival rate of irradiated mice and alleviated the weight loss

[0043] In this experiment, an abdominal irradiation method was used to establish a radiation-induced intestinal injury model to study the effect of exosomes encapsulated with sodium alginate on the survival of a radiation-induced intestinal injury mouse model after lethal-dose irradiation. The results showed that under abdominal irradiation at a dose of 17 Gy, the survival rate of the IR+SA-exo group was significantly higher than that of the IR group, the IR+exo group, and the IR+SA group. All the mice in the IR group and the IR+exo group died on the 10th day, while half of the mice in the IR+SA-exo group still survived. The weight change rate can reflect the vomiting and diarrhea conditions of mice with radiation-induced intestinal injury. It was observed that under lethal-dose abdominal irradiation, the weight of the mice in the IR+SA-exo group stopped decreasing on the 6th - 7th day, began to recover on the 8th day, and increased steadily thereafter. The weights of the mice in the IR group, the IR+EXO group, and the IR+SA group continued to decrease, and the number of surviving mice in the IR group, the IR+exo group, and the IR+SA-exo group was less than 3 on the 8th day, and the weight change rate could not be statistically analyzed ( Figure 3 and 4)。Compared with the IR group, SA-exo significantly improved the weight loss in mice after irradiation. In summary, these data indicate that intragastric administration of SA-exo after lethal-dose abdominal irradiation can improve the weight loss and death in mice.

[0044] 2. SA-exo can restore the integrity of the intestinal epithelium in irradiated mice

[0045] Studies have shown that radiation can cause the death of villous epithelial cells in mice and the loss of intestinal crypts, leading to the disruption of the intestinal mucosal barrier, bacterial invasion, secondary inflammation and sepsis, and ultimately resulting in the death of mice. In this experiment, the small intestine lesions of the IR group, IR+SA group, IR+exo group, and IR+SA-exo group were mainly evaluated 4 days after 14Gy abdominal irradiation to study the protective effect of intragastric administration of SA-exo on the small intestine of mice with radiation-induced intestinal injury. As Figure 5 shown are the HE staining of the small intestine of the CON group, IR group, IR+SA group, IR+exo group, and IR+SA-exo group on the 4th day after irradiation. Compared with the CON group, 4 days after abdominal irradiation, the small intestine villi of the IR group, IR+SA group, and IR+exo group were damaged, the villus-crypt height became shallower, and the villus structure was disordered, while the intestinal villi of the IR+SA-exo group were dense, and the villus-crypt height showed a recovery trend. After treatment with SA-exo, the number of intestinal crypts per millimeter in the IR+SA-exo group was significantly higher than that in the IR group, IR+exo group, and IR+SA group, and the difference between groups was statistically significant. The number of intestinal crypts per millimeter in the IR+SA-exo group was 81.3% of that in the CON group. The length of the long villi in the IR+SA-exo group was equivalent to the villus height in the CON group, and was significantly higher than that in the IR group, IR+exo group, and IR+SA group, and the difference between groups was statistically significant ( Figure 6 and Figure 7 ).

[0046] In the present invention, exo is encapsulated in SA for oral delivery of drugs for radiation-induced intestinal injury. Its SA shell is structurally stable under acidic conditions and dissolves under alkaline conditions. MSC-exo has a strong immunomodulatory effect on tissue injury repair. However, due to the action of a large amount of gastric acid and enzymes in the stomach, exo rarely reaches the intestinal site through different administration methods. Encapsulation with SA can protect exo from gastric acid corrosion and concentrate a large amount in the small intestine for release. Also, since exo inhibits inflammation through immunomodulation and promotes tissue injury repair. In this experiment, the SA-exo group promoted the repair of radiation-induced intestinal injury by protecting intestinal epithelial cells. Therefore, SA microcapsule-encapsulated delivery of MSC-exos has been proven to have excellent therapeutic effects on RIII. This oral SA microcapsule loaded with MSC-exos can be widely applied in various fields.

Claims

1. Use of a microcapsule in the preparation of a drug for treating radiation-induced intestinal injury, characterized in that, The average diameter of the microcapsules is 80 mm - 140 mm, and the preparation of the microcapsules comprises the following steps: Step 1: Dissolve sodium alginate in PBS to obtain a sodium alginate solution, then add exosomes and stir until uniform to obtain a mixed solution; Step 2: Transfer the mixed solution in Step 1 to a capillary tube, place it in an electric field, and under the action of the electric field, generate droplets, which are dropped into the CaCl2 solution below for solidification to obtain the microcapsules; Among them, in Step 1, the protein quantification of the exosomes is 10 mg / ml, the mass concentration of the sodium alginate solution is 1%, and the exosomes are added in a volume ratio of 2:1 of the exosomes to the sodium alginate solution.

2. The application according to claim 1, wherein In Step 2, the inner diameter of the capillary tube is 60 mm - 100 mm.

3. The application according to claim 1, wherein In Step 2, the flow rate of the mixed solution is 40 u / min - 60 u / min.

4. The application according to claim 1, characterized in that In Step 2, the collection distance is 4 cm - 6 cm.

5. The application according to claim 1, characterized in that, In Step 2, the voltage is 8 kv - 10 kv.

6. The application according to claim 1, wherein In Step 2, the mass concentration of the CaCl2 solution is 2% - 4%.

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