Magnetic / ultrasonic response microcapsule for repairing endometrial injury and preparation method thereof

Abstract

The invention discloses a magnetic / ultrasonic response microcapsule for repairing endometrial injury and a preparation method of the magnetic / ultrasonic response microcapsule. The method is based on an electronic injection micro-fluidic chip, monodisperse liquid drops with uniform size are generated, the monodisperse liquid drops are dropped into collected liquid, and meanwhile, the microcapsule with a core-shell structure is formed through ultraviolet irradiation and crosslinking. According to the core-shell structure microcapsule prepared by the invention, accurate adjustment of the core-shell size and performance can be realized by changing preparation conditions (internal phase and external phase flow rates and the proportion of components in the external phase), the preparation method is simple and easy to implement, the requirements on reaction conditions are low, and the size of the microcapsule can be easily and accurately adjusted; the microcapsule has magnetic field and ultrasonic response, and can effectively repair damaged endometrium to prevent intrauterine adhesion.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for repairing endometrial damage, in particular to a magnetic / ultrasound responsive microcapsule for repairing endometrial damage and a preparation method thereof. Background Art

[0002] Intrauterine adhesion (IUA), the most common cause is damage to the basal layer and even the superficial muscle layer of the endometrium caused by intrauterine operation. IUA often leads to female infertility and recurrent miscarriage, seriously endangering the reproductive health of women of childbearing age. The main treatment method at present is hysteroscopic intrauterine adhesion separation, and postoperative hormonal drugs or physical barriers inserted into the uterine cavity are used to prevent adhesion recurrence. However, estrogen therapy may increase the risk of thrombosis and breast masses. The introduction of physical barriers into the uterine cavity cannot induce endometrial repair and there is a risk of local inflammation, which limits the efficacy.

[0003] In recent years, some biomaterials have made new progress in the treatment of IUA: for example, the addition of growth factors, stem cells, and exosomes can promote endometrial regeneration, but the efficacy is limited because of low implantation and retention rates, and the risk of immune rejection or tumor formation. In addition, although biomaterials including hyaluronic acid, polytetrafluoroethylene, and chitosan have good biocompatibility, they degrade rapidly in vivo and have poor adaptability to the shape of the uterine cavity, which affects the clinical application of these materials. Therefore, there is an urgent need to develop new stable and multifunctional methods to treat and prevent IUA.

[0004] Microfluidics has been applied to fabricate polymer and composite particles with complex structures due to its precise microscale integrated channels. Micromotors are microscopic devices that can be driven by external factors such as light, ultrasound, and magnetic fields, and have advantages such as motion controllability and easy integration with other technologies. Magnetically driven iron-containing micromotors are environmentally friendly, easy to manufacture, and low-cost. They can accelerate with high precision without chemical fuels, providing a promising innovative paradigm for drug delivery. Selenium is a trace element essential for maintaining normal life activities of the human body. Selenium nanoparticles (SeNPs) inherit its antioxidant and anti-aging activities and have excellent biocompatibility and degradability. Inspired by the satisfactory therapeutic effects achieved by these materials, the combination of micromotors and SeNPs through microfluidics provides an opportunity to design new biomedical carriers with the potential to promote wound repair and prevent IUA. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing a new type of magnetic / ultrasound responsive microcapsules in view of the shortcomings of the prior art. The new type of microparticles can effectively promote endometrial repair and prevent intrauterine adhesions by utilizing their magnetic / ultrasound controllable properties and the biological activity of selenium nanoparticles.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage comprises the following steps: (1) Preparation of Fe3O4-Se: Monodisperse superparamagnetic Fe3O4 nanoparticles were synthesized by hydrothermal method, and then Fe3O4-Se was synthesized by in situ growth method and collected by magnetic attraction; (2) Generating monodisperse droplets: A microfluidic chip was constructed, and a mixed aqueous solution of sodium alginate (ALG), methacrylated gelatin (GelMA), phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and Fe3O4-Se was used as the external phase solution, and a hydrophobic decafluoropentane solution (DFP) was used as the internal phase solution. Monodisperse droplets of uniform size were obtained by electrospraying a microfluidic chip. (3) The monodisperse droplets in step (2) are collected in a fixed container filled with a collection liquid, and are simultaneously cured by ultraviolet light.

