Magnetic Janus hydrogel micro-robot loaded with double bioactive ions and preparation method of magnetic Janus hydrogel micro-robot

Abstract

The invention discloses a magnetic Janus hydrogel micro-robot loaded with double bioactive ions, which is characterized in that magnesium phosphate, ferroferric oxide and sodium alginate loaded with zinc oxide pass through a customized double-channel coaxial needle system; the magnetic Janus hydrogel micro-robot loaded with Zn < 2 + > and Mg < 2 + > is prepared by collecting the magnetic Janus hydrogel into a calcium chloride solution after being sheared by nitrogen and cross-linking, and the magnetic Janus hydrogel micro-robot loaded with Zn < 2 + > and Mg < 2 + > can reconstruct a natural gradient environment of Mg < 2 + > and Zn < 2 + > of a tendon-bone interface through magnetic control alignment after being implanted into a target part. The magnetic Janus hydrogel micro-robot is regular in shape, the size is easy to control, the magnetic Janus hydrogel micro-robot has good biocompatibility, and Mg < 2 + > and Zn < 2 + > can still be distributed in a gradient mode after 21 days of slow release. Compared with traditional operative treatment, the problem that the tendon-bone interface ion gradient is difficult to repair is solved to a great extent.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a magnetic Janus hydrogel micro-robot loaded with dual bioactive ions and a preparation method thereof. Background Art

[0002] RCT is a shoulder disease characterized by rotator cuff tendon tear, which is common in elderly patients. Due to its limited self-healing ability, surgical repair is often required for treatment. However, surgical treatment has limitations such as long postoperative recovery time, high treatment cost, and high recurrence rate. The main reason is the poor healing of the tendon-bone interface after repair, making it difficult to reconstruct the natural gradient of composition and microstructure. Therefore, the key to its clinical challenge lies in formulating a strategy to reconstruct the natural gradient of the tendon-bone interface.

[0003] Currently, studies have confirmed that bioactive metal ions are important components for promoting tissue and organ regeneration. Among them, Mg 2+ and Zn 2+ play key roles in bone development and tendon repair processes respectively. The Mg 2+ / Zn 2+ ion natural gradient at the tendon-bone interface, while promoting bone and tendon regeneration, gives specific growth directions to bone and tendon. Although Mg 2+ and Zn 2+ have been proven to promote bone and tendon regeneration respectively, combining the two alone is not sufficient to effectively promote tendon-bone interface repair. There is still an urgent need to develop an effective strategy to reconstruct the Mg 2+ and Zn 2+ gradient environment at the tendon-bone interface so that it can guide the synchronous regeneration of tendon and bone in RCT.

[0004] Janus hydrogel is a gel material with an asymmetric morphological structure or chemical composition. Its different sides have different or even completely opposite properties or functions. Compared with homogeneous or symmetric materials, Janus materials can provide simultaneous or staged properties or opposite properties in different regions. Its unique asymmetric structure and function make it have important research value in the biomedical field. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title. However, such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a magnetic Janus hydrogel micro-robot loaded with dual bioactive ions.

[0008] To solve the above technical problems, the present invention provides the following technical solutions:

[0009] A premixed solution obtained by stirring a Mg3(PO4)2 solution and a Fe3O4 solution evenly is added to a sodium alginate solution to obtain a mixed solution I. Additionally, a ZnO solution is added to the sodium alginate solution to obtain a mixed solution II;

[0010] The mixed solution I and the mixed solution II are respectively introduced into the two needle cores of a double-channel coaxial needle system, and nitrogen is simultaneously supplied to the outer needle to form micro-droplets;

[0011] The formed micro-droplets are collected in a CaCl2 solution to form magnetic alginate microspheres with a Janus structure, which are the magnetic Janus hydrogel micro-robots loaded with dual bioactive ions;

[0012] Among them, the mass concentration of Mg3(PO4)2 relative to the mixed solution I is 1-5%; the mass concentration of Fe3O4 relative to the mixed solution I is 0.1-0.5%; the mass concentration of ZnO relative to the mixed solution II is 0.1-0.5%; the flow rate ratio of the mixed solution I and the mixed solution II is 1:1.

[0013] As a preferred scheme of the method for preparing the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions of the present invention, among them: the mass concentration of Mg3(PO4)2 relative to the mixed solution I is 3%.

[0014] As a preferred scheme of the method for preparing the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions of the present invention, among them: the mass concentration of Fe3O4 relative to the mixed solution I is 0.25%.

[0015] As a preferred scheme of the method for preparing the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions of the present invention, among them: the mass concentration of ZnO relative to the mixed solution II is 0.1%.

[0016] As a preferred scheme of the method for preparing the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions of the present invention, among them: the mass concentration of sodium alginate in the mixed solution I or the mixed solution II is 1-5%.

