CN114712302A

CN114712302A - Exosome hydrogel and application thereof

Info

Publication number: CN114712302A
Application number: CN202210305501.5A
Authority: CN
Inventors: 张丹; 高建青; 林一峰; 蒋心驰; 李耀生; 刘娟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2022-07-08

Abstract

The invention discloses an exosome hydrogel and application thereof. The exosome hydrogel disclosed by the invention can be used for gelatinizing a lesion part in a uterine cavity after injection to inhibit adhesion, releasing exosomes for a long time, reducing loss of exosomes at the lesion part, improving a disease microenvironment, inhibiting fibrosis of endometrium and improving a treatment effect.

Description

Exosome hydrogel and application thereof

Technical Field

The invention belongs to the field of slow release and drug delivery of biological carriers, and particularly relates to an exosome hydrogel and application thereof.

Background

Intrauterine adhesion (IUA) is caused by repair disorder after damage of endometrial basal layer, and normal endometrial tissue is replaced by fibrous tissue, so that partial or total occlusion of uterine cavity or (and) cervical canal can be caused, clinically manifested by hypomenorrhea, amenorrhea, periodic abdominal pain, recurrent abortion, infertility and obstetrical complications such as placenta implantation, placenta adhesion and the like, and is one of public health problems seriously harming female reproductive health, also called Asherman Syndrome (Asherman Syndrome). Uterine cavity operation in gestation period such as induced abortion, incomplete abortion or abortive abortion and uterine clearing, uterine cavity infection (tuberculosis), uterine artery embolization, non-gestation period such as hysteromyoma excision, uterus septal incision and the like are common causes of uterine cavity adhesion. The treatment of the intrauterine adhesion is a difficult problem in the clinical practice at present, particularly the moderate and severe intrauterine adhesion, and the effect of the existing treatment means is not good enough. The method is a main treatment method of the existing IUA, but has poor treatment effect on moderate and severe uterine cavity adhesion patients, and the postoperative recurrence rate of the IUA can be up to 62.5%. The biggest influence of intrauterine adhesion on women with childbearing age is fertility, so that for women who urgently want fertility function, the scarring of uterine cavity and the loss of normal endometrium seriously interfere the fertility function, which not only brings dissonance to families, but also causes serious psychological and economic burden to patients, and therefore, the search for a new method for effectively treating intrauterine adhesion becomes a difficult problem to be solved clinically.

Exosome transplantation shows good curative effects in various wound repair diseases. Research finds that the exosome can regulate disease microenvironment at a focus part and promote injured tissues to repair and regenerate, and the exosome has huge potential in treatment of traumatic diseases. However, the exosome is directly and locally applied to the adhesion part of the uterine cavity, the exosome is seriously lost, the action time on the focus is short, and the treatment effect is still not ideal. Therefore, the exosome is loaded into the gel, so that the exosome can act on the focus position for a long time, the loss of the exosome in delivery is reduced, and the curative effect of the exosome is improved. In recent years, poloxamer gel has been widely applied in clinic, the poloxamer gel has good biocompatibility, and the formed gel has temperature-sensitive property and can be injected to form gel in situ at a focus position. In conclusion, the poloxamer temperature-sensitive gel delivery exosome has good application prospect and important scientific significance for treating intrauterine adhesion.

Disclosure of Invention

The invention aims to provide an exosome hydrogel and application thereof aiming at the defects of the prior art, and solves the problems of more loss and short action time in exosome local application in the prior art. The preparation method of the exosome hydrogel is simple to operate and convenient to control.

The purpose of the invention is realized by the following technical scheme: an exosome hydrogel prepared by the following steps:

(1) preparing poloxamer temperature-sensitive hydrogel: dissolving a proper amount of poloxamer 188 in a dispersion medium to enable the mass fraction of the poloxamer 188 in the solution to be 1-10%; and after the poloxamer 188 is completely dissolved, adding a proper amount of poloxamer 407, enabling the mass fraction of the poloxamer 407 in the solution to be 10-30%, standing overnight at 4 ℃, and completely dissolving the poloxamer 407 to obtain the poloxamer thermo-sensitive gel.