[0007] Specifically, in step (1), sodium selenite is reduced by L-ascorbic acid to in-situ grow SeNPs on the surface of Fe3O4, thereby synthesizing Fe3O4-Se.

[0008] Specifically, in step (1), the particle size of the monodisperse superparamagnetic Fe3O4 nanoparticles is 70-100 nm.

[0009] Specifically, in step (2), the microfluidic chip is assembled and bonded by a glass capillary, a glass slide and a needle; wherein the glass capillary is assembled by coaxially nesting an outer phase capillary and an inner phase capillary.

[0010] Specifically, the outer phase capillary tube diameter is 300-400 μm, and the inner phase capillary tube diameter is 100-150 μm; by changing the flow rates of the inner and outer phases, the inner and outer diameters of the polymer microcapsules of the monodisperse droplets can be adjusted.

[0011] Preferably, in step (2), in the external phase solution, the concentration of sodium alginate is 0.5-3wt%; the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.05-0.3wt%; the concentration of methacryloyl gelatin is 2.5-30wt%; and the concentration of Fe3O4-Se is 0.1-0.4 wt%.

[0012] Preferably, in step (2), the flow rate of the external phase solution is 1.2-10 mL / h; and the flow rate of the internal phase solution is 0.3-2 mL / h.

[0013] Preferably, in step (3), the collection liquid is a calcium chloride (CaCl2) aqueous solution with a concentration of 1.0-4.0 wt%.

[0014] Furthermore, the magnetic / ultrasound responsive microcapsules prepared by the above preparation method for repairing endometrial damage are also within the protection scope of the present invention.

[0015] Furthermore, the present invention also claims to protect the use of the above-mentioned magnetic / ultrasound responsive microcapsules in the preparation of drugs for repairing endometrial damage. Beneficial Effects

[0016] (1) Compared with other biomaterials in the field of IUA, the nanomaterial Fe3O4-Se synthesized by the present invention has good dispersibility, simple and easy preparation process, and low requirements on reaction conditions, in which Fe and Se are both trace elements of the human body.

[0017] (2) The present invention relies on microfluidic technology and uses a microfluidic chip to prepare droplets. The channel is simple, the construction steps are few, and no complicated mechanical processing is required. The process is simple. The size of the monodisperse droplets and the size of the corresponding microcapsules after solidification can be adjusted by the flow rate of the internal and external phases, and the operation is convenient. (3) The present invention can easily achieve precise adjustment of the size of microcapsules for repairing endometrial damage by changing the liquid flow rates of the inner phase and the outer phase. The vaporization of the inner core DFP under ultrasound can promote drug release. The Fe3O4-Se in the outer shell is responsive to the magnetic field and can flexibly change its distribution, which is expected to better match the irregular shape of the uterine cavity. In addition, Fe3O4-Se can promote the repair of damaged endometrium. Therefore, the microcapsules of the present invention have good application prospects in preventing intrauterine adhesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 The present invention is a flow chart of the preparation process of magnetic / ultrasound responsive microcapsules for repairing endometrial damage.

[0020] Figure 2 Light microscopic images of microcapsules. Figure (a) and Figure (b) are light microscopic images of microcapsules before and after ultrasound, respectively.

[0021] Figure 3The relationship curve between the flow rate of the internal and external phases and the inner and outer diameters of the microcapsules in the microfluidic chip, where Figure (a) is the relationship between the flow rate of the internal phase and the particle size, and Figure (b) is the relationship between the flow rate of the external phase and the particle size.

[0022] Figure 4 A camera observes the process of microcapsules moving in different directions under the guidance of magnets.

[0023] Figure 5 Statistical graph of SeNPs release from microcapsules with and without ultrasound stimulation, which was applied once every hour for 10 seconds.