[0017] As a preferred embodiment of the preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to the present invention, wherein: the flow rate of the mixed solution I or the mixed solution II through the double-channel coaxial needle system is 1-1.2 mL / h.

[0018] As a preferred embodiment of the preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to the present invention, wherein: the flow rate of the nitrogen gas delivered to the outer needle is 3-7 L / min.

[0019] As a preferred embodiment of the preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to the present invention, wherein: the mass concentration of the CaCl2 solution is 1-3%.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide a magnetic Janus hydrogel micro-robot prepared by the preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions.

[0021] As a preferred embodiment of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to the present invention, wherein: the micro-robot has a Janus structure and simultaneously loads Mg 2+ and Zn 2+ ions, and the micro-robot has directional adjustability through an external magnetic field.

[0022] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions in the treatment of shoulder diseases characterized by rotator cuff tendon tears.

[0023] As a preferred embodiment of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to the present invention, wherein: the micro-robot can still make Mg 2+ and Zn 2+ show a gradient distribution after 21 days of slow release.

[0024] Advantages of the present invention:

[0025] (1) The present invention constructs a magnetic Janus hydrogel micro-robot loaded with magnesium ions (Mg 2+ ) and zinc ions (Zn 2 + ) through a gas-shearing microfluidic platform. The magnetic Janus hydrogel micro-robot has good biocompatibility, size controllability, and magnetic controllability.

[0026] (2) Orient the Janus hydrogel micro-robots through magnetic fields, and Mg can still show a gradient distribution after 21 days of sustained release 2+ and Zn 2+ Compared with traditional surgical treatments, the tendon-bone interface Mg 2+ and Zn 2+ concentration gradient repair problem of rotator cuff tear (RCT) is largely solved. With the drug delivery characteristics of minimally invasive surgery and non-contact operation, this micro-robot is expected to become an accurate, effective and convenient RCT reconstruction carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0028] Figure 1 It is a diagram of an oil-free gas-shearing microfluidic platform for preparing magnetic Janus hydrogel micro-robots in Embodiment 1 of the present invention;

[0029] Figure 2 It is a diagram of the magnetic Janus hydrogel micro-robot prepared in Embodiment 1 of the present invention performing spin motion under the action of an external magnetic field;

[0030] Figure 3 It is a scanning electron microscope (SEM) diagram (left) and an EDS diagram (right) of the magnetic Janus hydrogel micro-robot prepared in Embodiment 1 of the present invention;

[0031] Figure 4 It is a degradation curve diagram of the magnetic Janus hydrogel micro-robot prepared in Embodiment 1 of the present invention;

[0032] Figure 5 It is a CLSM image, bright field image and size distribution diagram of the micro-robots prepared under different nitrogen gas flow rates in Embodiments 1-2 of the present invention;

[0033] Figure 6 It is a live / dead staining diagram of BMSCs in the Ms and Mg-Ms groups prepared in Comparative Examples 1 and 4 of the present invention;

[0034] Figure 7 It is a CCK-8 quantitative data diagram of BMSCs in the Ms and Mg-Ms groups prepared in Comparative Examples 1 and 4 of the present invention;

[0035] Figure 8 It is a scratch healing image of BMSCs in the Ms and Mg-Ms groups prepared in Comparative Examples 1 and 4 of the present invention;

[0036] Figure 9 Migration cell diagrams of BMSCs in the Ms and Mg-Ms groups prepared in Comparative Examples 1 and 4 in the Transwell chemotaxis experiment of the present invention, stained with crystal violet;

[0037] Figure 10 ARS staining diagrams (upper) and ALP staining diagrams (lower) of BMSCs in the Ms and Mg-Ms groups prepared in Comparative Examples 1 and 4 of the present invention;

[0038] Figure 11 RT-qPCR data diagrams of BMSCs in the Ms and Mg-Ms groups prepared in Comparative Examples 1 and 4 of the present invention;

[0039] Figure 12 Live / dead staining diagrams of TCs in the Ms and Zn-Ms groups prepared in Comparative Examples 2 and 4 of the present invention;

[0040] Figure 13 CCK-8 quantitative data diagrams of TCs in the Ms and Zn-Ms groups prepared in Comparative Examples 2 and 4 of the present invention;

[0041] Figure 14 Migration diagrams of TCs in the Ms and Zn-Ms groups prepared in Comparative Examples 2 and 4 in the Transwell chemotaxis experiment of the present invention;

[0042] Figure 15 Wound healing diagrams of TCs in the Ms and Zn-Ms groups prepared in Comparative Examples 2 and 4 of the present invention;

