Magnetic micro-robot with ros response and shape adaptability and preparation method and application thereof

Abstract

The application belongs to the technical field of micro-robot preparation, and provides a magnetic micro-robot with ROS response and shape self-adaptability, a preparation method and application thereof. The preparation method comprises the following steps: mixing eight-arm polyethylene diamine, eight-arm succinimidyl carboxylate, iron oxide nanoparticles, antioxidant material, cell adhesion molecule and PBS buffer to obtain a mixture; mixing the mixture with n-hexane, liquid paraffin and span 80, and performing magnetic stirring, and then standing to obtain a magnetic micro-robot mixture; and separating the magnetic micro-robot mixture by using a permanent magnet to obtain magnetic gel microspheres, i.e. the magnetic micro-robot. The magnetic micro-robot prepared by the application has ROS response and good shape self-adaptability, and can be widely applied to the medical field.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of micro-robot preparation, in particular to a magnetic micro-robot with ROS response and shape adaptability, and a preparation method and application thereof. BACKGROUND

[0002] Due to the unique active motion ability of micro-robots, they have good application prospects in biomedical fields such as targeted delivery of drugs, genes and cells, biological separation, detoxification, etc. In particular, magnetic field driven micro-robots have high depth penetration and safety, providing the best choice for in vivo applications. In order to realize the active propulsion ability of micro-robots, Zhang et al. reported that magnetic nanoparticles with a diameter of 100 nm can realize their motion in plasma, serum, gastric acid, hyaluronic acid, gastric acid, blood and vitreous body through swarm behavior (nature communications. 2019; 10: 5631). Alapan et al. achieved the reverse flow behavior of micro-robots in blood through a leukocyte-like strategy (science robotics. 2020; 5 (42): eaba5726.). Although the above reported micro-robots can realize active propulsion, they are all based on hard metal materials. These materials have poor biocompatibility and may easily cause some damage to tissues. Moreover, the in vivo environment is complex (such as branches, twists and restricted environments). Therefore, it is of great significance to construct soft micro-robots with shape adaptability under the condition of realizing active propulsion.

[0003] At present, soft micro-robots with shape adaptability are mainly biological hybrid micro-robots prepared by combining the self-deformability of cells (such as red blood cells) with magnetic materials (such as iron oxide nanoparticles). For example, Zhang et al. constructed a soft micro-robot with shape adaptability by using three-dimensional (3D) self-assembled stem cells doped with a low dose of magnetic particles (sciencerobotics. 2021; 6 (52): eaba2813.). However, there may be problems of uncontrollable micro-robot size. Therefore, although soft micro-robots with shape adaptability have broad application prospects, most of the current demonstrations are mainly carried out in vitro conditions, and the propulsion mechanism is based on the assumption of uniform Newtonian environment, while the real biological environment is much more complex. In order to better apply micro-robots in vivo and realize clinical transformation, the following three key challenges need to be overcome: 1. shape adaptability; 2. micro-robots can effectively adjust the pathological microenvironment; 3. magnetic field penetration ability to tissues. Hydrogel materials have good biocompatibility, are diverse, and have adjustable mechanical properties. Therefore, it is of great practical significance to construct a soft hydrogel micro-robot with active propulsion and shape adaptability and find suitable application scenarios for its broad application in the biomedical field. SUMMARY

[0004] To overcome the above-mentioned defects in the prior art, the application provides a magnetic micro robot with ROS response and shape adaptability as well as a preparation method and application thereof.

[0005] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions:

[0006] The application provides a preparation method of a magnetic micro robot with ROS response and shape adaptability, comprising the following steps:

[0007] (1) mixing eight-arm polyethylene diamine with eight-arm succinimidyl carboxymethyl ester, iron oxide nanoparticles, antioxidant material, cell adhesion molecule and PBS buffer to obtain a mixture;

[0008] (2) mixing the mixture with n-hexane, liquid paraffin and span 80 and performing magnetic stirring, and then standing to obtain a magnetic micro robot mixture;

[0009] (3) separating the magnetic micro robot mixture by using a permanent magnet to obtain a magnetic gel microsphere, namely the magnetic micro robot.