(2) Culturing the mesenchymal stem cells: cells were cultured in DMEM/F12 basal medium containing 10% fetal bovine serum, 1% penicillin. The normal oxygen culture conditions are as follows: the oxygen concentration is 21%, the carbon dioxide concentration is 5%, and the temperature is 37 ℃; the hypoxic culture conditions are as follows: the oxygen concentration is 1-5%, the carbon dioxide concentration is 5%, the temperature is 37 ℃, and both culture conditions can be used.

(3) Extracting and separating exosomes secreted by the mesenchymal stem cells: collecting the mesenchymal stem cell culture solution, centrifuging for 15-40min by 3000g of 1000-. The centrifuged culture solution is centrifuged for 60-90min by 80000-120000 g. Centrifugation was then repeated twice at 80000-120000g for 60-90 min. The exosome was resuspended in PBS and then stored in PBS buffer at-60 deg.C to 100 deg.C. (4) Preparation of exosome hydrogel: taking 1mL of the poloxamer hydrogel prepared in the step 1, adding 1000-2000 mu g of the exosome extracted in the step 2 into the poloxamer hydrogel at the temperature of between 0 and 4 ℃ in an ice-water bath, and uniformly mixing the system by using a 1mL pipette to obtain the exosome hydrogel.

Further, in the step (1), the mass fraction of poloxamer 188 in the solution is 5%, and the mass fraction of poloxamer 407 in the solution is 20%.

Further, in the step (1), the dispersion medium is normal saline or normal saline containing 1-5% by mass of ethylene glycol.

Further, in the step (2), the source of the mesenchymal stem cells is placenta, bone marrow, umbilical cord blood or adipose tissue.

Further, in the step (2), the culturing conditions of the mesenchymal stem cells include culturing under an ordinary oxygen culturing condition at an oxygen concentration of 21% and a carbon dioxide concentration of 5% at a temperature of 37 ℃ and under a hypoxic condition at an oxygen concentration of 1-5%, a carbon dioxide concentration of 5% and a temperature of 37 ℃.

An application of the exosome hydrogel in preparing a composite preparation for treating intrauterine adhesion.

Compared with the prior art, the invention has the beneficial effects that:

1. the exosome hydrogel can realize in-situ gelling at the lesion site of the uterine cavity to play a role in protecting, and simultaneously prolong the acting time of exosomes at the lesion site.

2. The exosome hydrogel disclosed by the invention is simple to synthesize, can be directly implanted into a lesion site in a uterine cavity through injection, and can be completely degraded without residues after a period of time, so that adverse effects on patients are reduced.

Drawings

FIG. 1 is a transmission electron micrograph of exosomes secreted by mesenchymal stem cells;

FIG. 2 is a bar graph of gelation temperature of poloxamer hydrogels before and after addition to exosomes;

FIG. 3 is a graph of the in vitro release profile of poloxamer hydrogels;

FIG. 4 is a histogram of the statistical thickness of the endometrium on the control side of the rat uterus and on the adhesion side of the uterine cavity;

FIG. 5 is a bar graph of the statistics of Ki67 scores on the control side of the rat uterus and on the adhesion side of the uterine cavity;

FIG. 6 shows embryo planting on the control side of the uterus and the adhesion side of the uterine cavity of a mouse;

FIG. 7 is a histogram of statistics of embryo implantation numbers on the control side and the intrauterine adhesion side of a mouse uterus;

figure 8 is a graph of poloxamer hydrogel prolonging the residence time of exosomes in the uterus of rats.