[0043] Figure 16 Immunostaining diagrams of TCs in the Ms and Zn-Ms groups prepared in Comparative Examples 2 and 4 of the present invention;

[0044] Figure 17 RT-qPCR data diagrams of TCs in the Ms and Zn-Ms groups prepared in Comparative Examples 2 and 4 of the present invention;

[0045] Figure 18 Micro-CT diagrams of the humeral head and representative MRI diagrams of the tendon regeneration of the supraspinatus-humerus complex at the 4th and 8th weeks of repair by the micro-robots prepared in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0046] Figure 19 Gait analysis diagrams of rats treated by non-contact drug delivery with Mg / Zn-Janus-Mr prepared in Example 1 of the present invention;

[0047] Figure 20 Gait analysis comparison diagrams of rats treated with drug delivery by the micro-robots prepared in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0048] Figure 21Histological images and magnified views of HE, Masson, and Sirius red staining of the regenerated tendon-bone interface at the 4th and 8th weeks after repair by the micro-robots prepared in Example 1 and Comparative Examples 1-3 of the present invention;

[0049] Figure 22 Statistical charts of histological scores of the regenerated tendon-bone interface at the 4th and 8th weeks after repair by the micro-robots prepared in Example 1 and Comparative Examples 1-3 of the present invention;

[0050] Figure 23 Representative images of multiplex immunofluorescence of BMP-2 and OCN at the tendon-bone interface at the 4th and 8th weeks after repair by the micro-robots prepared in Example 1 and Comparative Examples 1-3 of the present invention;

[0051] Figure 24 Multiplex immunofluorescence images of COL-I (green), COL-III (red), and TNC (yellow) of the regenerated tendon at the 4th and 8th weeks after repair by the micro-robots prepared in Example 1 and Comparative Examples 1-3 of the present invention. Detailed implementation manners

[0052] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following describes the detailed implementation manners of the present invention in conjunction with the embodiments of the specification.

[0053] In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0054] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that can be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or selectively exclusive embodiments from other embodiments.

[0055] The sodium alginate used in the present invention is A2033, Sigma-Aldrich; Mg3(PO4)2 is M341105, Aladdin; Fe3O4 is XFJ119, XFNANO; ZnO is Z112848, Aladdin; CaCl2 is C299717, Aladdin.

[0056] The oil-free and gas-free shear microfluidic platform used in the present invention mainly consists of four parts, including an electronic injection pump for providing alginate solution; a nitrogen gas cylinder for supplying nitrogen, and the nitrogen gas flow is regulated and controlled by a flow meter; a customized double-channel coaxial needle system; and a collection tank containing CaCl2 solution.

[0057] The double-channel coaxial needle system used in the present invention is a customized double-channel coaxial needle system, which includes two inner needles with a size of 30G fixed by rubber tubes and inserted into an outer needle with a size of 1 mL from the inside for transporting liquids, and a needle with a size of 14G is inserted into the outer needle from the side for transporting nitrogen.

[0058] In the present invention, the mass concentration represents the mass of the solute divided by the volume of the solution.

[0059] The biocompatibility test method in the present invention is as follows: Bone marrow mesenchymal stem cells (BMSCs) are loaded into the Transwell chamber and co-cultured with micro-robots (Mg-Ms) loaded with Mg3(PO4)2 and Fe3O4, and tendon cells (TCs) are co-cultured with micro-robots (Zn-Ms) loaded with ZnO for 3 days. Then, the cells are stained with a live / dead staining kit, and green live cells and red dead cells are observed with an inverted fluorescence microscope.

[0060] The test method for cell proliferation in the present invention is as follows: The Cell Counting Kit-8 (CCK-8) is used to detect the effect of micro-robots on cell proliferation. BMSCs are injected into the lower chamber of the Transwell, and Mg-Ms are injected into the upper chamber. After co-culturing for 1, 3, and 5 days, the cells are incubated with the CCK-8 solution at 37°C for 1 h, and then the OD 450 value is recorded with an enzyme-linked immunosorbent assay (ELISA) reader.

[0061] The method for measuring cell migration in the present invention is as follows: In the Transwell experiment, BMSCs are seeded in the upper well of the Transwell after serum starvation, and Mg-Ms are seeded in the lower well. The upper chamber is filled with a medium containing 1% fetal bovine serum, and the lower chamber is filled with a medium containing 10% fetal bovine serum. TCs are co-cultured with Zn-Ms. After co-culturing the cells with the microspheres for 12 h, the migrated cells on the membrane are fixed with paraformaldehyde and stained with crystal violet dye, and then observed under an inverted light microscope.