[0010] Preferably, in step (1), the mixing ratio of the eight-arm polyethylene diamine, the eight-arm succinimidyl carboxymethyl ester, the iron oxide nanoparticles, the antioxidant material, the cell adhesion molecule and the PBS buffer is 12-25 mg: 16-18 mg: 4-6 mg: 0.5-1.5 mg: 0.5-1.5 mg: 1-3 mL.

[0011] Preferably, in step (1), the purity of the eight-arm polyethylene diamine is 96-97%, the purity of the eight-arm succinimidyl carboxymethyl ester is 98.5-99.5%, the diameter of the iron oxide nanoparticles is 10-50 nm, the antioxidant material comprises one of melanin nanoparticles, 4-(benzyl aminoacyl) phenylboronic pinacol ester and tea polyphenol, and the cell adhesion molecule comprises one of dopamine, collagen and RGD.

[0012] Preferably, in step (1), the volume ratio of the PBS buffer to the n-hexane, the liquid paraffin and the span 80 in step (2) is 1-3: 48-52: 48-52: 1-3, the rotating speed of the magnetic stirring is 3000-8000 rpm, the magnetic stirring time is 25-35 min, and the standing time is 0.5-2 h.

[0013] Preferably, in step (3), the permanent magnet is a neodymium-iron-boron magnet, and the diameter of the magnetic gel microsphere is 50-500 µm.

[0014] The application further provides the ROS-responsive and shape-adaptive magnetic micro-robot prepared by the preparation method.

[0015] The application further provides application of the ROS-responsive and shape-adaptive magnetic micro-robot in preparation of a medicine for treating male erectile dysfunction.

[0016] The application further provides application of the ROS-responsive and shape-adaptive magnetic micro-robot in preparation of a medicine for treating maculopathy.

[0017] The application further provides application of the ROS-responsive and shape-adaptive magnetic micro-robot in preparation of a medicine for dissolving thrombus.

[0018] The application further provides application of the ROS-responsive and shape-adaptive magnetic micro-robot in preparation of a medicine for treating prostate cancer.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1. The micro-robot prepared by the application has good biological safety and can be biodegraded, thereby avoiding accumulation in organs and causing chronic inflammatory reactions.

[0021] 2. The micro-robot prepared by the application shows cell reactive oxygen species (ROS) response, thereby removing excess ROS in tissues and accelerating tissue repair.

[0022] 3. The micro-robot prepared by the application only needs to adjust the concentration of the reactant (eight-arm polyethylene diamine), so as to adjust the arrangement of the magnetic nanoparticles in the micro-robot.

[0023] 4. The preparation method of the application is simple and fast, and can be completed only by an emulsification method.

[0024] 5. The micro-robot prepared by the application is driven by a magnetic field, and the magnetic field has wide prospects in medical applications due to its minimally invasive tissue penetration and good clinical safety.

[0025] 6. The micro-robot prepared by the application shows super softness, and the modulus is lower than 1 kPa. The super low modulus makes the micro-robot show shape adaptability, and the micro-robot can pass through some blood vessel tissues which are narrower than itself under the driving of the magnetic field, thereby avoiding causing blood vessel blockage.

[0026] 7. The micro-robot prepared by the application has controllability in complex biological fluids (PBS, blood, DMEM), shape adaptability and reverse flow characteristics in blood.

[0027] 8. The application focuses on the treatment of erectile dysfunction of the penis, and the application site shows particularity. First, due to the large blood flow in the cavernous tissue of the penis, it is urgent to solve the problem of stem cell retention with the aid of micro-robots. Second, compared with the brain, heart and other deep organs, the cavernous body of the penis is a superficial organ, which requires less magnetic force, making it more potential for clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0029] Figure 1 Infrared spectrum of the magnetic micro-robot prepared in Example 1 of the present application;

[0030] Figure 2 The determination result of Young's modulus of the magnetic micro-robot prepared in Example 1 of the present application;