[0024] Figure 6 HE staining images of the control group and the experimental group treated with microcapsule, where (a) is the HE staining result of the wound surface of the control group, and (b) is the HE staining result of the wound surface of the experimental group. DETAILED DESCRIPTION

[0025] The present invention can be better understood with reference to the following examples.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0027] The present invention provides a method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage, comprising the following steps: (1) Preparation of Fe3O4-Se: Monodisperse superparamagnetic Fe3O4 nanoparticles were synthesized by a hydrothermal method, and then Fe3O4-Se was synthesized by an in situ growth method, which was finally collected by magnetic attraction.

[0028] (2) Generate monodisperse droplets: Build a microfluidic chip, select DFP solution as the inner phase, select ALG and LAP, GelMA and Fe3O4-Se mixed solution of different concentrations as the outer phase, and prepare monodisperse droplets of uniform size through electrospray microfluidic chip; (3) Preparation of core-shell structured microcapsules: The monodisperse droplets prepared in step 1) are collected in a fixed container filled with a collection liquid, and simultaneously irradiated with ultraviolet light to form microcapsules with a core-shell structure for repairing endometrial damage. The size and performance of the microcapsules are adjusted by changing the flow rates of the inner and outer phases or the concentration of the outer phase.

[0029] In step (1), sodium selenite (Na2SeO3) is reduced by L-ascorbic acid (AA) to in situ grow SeNPs on the surface of Fe3O4 to synthesize Fe3O4-Se.

[0030] In step (2), the microfluidic chip is assembled from glass capillaries, a glass slide, a spotting needle and quick-drying glue, wherein the glass capillaries are assembled by coaxially nesting an outer phase capillary with a diameter of 300-400 μm and an inner phase capillary with a diameter of 100-150 μm. The particle size of the microcapsule can be adjusted by changing the flow rate of the inner and outer phases or the diameter of the inner and outer phase capillaries.

[0031] In step (2), the hydrophobic phase is selected as DFP solution, and the external phase is composed of a certain concentration of ALG and LAP and different concentrations of GelMA and Fe3O4-Se dissolved in ultrapure water, wherein the specific concentration of ALG dissolved in ultrapure water is 1.5wt%, the specific concentration of LAP is 0.1wt%, the concentration of GelMA is 0, 2.5, 5 and 10wt%, and the concentration of Fe3O4-Se is 0, 0.1, 0.2 and 0.4wt%.

[0032] In step (2), the solution is formed into monodisperse droplets by adjusting the high voltage connected to the microfluidic chip.

[0033] In step (3), the collecting liquid is a CaCl2 solution with a concentration of 2.0 wt%, and the monodispersed droplets are collected in a fixed container filled with the collecting liquid, and are simultaneously cured by ultraviolet light. By changing the flow rate of the inner and outer phases, the inner and outer phase sizes of the microcapsules can be adjusted.

[0034] Furthermore, the fixed container is a round glass culture dish, and the inner diameter of the round glass culture dish is 2.2 cm. Example 1

[0035] A magnetic / ultrasound responsive microcapsule for repairing endometrial damage, the preparation process is as follows Figure 1 As shown, the following steps are included: (1) Preparation of internal and external phase solutions and collection solution: 1.1) Internal phase solution: composed of pure DFP solution; 1.2) External phase solution: composed of ALG / LAP / GelMA / Fe3O4-Se aqueous solution; ALG uses sodium alginate solid particles, which are dissolved in ultrapure water to form a 1.5wt% ALG aqueous solution; LAP is used as a photocuring agent, and LAP solid particles are selected and dissolved in ultrapure water to form a 0.1wt% LAP aqueous solution; GelMA is dissolved in ultrapure water, and GelMA aqueous solutions with concentration gradients of 0, 2.5, 5 and 10wt% are prepared respectively; Fe3O4-Se is selected as freeze-dried powder, which is dissolved in ultrapure water, and Fe3O4-Se aqueous solutions with concentration gradients of 0, 0.1, 0.2 and 0.4wt% are prepared respectively; 1.3) Collection liquid: CaCl2 solid particles dissolved in ultrapure water, using 2.0wt% CaCl2 aqueous solution; (2) Assembling the microfluidic chip: Use a microelectrode puller or an acetylene torch to pull two glass capillaries of different sizes. The diameter of the inner glass capillary is 100-150 μm, and the diameter of the outer glass capillary is 300-400 μm. The microfluidic chip is assembled by the inner and outer glass capillaries, a glass slide, a spotting needle and quick-drying glue. The inner and outer glass capillaries are coaxially nested.