[0062] The scratch wound healing experiment in the present invention is as follows: BMSCs are cultured in the lower part of the Transwell chamber. After reaching 100% confluence, a scratch is made on the BMSCs. After washing with PBS and filling the lower chamber with a medium supplemented with fetal bovine serum (FBS), Mg-Ms are added to the upper part of the Transwell chamber. TCs are co-cultured with Zn-Ms. The wound closure time is recorded at 0, 12, 24, and 36 h under an inverted optical microscope, and the migration area is measured using ImageJ software.

[0063] In the present invention, the method for evaluating the osteogenic ability of early and late BMCSs is as follows: alkaline phosphatase (ALP) staining and alizarin red (ARS) staining are used to evaluate the effect of Mg-Ms on the osteogenic differentiation of BMSCs. BMSCs and Mg-Ms are co-cultured in an osteogenic conditioned medium in a Transwell system. After 7 and 14 days of culture, ALP staining solution is added and incubated at room temperature for 40 min, and the ALP activity is measured. After 14 and 21 days, ARS solution is added and incubated for 30 min, the mineralized nodules are stained and the staining conditions are recorded with a digital camera.

[0064] In the present invention, the method for immunofluorescence staining of cells is as follows: after BMSCs and Mg-Ms are co-cultured in a Transwell system with an osteogenic differentiation medium for 7 days, the BMSCs are digested and seeded in a confocal dish. After the cells adhere to the wall, the cells are washed with PBS, fixed with paraformaldehyde for 15 min, blocked with goat serum for 15 min, permeabilized with Triton X-100 for 20 min, and then BMP-2 (bone morphogenetic protein 2), RUNX-2 (RUNX family transcription factor 2), and OCN (osteocalcin) are added and incubated overnight at 4°C. After washing with PBS, goat anti-rabbit IgG is added and incubated in the dark at 37°C for 1 h. The cytoskeleton and nucleus are stained with Rhodamine-Phalloidin and DAPI respectively, and the average fluorescence intensity is analyzed by ImageJ after observing the immunofluorescence staining of the cells by CLSM. TCs and Zn-Ms are co-cultured in DMEM for 3 days, and COL-I (type I collagen), COL-III (type III collagen), and TNC (tumor necrosis factor) staining are performed using the same protocol as above.

[0065] In the present invention, real-time quantitative polymerase chain reaction (RT-qPCR) is as follows: after BMSCs and Mg-Ms are co-cultured in a Transwell system with an osteogenic differentiation medium for 7 days, the cell mRNA is extracted using the Eastep TM Super Total RNA Extraction Kit (RNA extraction kit). Then use The Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR Kit (RNA extraction kit) reversely transcribes mRNA into complementary DNA. RT-qPCR of BMP-2, RUNX-S, OCN, and ALP was performed on a quantistudio 3 (Applied Biosystems) using gene-specific primers and SYBR Green. TCs were co-cultured with Zn-Ms in DMEM for 3 days, and RT-qPCR detection of COL-I, COL-III, TNC, and MKX (Mohawk homeobox) was performed using the same method as above.

[0066] The method for evaluating the regeneration of the supraspinatus tendon in vivo in the present invention is as follows: perform radiological examinations. At the 4th and 8th weeks after surgery, the rats were anesthetized by inhaling isoflurane and scanned using a 7T animal magnetic resonance imaging (MRI) system (Bruker BioSpec 70 / 30 USR). The sagittal T2WI images were analyzed using Materialise Mimics Innovation Suite Medical 21.0 software.

[0067] After euthanizing the rats, the humeral complex was collected and fixed with paraformaldehyde. Micro-computed tomography (micro-CT) (Quantum GX micro-CT imaging system) was used to evaluate the formation of new bone in the humerus. The CTAn μ-CT software was used to analyze the images, and parameters such as bone mineral density (BMD) and bone volume / total volume fraction (BV / TV) were calculated.

[0068] The method for evaluating the functional recovery of the rat forelimb in the present invention is as follows: perform gait analysis. Prepare a blank paper of 100 cm × 20 cm with a dark box at the end of the paper. Place the rats at the beginning of the paper and let them freely walk into the dark box. The front paws of the rats were stained with black ink, and the footprints of the front paws of the rats were recorded. Stride 1 (stride refers to the distance from one footprint to the next footprint of the same paw) and stride 2 (step refers to the distance from the footprint of one paw to the footprint of the other paw) were recorded.

[0069] The method for evaluating the quality of the tendon-bone interface in the present invention is as follows: perform histological staining. After the supraspinatus-humeral complex was completely fixed, it was decalcified with an ethylenediaminetetraacetic acid solution for 4 weeks, dehydrated, and embedded in paraffin to make continuous tissue sections with a thickness of 5 μm. HE, Masson staining, and Sirius Red staining were performed to observe the tissue structure and composition of the specimens. A modified histological scoring system was used to quantitatively evaluate the quality of the tendon-bone interface.