Detailed Description

Example 1: preparing an exosome temperature sensitive gel

The exosome temperature sensitive gel is prepared by the following steps:

1. preparation of poloxamer temperature-sensitive hydrogel

Dissolving a proper amount of poloxamer 188 in ice physiological saline at 0-4 ℃ to ensure that the mass fraction of the poloxamer 188 is 5%; and after the poloxamer 188 is completely dissolved, adding a proper amount of poloxamer 407 to enable the mass fraction of the poloxamer to be 20%, standing overnight at 4 ℃, and completely dissolving the poloxamer 407 to obtain the poloxamer thermo-sensitive gel.

2. Culturing mesenchymal stem cells

Cells were cultured aerobically in DMEM/F12 basal medium containing 10% fetal bovine serum, 1% penicillin. The normal oxygen culture conditions are as follows: the oxygen concentration was 21%, the carbon dioxide concentration was 5%, and the temperature was 37 ℃.3. Exosome secreted by extraction and separation mesenchymal stem cells

Collecting mesenchymal stem cell culture solution, centrifuging for 30min by 3000g, collecting supernatant, centrifuging for 30min by 10000g, collecting supernatant, and removing cell debris and organelle precipitate. The centrifuged broth was centrifuged at 100000g for 70min, then the particles were washed with PBS and centrifuged at 100000g for 70min, repeated twice. The exosomes were resuspended in PBS and then stored at-80 ℃ in PBS buffer. The particle size of the final exon was measured by Nanoparticle Tracking Analysis (NTA). The morphology of the exosomes was verified by transmission electron microscopy (TEM, JEM1400PLUS), 20 μ L of exosomes were taken, absorbed with carbon film, then incubated at room temperature for 5 minutes and negatively stained with 2% mass fraction phosphotungstic acid, and then the sample was air-dried by TEM to obtain images. To quantify the final exon particles suspended in PBS, protein concentration was measured using BCA protein assay kit (Applygen). Exon-associated markers were detected by western blot analysis.

4. Preparation of exosome hydrogel

And (2) taking 1mL of the poloxamer hydrogel prepared in the step (1), adding 1000 mu g of the exosome extracted in the step (2) into the poloxamer hydrogel at the temperature of 0-4 ℃ in an ice-water bath, and uniformly mixing the system by using a 1mL pipette to obtain the exosome hydrogel.

As shown in figure 2, the addition of exosomes did not affect the gelling temperature of poloxamer gel, and poloxamer hydrogel containing exosomes automatically transformed into gel state at around 35 ℃. As shown in figure 3, exosomes, after gelling with hydrogel at 37 ℃, can be slowly released within 72h at body fluid pH. By combining the results, the exosome can be loaded into poloxamer hydrogel, and temperature-sensitive gelling and slow release are realized.

Example 2: preparation of exosome temperature sensitive gel

The exosome temperature sensitive gel is prepared by the following steps:

1. preparation of poloxamer temperature-sensitive hydrogel

Dissolving a proper amount of poloxamer 188 in ice physiological saline at 0-4 ℃ to ensure that the mass fraction of the poloxamer 188 is 5%; and after the poloxamer 188 is completely dissolved, adding a proper amount of poloxamer 407 to make the mass fraction of the poloxamer be 20%, standing overnight at the temperature of below 4 ℃, and after the poloxamer 407 is completely dissolved, obtaining the poloxamer temperature-sensitive gel.

2. Culturing mesenchymal stem cells

Cells were cultured hypoxic in DMEM/F12 basal medium containing 10% fetal bovine serum, 1% penicillin. The hypoxic culture conditions are as follows: the oxygen concentration is 1-5%, the carbon dioxide concentration is 5%, and the temperature is 37 ℃.