[0036] (3) Preparation of core-shell microcapsules: The inner and outer phase solutions were extracted into glass syringes of corresponding specifications and placed on two peristaltic pumps respectively. The glass syringes and microfluidic chips were connected by polyethylene tubes. The flow rates of the inner and outer phases were set. The high voltage electricity was connected to the spotting needle, the peristaltic pump was started, and the voltage was turned on. At the mouth of the glass capillary, when the inner and outer phase fluids were pumped out, the fluid was cut off by the voltage under the action of the high voltage electricity to form monodisperse droplets. The monodisperse droplets were collected in a circular glass culture dish with an inner diameter of 2.2 cm filled with the collection liquid (2.0wt% CaCl2 aqueous solution), and at the same time, they were cured by ultraviolet light. The optical microscopy image of the surface core-shell structure microcapsules was observed using a stereo microscope, as shown in the figure. Figure 2 As shown in (a), it can be seen that the microcapsules are uniform in size. The above microcapsules are treated in ultrasound for 40 seconds to obtain hollow microcapsules. The morphology of the microcapsules is observed under a light microscope, as shown in FIG. Figure 2 As shown in (b).

[0037] It can be seen that the outward flow rate is controlled to be 7.2mL / h, other factors remain unchanged, and the internal phase flow rates are set to 0.6mL / h, 0.9mL / h, 1.2mL / h, and 1.5mL / h respectively. Figure 3 As can be seen in (a), in the range of 0.6~1.5mL / h, the inner diameter of the microcapsule increases with the increase of the internal phase flow rate; the inward flow rate is controlled to be 0.9mL / h, other factors remain unchanged, and the internal phase flow rates are set to 2.4mL / h, 4.8mL / h, 7.2mL / h, and 9.6mL / h, respectively. Figure 3 As can be seen in (b), in the range of 2.4~9.6mL / h, the outer diameter of the microcapsule increases with the increase of the external phase flow rate; the above results show that the inner and outer diameters of the microcapsules can be adjusted by changing the internal and external phase flow rates. The inner and outer diameters of the microcapsules prepared by this method are linearly related to the internal and external phase flow rates; therefore, changing the internal and external phase flow rates can achieve the adjustment of the inner and outer diameters of the microcapsules. Example 2: Magnetic field / ultrasound response experiment of core-shell structure microcapsules

[0038] In order to adapt the microcapsules to a wider range of applications, the core-shell structure microcapsules for repairing endometrial damage prepared in Example 1 were placed in an aqueous solution, and a magnet was placed under the container. Figure 4Figure 3 is a diagram of the magnetic response process of the microcapsules. It can be found that after adding the magnetic field, the microcapsules move along the moving trajectory of the magnetic field, indicating that the microcapsules have strong magnetic responsiveness.