[0070] Stain osteogenic markers such as BMP-2 and OCN, and osteogenic markers such as COL-I, COL-III, and TNC using a tyramide signal amplification (TSA) kit. After antigen retrieval, block the sections and incubate with the anti-COL-I or anti-BMP-2 working solution at 37 °C for 40 min. Incubate with Opal Polymer HRP at 37 °C for 10 min. After washing twice with PBS, add Opal 520 and stain at room temperature for 10 minutes. Perform another round of antigen retrieval and blocking, incubate the sections with the anti-COL-III or anti-ocn working solution in sequence, and then stain with Opal Polymer HRP and Opal 570. Incubate in the anti-tnc working solution and perform Opal Polymer HRP and Opal 650 staining. After DAPI staining and sealing with an anti-fading solution, scan the sections with an Olympus VS200 system and analyze the images with OlyVIA 3.4.1 software.

[0071] Example 1

[0072] This example provides a method for preparing a Janus hydrogel micro-robot, specifically as follows:

[0073] A premixed solution obtained by stirring the Mg3(PO4)2 solution and the Fe3O4 solution evenly is added to the sodium alginate solution to obtain a mixed solution I. Additionally, the ZnO solution is added to the sodium alginate solution to obtain a mixed solution II; wherein, the mass concentration of Mg3(PO4)2 relative to the mixed solution I is 3% (w / v); the mass concentration of Fe3O4 relative to the mixed solution I is 0.25% (w / v); the mass concentration of ZnO relative to the mixed solution II is 0.1% (w / v); the mass concentration of sodium alginate in the mixed solution I or the mixed solution II is 2.5% (w / v).

[0074] The mixed solution I and II are respectively introduced into two needle cores, and both mixed solutions pass through a double-channel coaxial needle system at a flow rate of 1 mL / h, that is, the flow rate ratio of the mixed solution I to the mixed solution II is 1:1. At the same time, nitrogen is transported to the outer needle at a flow rate of 5 L / min to provide a shear force for the formation of micro-droplets, thereby controlling the size of the micro-droplets and forming micro-droplets;

[0075] Collect the formed micro-droplets in a 2% (w / v) CaCl2 solution to form magnetic alginate microspheres with a Janus structure, namely magnetic Janus hydrogel micro-robots (Mg / Zn-Janus-Mr).

[0076] In a culture dish and an ex vivo tendon-bone interface model using rat Achilles tendon and tibia as materials, the micro-robots prepared in Example 1 are made to perform spinning motion under the action of an external magnetic field, asFigure 2 As shown, a scanning electron microscope (SEM) and an energy dispersive spectrometer (EDS) were used to analyze the composition of the freeze-dried micro-robots at different degradation times, as Figure 3 shown. In addition, the degradation time of the micro-robots prepared in Example 1 was analyzed, and the results are as Figure 4 shown.

[0077] From Figure 2 it can be seen that the magnetic Janus hydrogel micro-robots can perform spin motion and align in the same direction under the action of an external magnetic field, and their bone side can rotate and orient towards the bone direction. Correspondingly, the tendon side of the micro-robots can also be oriented towards the tendon direction.

[0078] According to Figure 3 it can be seen that the micro-robots gradually degrade, and the unique morphology of the bone or tendon side micro-robots with two compartments is still clearly distinguishable. The EDS spectrum shows that the degraded micro-robots still exhibit a gradient distribution in terms of Mg 2+ / Zn 2+ content. In addition, during the entire degradation process, Fe 2+ and Mg 2+ are co-localized on the bone side of the micro-robots, ensuring the directional adjustability of the micro-robots through an external magnetic field after implantation.

[0079] From Figure 4 it can be seen that the degradation curve of the micro-robots is slow. In the ion release experiment, the release curves of Mg 2+ and Zn 2+ from the micro-robots both extend to more than 21 days, which is beneficial for promoting the Mg 2+ and Zn 2+ concentration gradient repair at the tendon-bone interface of rotator cuff tear (RCT).

[0080] Example 2

[0081] This example provides a preparation method for Janus hydrogel micro-robots. The flow rate of nitrogen in Example 1 was adjusted to 7 L / min and 3 L / min respectively to prepare the micro-robots of this example.

[0082] Fluorescent polystyrene nanoparticles were added to the alginate solution, and the morphology and size of the magnetic Janus hydrogel micro-robots prepared in Examples 1-2 were observed. The results are as Figure 5 shown, Figure 5 including the CLSM images, bright field images and size distribution diagrams of the micro-robots respectively. It can be seen that the size of the micro-robots can be easily controlled by adjusting the nitrogen flow rate. There is a substantial contrast in the morphology of the two separated chambers in the micro-robots, with an obvious dividing line.