3. Exosome secreted by extraction and separation mesenchymal stem cells

Collecting mesenchymal stem cell culture solution, centrifuging for 40min at 3000g, collecting supernatant, centrifuging for 40min at 12000g, collecting supernatant, and removing cell debris and organelle precipitate. The centrifuged culture solution was centrifuged at 120000g for 90 min. Then, the particles are added into a dextran exclusion chromatographic column after being resuspended, and the components with the retention time of 2-10min are collected. Finally, centrifugation was carried out at 120000g for 90min, and repeated twice. The exosomes were resuspended in PBS and then stored at-80 ℃ in PBS buffer. The particle size of the final exon was measured by Nanoparticle Tracking Analysis (NTA). The morphology of the exosomes was verified by transmission electron microscopy (TEM, JEM1400PLUS), 20 μ L of exosomes were taken, absorbed with carbon film, then incubated at room temperature for 5 minutes and negatively stained with 2% mass fraction phosphotungstic acid, and then the sample was air-dried by TEM to obtain images. To quantify the final exon particles suspended in PBS, protein concentration was measured using BCA protein assay kit (Applygen). Exon-associated markers were detected by western blot analysis.

4. Preparation of exosome hydrogel

And (3) taking 1mL of the poloxamer hydrogel prepared in the step (1), adding 1000 μ g of the exosomes extracted in the step (2) into the poloxamer hydrogel at the temperature of 0-4 ℃ in an ice-water bath, and uniformly mixing the system by using a 1mL pipette to obtain the exosome hydrogel.

The experimental results obtained were the same as in example 1, i.e. the addition of exosomes did not affect the gelling temperature of the poloxamer gel, and the poloxamer hydrogel containing exosomes automatically transformed into the gel state at around 35 ℃. The exosome can be slowly released within 72h at body fluid pH after gelling with hydrogel at 37 ℃. By combining the results, the exosome can be loaded into poloxamer hydrogel, and temperature-sensitive gelling and slow release are realized.

Example 3: preparing an exosome temperature sensitive gel

The exosome temperature sensitive gel is prepared by the following steps:

1. preparation of poloxamer temperature-sensitive hydrogel

2. Culturing mesenchymal stem cells

2. Exosome secreted by extraction and separation mesenchymal stem cells

Collecting mesenchymal stem cell culture solution, centrifuging for 15min by 1000g, collecting supernatant, centrifuging for 20min by 8000g, collecting supernatant, and discarding cell debris and organelle precipitate. The centrifuged culture solution was centrifuged at 80000g for 60 min. Finally, centrifugation was carried out at 80000g for 60min, which was repeated twice. The exosomes were resuspended in PBS and then stored at-80 ℃ in PBS buffer. The particle size of the final exons was measured by Nanoparticle Tracking Analysis (NTA) using zetaview (particle metric). Morphology of the exosomes was verified by transmission electron microscopy (TEM, JEM1400PLUS), 20 μ L of exosomes were taken, absorbed with a carbon film, then incubated at room temperature for 5 minutes, and negatively stained with 2% mass fraction of phosphotungstic acid, and then the sample was air-dried by TEM to obtain an image. To quantify the final exon particles suspended in PBS, protein concentration was measured using BCA protein assay kit (Applygen). Exon-associated markers were detected by western blot analysis.