[0031] Figure 3 Optical microscope image of the magnetic micro-robot prepared in Example 1 of the present application;

[0032] Figure 4 Optical microscope image of the magnetic micro-robot prepared in Example 2 of the present application;

[0033] Figure 5 Optical microscope image of the magnetic micro-robot prepared in Example 3 of the present application;

[0034] Figure 6 Optical microscope image of the micro-robot prepared in Comparative Example 1 of the present application;

[0035] Figure 7 Motion behavior of the magnetic micro-robot prepared in Example 2 of the present application under magnetic field (Note: A represents that a single micro-robot moves along the set route; B represents that multiple micro-robots move along the set route under the action of magnetic field; C represents the triangular motion trajectory of the micro-robot; D represents the motion of the micro-robot in different physiological environments (including PBS, DMEM culture medium and blood));

[0036] Figure 8 ROS scavenging ability of the magnetic micro-robot prepared in Example 1 of the present application;

[0037] Figure 9A graph of the intracavernous pressure (ICP) of the penis of a rat (Note: ED model represents a rat with erectile dysfunction; MSC represents injection of stem cells into a rat with erectile dysfunction; MSC+Microrobot represents injection of stem cells-loaded microrobots into a rat with erectile dysfunction). DETAILED DESCRIPTION

[0038] The application provides a preparation method of a magnetic microrobot with ROS response and shape self-adaptability, comprising the following steps:

[0039] (1) mixing eight-arm polyethylene diamine with eight-arm succinimidyl carboxymethyl ester, iron oxide nanoparticles, antioxidant material, cell adhesion molecule and PBS buffer to obtain a mixture;

[0040] (2) mixing the mixture with n-hexane, liquid paraffin and span 80, and performing magnetic stirring, and then standing to obtain a magnetic microrobot mixture;

[0041] (3) separating the magnetic microrobot mixture by a permanent magnet to obtain a magnetic gel microsphere, namely the magnetic microrobot.

[0042] In the application, the mixing ratio of the eight-arm polyethylene diamine, the eight-arm succinimidyl carboxymethyl ester, the iron oxide nanoparticles, the antioxidant material, the cell adhesion molecule and the PBS buffer in step (1) is preferably 12-25 mg: 16-18 mg: 4-6 mg: 0.5-1.5 mg: 0.5-1.5 mg: 1-3 mL, and further preferably 20 mg: 17 mg: 5 mg: 1 mg: 1 mg: 2 mL.

[0043] In the application, the purity of the eight-arm polyethylene diamine in step (1) is preferably 96-97%, and further preferably 96.8%; the purity of the eight-arm succinimidyl carboxymethyl ester is preferably 98.5-99.5%, and further preferably 99%; the diameter of the iron oxide nanoparticles is preferably 10-50 nm, and further preferably 20-40 nm, and more further preferably 25 nm; the antioxidant material preferably comprises one of melanin nanoparticles, 4-(benzyl aminoacyl) phenylboronic pinacol ester and tea polyphenol, and further preferably 4-(benzyl aminoacyl) phenylboronic pinacol ester; the cell adhesion molecule preferably comprises one of dopamine, collagen and RGD, and further preferably RGD; and the PBS buffer is preferably 1x PBS buffer.

[0044] In the application, the volume ratio of the PBS buffer in step (1) to the n-hexane, liquid paraffin and span 80 in step (2) is preferably 1-3:48-52:48-52:1-3, further preferably 2:50:50:2; the rotating speed of the magnetic stirring is preferably 3000-8000 rpm, further preferably 4000-6000 rpm, and more further preferably 5000 rpm; the time of the magnetic stirring is preferably 25-35 min, further preferably 28-32 min, and more further preferably 30 min; and the time of the standing is preferably 0.5-2 h, further preferably 0.5-1.5 h, and more further preferably 1 h.

[0045] In the application, the permanent magnet in step (3) is preferably a neodymium-iron-boron magnet; and the diameter of the magnetic gel microspheres is preferably 50-500 μm, further preferably 100-300 μm, and more further preferably 200 μm.