[0083] Comparative Example 1

[0084] In this comparative example, only the premixed solution obtained by stirring Mg3(PO4)2 and Fe3O4 evenly was added to the sodium alginate solution, and the resulting mixed solution was passed through a single-channel needle. The remaining preparation processes were the same as those in Example 1, and the micro-robots (Mg-Ms) loaded with Mg3(PO4)2 and Fe3O4 of this comparative example were prepared.

[0085] Comparative Example 2

[0086] In this comparative example, only the mixed solution obtained by adding the ZnO solution to the sodium alginate solution was passed through a single-channel needle. The remaining preparation processes were the same as those in Example 1, and the micro-robots (Zn-Ms) loaded with ZnO of this comparative example were prepared.

[0087] Comparative Example 3

[0088] This comparative example provides a preparation method for uniformly mixed microspheres (Mg / Zn-Mixed-Ms) loaded with Mg 2+ and Zn 2+ , specifically as follows:

[0089] The Mg3(PO4)2 and ZnO solutions were simultaneously added to the sodium alginate solution to obtain a mixed solution. Among them, the mass concentration of Mg3(PO4)2 relative to the mixed solution was 3% (w / v), the mass concentration of ZnO relative to the mixed solution was 0.1% (w / v), and the mass concentration of sodium alginate relative to the mixed solution was 2.5% (w / v). The mixed solution was passed through a single-channel needle, and the remaining preparation processes were the same as those in Example 1, and the micro-robots (Mg / Zn-Mixed-Ms) of this comparative example were prepared.

[0090] Comparative Example 4

[0091] In this comparative example, only the sodium alginate solution with a mass concentration of 2.5% (w / v) was passed through a single-channel needle. The remaining preparation processes were the same as those in Example 1, and the pure sodium alginate micro-robots (Ms) of this comparative example were prepared.

[0092] The ability of the micro-robots prepared in Comparative Examples 1 and 4 to promote bone formation in vivo was evaluated, and the results are as Figures 6 - 11 shown.

[0093] Figure 6 It is the live / dead staining map of BMSCs in the control group (control), Ms group, and Mg-Ms group. The results showed that after co-culturing Mg-Ms and BMSCs in the Transwell system for 3 days, live / dead staining showed that there was almost no cell death in each group.

[0094] Figure 7Quantitative CCK-8 data graphs of BMSCs in the control (conventional medium), Ms, and Mg-Ms groups. Similarly, the CCK-8 test results showed that Mg-Ms significantly promoted the proliferation of BMSCs over time.

[0095] Figure 8 Scratch healing diagrams of BMSCs in the control (conventional medium), Ms, and Mg-Ms groups. In the scratch healing experiment, BMSCs in the Mg-Ms group migrated to the scratch area faster throughout the experiment in response to Mg released by Mg-Ms. 2+ 。

[0096] Figure 9 Diagrams of migrated cells stained with crystal violet of BMSCs in the control (conventional medium), Ms, and Mg-Ms groups. In the Transwell experiment, due to the chemotactic effect of Mg 2+ , BMSCs in the upper chamber migrated to Mg-Ms in the lower chamber, and there were more migrated cells stained with crystal violet in the Mg-Ms group than in other groups.

[0097] Figure 10 ARS staining diagrams (upper) and ALP staining diagrams (lower) of BMSCs in the control (conventional medium), Ms, and Mg-Ms groups. Among them, ALP staining and ARS staining were used to evaluate the osteogenic ability of early and late BMCSs, respectively. It was found that after 7 days and 14 days of induction in osteogenic conditioned medium, the staining area and staining depth of BMSCs in the Mg-Ms group were significantly larger than those in other groups, indicating that Mg 2+ induced enhanced ALP activity. ARS staining of BMSCs co-cultured with Mg-Ms for 14 days and 21 days also showed a larger staining area and a larger staining depth, indicating the highest calcium deposition efficiency.

[0098] Figure 11 RT-qPCR data graphs of BMSCs in the control (conventional medium), Ms, and Mg-Ms groups. This graph was consistent with the above experimental results. Mg-Ms upregulated the mRNA expression of BMP-2, RUNX-2, OCN, and ALP. BMSCs in the Mg-Ms group showed stronger osteogenic differentiation ability compared with the control (conventional medium) and Ms control groups.

[0099] Thus, it can be seen that Mg-Ms can promote the proliferation, migration, and osteogenic differentiation of BMCSs, ensuring the effectiveness of new bone formation on the bone side of the micro-robot during tendon-bone healing.