[0039] In order to verify the ultrasonic responsiveness of the microcapsules, the core-shell structure microcapsules for repairing endometrial damage prepared in Example 1 were immersed in 1 mL of PBS and incubated in an oscillator at 150 rpm and 37°C. The ultrasound group was ultrasonically treated for 10 seconds every hour, and 100 μL of liquid was collected at predetermined time intervals. The fluorescence intensity was quantitatively detected using a microplate reader, and then an equal volume of PBS solution was supplemented. Figure 5 The release statistics of microcapsules with and without ultrasonic stimulation are shown in Figure 2. The results show that ultrasound can promote the release of Fe3O4-Se in microcapsules, indicating that the microcapsules are ultrasonically responsive. Example 3: Core-shell microcapsules used in endometrial damage repair experiment

[0040] Taking the core-shell structure microcapsules prepared in Example 1 as an example, 6 female rats weighing 190-250 g in the same estrous cycle were anesthetized by intraperitoneal injection of 10% (w / v) chloral hydrate, and the "Y"-shaped uterus was surgically exposed under a sterile environment, and the uterine wound surface was scraped with an endometrial scraper until it was rough; The rats with uterine trauma were randomly divided into a control group and an experimental group, with 3 rats in each group: the control group did not receive any treatment; the experimental group was filled with core-shell structure microcapsules into the rat uterus, and the uterus, abdominal wall and skin were sutured layer by layer with surgical sutures, and antibiotics were given to the rats to prevent infection; after 7 days of treatment, samples were taken from the uterine wound of the rats for HE staining; Depend on Figure 6 The HE staining results shown in (a) show that the endometrial wound surface of the control group was severely damaged, with a thickness of only 363.1±32.7μm; Figure 6 (b) The wound area of ​​the experimental group was small, the epithelium was regenerated in large quantities, the endometrium was basically restored, and the thickness was 723.3±27.2μm, indicating that the microcapsules used for endometrial damage repair are beneficial to regulating cell proliferation and other behaviors, thereby actively promoting wound repair. Compared with the self-repair of the control group, this conductive microneedle patch has a good ability to promote uterine wound repair.

[0041] The present invention provides a magnetic / ultrasound responsive microcapsule for repairing endometrial damage and a method for preparing the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage, characterized in that: The steps include: (1) Preparation of Fe3O4-Se: Monodisperse superparamagnetic Fe3O4 nanoparticles were synthesized by hydrothermal method, and then Fe3O4-Se was synthesized by in situ growth method; (2) Generating monodisperse droplets: construct a microfluidic chip, use a mixed aqueous solution of sodium alginate, methacrylated gelatin, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and Fe3O4-Se as the external phase solution, and a hydrophobic decafluoropentane solution as the internal phase solution, and obtain monodisperse droplets of uniform size through an electrospray microfluidic chip; (3) The monodisperse droplets in step (2) are collected in a fixed container filled with a collection liquid, and are simultaneously cured by ultraviolet light.

2. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 1, characterized in that: In step (1), sodium selenite is reduced by L-ascorbic acid to in situ grow SeNPs on the surface of Fe3O4, thereby synthesizing Fe3O4-Se.

3. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 1, characterized in that: In step (1), the particle size of the monodisperse superparamagnetic Fe3O4 nanoparticles is 70-100 nm.

4. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 1, characterized in that: In step (2), the microfluidic chip is assembled and bonded by a glass capillary, a glass slide and a needle; wherein the glass capillary is assembled by coaxially nesting an outer phase capillary and an inner phase capillary.

5. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 4, characterized in that: The outer capillary tube diameter is 300-400 μm, and the inner capillary tube diameter is 100-150 μm.

6. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 1, characterized in that: In step (2), in the external phase solution, the concentration of sodium alginate is 0.5-3wt%; the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.05-0.3wt%; the concentration of methacryloyl gelatin is 2.5-30 wt%; and the concentration of Fe3O4-Se is 0.1-0.4 wt%.

7. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 1, characterized in that: In step (2), the flow rate of the external phase solution is 1.2-10 mL / h; the flow rate of the internal phase solution is 0.3-2 mL / h.

8. The method for preparing magnetic / ultrasound responsive microcapsules for repairing endometrial damage according to claim 1, characterized in that: In step (3), the collection liquid is a calcium chloride aqueous solution with a concentration of 1.0-4.0 wt%.

9. Magnetic / ultrasound responsive microcapsules for repairing endometrial damage prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the magnetic / ultrasound responsive microcapsule according to claim 9 in the preparation of a drug for repairing endometrial damage.

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