3. Preparation of exosome hydrogel

Example 4: animal experiments

1. And (3) establishing a uterine cavity adhesion (IUA) model.

An ICR female mouse (the number of mice per group is represented by n) at the same estrus cycle, 8 weeks old, weighing about 25g, was selected, and a stable endometrial injury mouse model was established by ethanol intrauterine injection: mice were tested for their effect on local injury to the endometrium by local injection of 25 μ L95% ethanol into the unilateral uterine horn (contralateral injection of an equal amount of saline). Mouse model of endometrial injury was assessed by HE staining for endometrial thickness (average endometrial thickness area/circumference, endometrial area and circumference calculated using Image analysis software (Image Pro-Plus)), immunohistochemical detection of cell proliferation antigen (Ki67) expression for assessment of glandular epithelial proliferation, number of embryos implanted at early in vivo for assessment of intimal tolerance, and thus for stable establishment of mouse model of endometrial injury. As shown in fig. 4, HE staining statistics showed significant thinning of endometrial thickness on the lesion side (ethanol treatment) compared to the control side (saline treatment) in model mice (342.1 ± 30.2 μm vs.215.1 ± 32.4 μm; p <0.05, n ═ 6); FIG. 5 is a score statistic of immunohistochemical staining Ki67, and the result shows that the average immunoreactivity score of glandular epithelial cells Ki67 on the side of endometrial injury is significantly lower than that on the side of control (IRS: 11.7 + -0.2 vs.7.5 + -1.2; p <0.05, n ═ 5), indicating that the proliferation capacity of glandular epithelium is reduced; fig. 6 shows the number of embryos implanted on the damaged side of the endometrium and on the control side, and fig. 7 is a histogram showing the statistical number of embryos implanted on the damaged side of the endometrium and on the control side. The results showed a significant decrease in the number of embryos planted on the side of the endometrial lesions (7.7 + -0.8 vs.3.2 + -1.1; p <0.01, n ═ 6). The results show that a stable mouse model of the endometrial injury can be established by injecting ethanol into the uterine cavity, and the mouse does not have other obvious adverse reactions by observing the model, so that the treatment of the exosome hydrogel injected into the uterine cavity is further carried out on the basis.

2. In vitro exploration of the residence time of exosome hydrogels in endometrium

Optimization of the therapeutic effect requires that the retention time of the exosomes in the uterus be kept to a maximum. We labeled the exosomes extracted in step 2 using a commercial cell membrane fluorescent probe CM-DiD (ex/em: 644/663). As shown in fig. 8, the left mouse was used to eliminate interference of autofluorescence, and the right mouse was the treatment group. Equal amounts of CM-DiD labeled exosomes and CM-DiD labeled exosome hydrogels were injected into the left and right uteri, respectively, of treated mice. On day 3 post-injection, the residence time of exosomes in the mouse uterus was observed by small animal in vivo imaging. The results show that only a small amount of exosomes stay in the uterus of the left-side single exosome injection group, and a large amount of exosomes stay in the right-side exosome hydrogel group, which suggests that the poloxamer hydrogel can effectively prolong the stay of the exosomes in the uterus.

Claims

1. An exosome hydrogel, prepared by the steps of:

(2) Extracting and separating exosomes secreted by mesenchymal stem cells: collecting mesenchymal stem cell culture solution, centrifuging for 15-40min by 3000g of 1000-; centrifuging the centrifuged culture solution for 60-90min by 80000-120000 g; then centrifuging for 60-90min at 80000-120000g, and repeating twice; the exosome was resuspended in PBS and then stored in PBS buffer at-60 deg.C to 100 deg.C.

(3) Preparation of exosome hydrogel: taking 1mL of the poloxamer hydrogel prepared in the step 1, adding 1000-2000 mu g of the exosome extracted in the step 2 into the poloxamer hydrogel in an ice-water bath at the temperature of 0-4 ℃, and uniformly mixing to obtain the exosome hydrogel.

2. An exosome hydrogel according to claim 1, wherein in step (1), the mass fraction of poloxamer 188 in solution is 5% and the mass fraction of poloxamer 407 in solution is 20%.

3. The exosome hydrogel according to claim 1, wherein in the step (1), the dispersion medium is a physiological saline or a physiological saline containing 1-5% by mass of ethylene glycol.

4. The method of claim 1, wherein in the step (2), the source of the mesenchymal stem cells is placenta, bone marrow, umbilical cord blood or adipose tissue.

5. The preparation method according to claim 4, wherein in the step (2), the normoxic culture conditions of the mesenchymal stem cells are as follows: the oxygen concentration was 21%, the carbon dioxide concentration was 5%, and the temperature was 37 ℃.

6. The preparation method according to claim 4, wherein in the step (2), the hypoxic culture condition of the mesenchymal stem cells is: the oxygen concentration is 1-5%, the carbon dioxide concentration is 5%, and the temperature is 37 ℃.

7. Use of the exosome hydrogel according to claim 1 in the preparation of a composite formulation for the treatment of intrauterine adhesions.