[0100] The ability of the micro-robots prepared in Comparative Examples 2 and 4 to promote tendon regeneration in vivo was evaluated, and the results were as follows Figures 12 - 17As shown

[0101] Figure 12 are the live / dead staining images after co-culturing control (conventional medium), Ms, and Zn-Ms with TCs respectively. Figure 13 are the CCK-8 quantitative data graphs after co-culturing control (conventional medium), Ms, and Zn-Ms with TCs respectively. The results of live / dead staining and CCK-8 experiments show that Zn-Ms has no cytotoxicity to TCs and can accelerate the proliferation of TCs.

[0102] Figure 14 are the migration images of TCs in the control (conventional medium), Ms, and Zn-Ms groups in the Transwell chemotaxis experiment. Figure 15 are the wound healing images of TCs in the control (conventional medium), Ms, and Zn-Ms groups. From the results of the Transwell chemotaxis experiment and the wound healing experiment, it is found that Zn-Ms significantly enhances the migration ability of TCs.

[0103] Figure 16 are the immunostaining images of TCs in the control (conventional medium), Ms, and Zn-Ms groups. In cell immunofluorescence staining, the higher the ratio of COL-I / COL-III, the better the tendon healing effect. Figure 16 shows that Zn-Ms can significantly increase the expression of COL-I and at the same time inhibit the expression of COL-III. In addition, the expression of TNC (Tenascin-C), an important regulatory factor for tendon regeneration and remodeling, is up-regulated with the release of Zn 2+

[0104] Figure 17 are the RT-qPCR data graphs of the control (conventional medium), Ms, and Zn-Ms groups. RT-qPCR shows that Zn-Ms can up-regulate the expression of COL-I and TNC mRNAs and down-regulate the expression of COL-III mRNA. In addition, the key tendon-specific transcription factor Mohawk (MKX) is significantly up-regulated at the transcriptional level, and its expression is positively correlated with tendon injury repair.

[0105] Thus, it can be seen that Zn-Ms can promote the proliferation, accumulation and function of TCs, ensuring the effectiveness of reconstructing tendons on the tendon side of the micro-robot during tendon-bone healing.

[0106] The micro-robots prepared in Example 1 and Comparative Examples 1-3 were evaluated for the repair and regeneration of the RCT gradient structure of rats, and the results are as Figures 18 - 24 shown

[0107] Figure 18 ​Micro-CT images of the humeral head at the 4th and 8th weeks (upper), and representative MRI images of the supraspinatus-humerus complex tendon regeneration in vivo at the 4th and 8th weeks (lower). It can be seen from the CT images that the bone line of the humeral head in the Mg / Zn-Janus-Mr group was continuous and without defect after 4 weeks of repair, and more bone formation was shown in the repair area. At the 8th week, the greater tubercle in the Mg / Zn-Janus-Mr group was similar to the normal anatomy. Quantitative analysis showed that during the whole repair process, the bone mineral density (BMD) and bone volume / total volume fraction (BV / TV) of the humeral head in the Mg / Zn-Janus-Mr group were significantly higher than those in other groups.

[0108] It can be seen from the MRI images that the tendon signal intensity in the Mg / Zn-Janus-Mr group was lower, indicating less tendon edema and inflammation. At the 8th week, the signal intensity in the tendon area of each group showed a downward trend compared with the 4th week, indicating a promoting progress during the healing process. The Mg / Zn-Janus-Mr group showed relatively continuous signals and the lowest intensity among all groups, indicating that Mg / Zn-Janus-Mr played the best role in tendon regeneration.

[0109] The Mg / Zn-Janus-Mr prepared in Example 1 was used to treat rats by non-contact drug delivery and gait analysis was performed, as Figure 19 shown. The micro-robots prepared in Example 1 and Comparative Examples 1-3 were used to treat rats and gait analysis was performed, and the results are as Figure 20 shown. It can be seen that the step length 1 and step length 2 of the Mg / Zn-Janus-Mr group at the 4th and 8th weeks recovered more significantly compared with other groups.

[0110] Thus, it can be seen that the Janus hydrogel micro-robot can synchronously induce new bone formation and tendon regeneration, thereby promoting the regeneration of the tendon-bone interface gradient structure.

[0111] Figure 21 Histological images of the regenerated tendon-bone interface repaired by different micro-robots at the 4th and 8th weeks, and enlarged views of HE, Masson, and Sirius red staining. It can be seen that at the 4th week, the tendon tissue morphology and arrangement in the Mg / Zn-Janus-Mr group were relatively regular. Compared with other groups, mature new bone formation was presented, and it was well combined with the regenerated tendon in structure, and denser and well-oriented collagen could be found. Using a modified histological scoring system to evaluate ( Figure 22 ), the scores of the Mg / Zn-Janus-Mr group at the 4th and 8th weeks were the highest, indicating that it promoted the healing and regeneration gradient structure of the tendon-bone interface, including tendon maturation and bone formation.

[0112] Figure 23Representative images of multiple immunofluorescence of BMP-2 and OCN at the tendon-bone interface at the 4th and 8th weeks of repair for different micro-robots. BMP-2 is an important osteogenic factor, and the Mg / Zn-Janus-Mr group had the highest BMP-2 protein signal intensity and the widest distribution among all groups. In addition, OCN is another important osteogenic marker, mainly expressed in the late stage of bone formation. It can be seen that the OCN positive staining in the Mg / Zn-Janus-Mr group was significantly higher than that in any other group. This is because this micro-robot promoted the expression of COL-I and simultaneously inhibited the deposition of COL-III. Therefore, it is speculated that the micro-robot successfully reconstructed the matrix components and stimulated tendon generation.

[0113] Figure 24 Multiple immunofluorescence images of COL-I (green), COL-III (red), and TNC (yellow) of regenerated tendons at the 4th and 8th weeks of repair for different micro-robots. The effects of Mg / Zn-Janus-Mr on COL-I and COL-III were very different: the micro-robot promoted the expression of COL-I and simultaneously inhibited the deposition of COL-III, clearly evaluating the type and deposition of collagen during tendon regeneration.

[0114] In summary, the present invention prepares magnetic Janus hydrogel micro-robots loaded with Zn 2+ and Mg 2+ through an oil-free gas-shearing microfluidic platform, which can be magnetically aligned after being implanted into the target site, and can reconstruct the natural gradient environment of Mg 2+ and Zn 2+ at the tendon-bone interface.

[0115] This magnetic Janus hydrogel micro-robot has a regular shape, easy-to-control size, good biocompatibility, and can still make Mg 2+ and Zn 2+ show a gradient distribution after 21 days of slow release. Compared with traditional surgical treatments, it largely solves the problem of difficult repair of the ion gradient at the tendon-bone interface.

[0116] In short, the present invention proposes a method for preparing magnetic Janus hydrogel micro-robots loaded with Zn 2+ and Mg 2+ using an oil-free gas-shearing microfluidic platform, and adopts drug delivery characteristics such as minimally invasive surgery and non-contact operation. This micro-robot is expected to become an accurate, effective and convenient RCT reconstruction carrier.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A preparation method of a magnetic Janus hydrogel micro-robot loaded with dual bioactive ions, characterized in that: Including, A premixed solution obtained by stirring a Mg3(PO4)2 solution and a Fe3O4 solution evenly, adding the premixed solution into a sodium alginate solution to obtain a mixed solution I, and additionally adding a ZnO solution into the sodium alginate solution to obtain a mixed solution II; The mixed solution I and the mixed solution II are respectively introduced into the two needle cores of a double-channel coaxial needle system, and nitrogen is simultaneously supplied to the outer needle to form microdroplets; The formed microdroplets are collected in a CaCl2 solution to form magnetic alginate microspheres with a Janus structure, which are magnetic Janus hydrogel micro-robots loaded with double bioactive ions; Among them, the mass concentration of the Mg3(PO4)2 relative to the mixed solution I is 1-5%; the mass concentration of the Fe3O4 relative to the mixed solution I is 0.1-0.5%; the mass concentration of the ZnO relative to the mixed solution II is 0.1-0.5%; the flow rate ratio of the mixed solution I and the mixed solution II is 1:

1.

2. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions as described in claim 1, characterized in that: The mass concentration of the Mg3(PO4)2 relative to the mixed solution I is 3%.

3. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to claim 1, characterized in that: The mass concentration of the Fe3O4 relative to the mixed solution I is 0.25%.

4. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to claim 1, characterized in that: The mass concentration of the ZnO relative to the mixed solution II is 0.1%.

5. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to claim 2, characterized in that: The mass concentration of sodium alginate in the mixed solution I or the mixed solution II is 1-5%.

6. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to claim 1, characterized in that: The flow rate of the mixed solution I or the mixed solution II through the double-channel coaxial needle system is 1-1.2 mL / h.

7. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions according to claim 1, wherein: The flow rate of supplying nitrogen to the outer needle is 3-7 L / min.

8. The preparation method of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions as claimed in claim 1, wherein: The mass concentration of the CaCl2 solution is 1-3%.

9. A magnetic Janus hydrogel micro-robot loaded with dual bioactive ions prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The micro-robot has a Janus structure and is simultaneously loaded with Mg 2+ and Zn 2+ ions, and the micro-robot has directional adjustability through an external magnetic field.

10. Use of the magnetic Janus hydrogel micro-robot loaded with dual bioactive ions in the treatment of shoulder diseases characterized by rotator cuff tendon tears, characterized in that: The micro-robot can still make Mg 2+ and Zn 2+ show a gradient distribution after 21 days of sustained release.

Images