[0046] In the application, the specific operation steps of step (3) are preferably as follows: the permanent magnet is put into the magnetic micro-robot mixed solution to obtain the magnetic gel microspheres under the adsorption of the magnetic force, the solution is discarded, 50 ml of PBS buffer is added for cleaning, the mixture is mixed with a rubber suction tube, and then the permanent magnet is used for separation, the step is repeated 5 times, and the finally obtained magnetic gel microspheres are the magnetic micro-robots.

[0047] The application further provides the magnetic micro-robots with ROS response and shape self-adaptability prepared by the preparation method.

[0048] The application further provides the application of the magnetic micro-robots with ROS response and shape self-adaptability in the preparation of a medicine for treating male erectile dysfunction.

[0049] The application further provides the application of the magnetic micro-robots with ROS response and shape self-adaptability in the preparation of a medicine for treating maculopathy.

[0050] The application further provides the application of the magnetic micro-robots with ROS response and shape self-adaptability in the preparation of a medicine for dissolving thrombus.

[0051] The application further provides the application of the magnetic micro-robots with ROS response and shape self-adaptability in the preparation of a medicine for treating prostate cancer.

[0052] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0053] 8-arm-PEG-NH2, 8-arm-PEG-NHS in the following examples were purchased from Xiamen Sino-Biongen Biotech Co., Ltd.; 4-(benzylcarbonyl) phenylboronic acid pinacol ester was purchased from Macklin; RGD (arginyl-glycyl-aspartic acid) was purchased from Jier Biochemical Co., Ltd.; n-hexane was purchased from Macklin; liquid paraffin was purchased from Macklin; span80 was purchased from Aldrin; the rest of the raw materials were all ordinary commercially available.

[0054] Example 1

[0055] A method for preparing a magnetic micro-robot with ROS response and shape adaptability, the steps are as follows:

[0056] Take 8-arm-PEG-NH2 with a purity of 96.8% (12 mg), 8-arm-PEG-NHS with a purity of 99% (17 mg), iron oxide nanoparticles (5 mg), 4-(benzylcarbonyl) phenylboronic acid pinacol ester (1 mg), RGD (1 mg) into 2 ml PBS buffer, slowly drop into the prepared solution (50 ml n-hexane, 50 ml liquid paraffin, 2 ml span80), magnetic stirrer 3000 rpm stirring for half an hour, standing solution 1 h, using magnet to separate the magnetic microspheres, then, wash 5 times with PBS buffer, get magnetic micro-robot.

[0057] Figure 1 The infrared spectrum of the magnetic micro-robot prepared in Example 1.

[0058] Figure 2 The determination results of the Young's modulus of the magnetic micro-robot prepared in Example 1.

[0059] Figure 3 The optical microscope image of the magnetic micro-robot prepared in Example 1. Figure 3 It can be seen that the magnetic particles of the micro-robot are arranged as a single chain and located in the middle of the gel microspheres.

[0060] Example 2

[0061] A method for preparing a magnetic micro-robot with ROS response and shape adaptability, the steps are as follows:

[0062] Take the purity of 96.8% 8-arm-PEG-NH2(20mg), the purity of 99% 8-arm-PEG-NHS(17mg), iron oxide nanoparticles(5mg), 4-(benzyl aminoacyl) phenylboronic pinacol ester(1mg), RGD(1mg) into 2ml PBS buffer solution, slowly drop into the prepared solution(50ml n-hexane, 50ml liquid paraffin, 2ml span80), magnetic stirrer 5000rpm stirring for half an hour, the solution is placed for 1h, the magnetic microspheres are separated by magnet, then, washed with PBS buffer solution for 5 times, get magnetic micro robot.

[0063] Figure 4 The optical microscope of the above magnetic micro robot is shown in the figure. Figure 4 It can be seen that the magnetic particles of the micro robot are arranged as multiple chains distributed in the microspheres.

[0064] Example 3

[0065] A method for preparing a magnetic micro robot with ROS response and shape adaptability, the steps are as follows:

[0066] Take the purity of 96.8% 8-arm-PEG-NH2(25mg), the purity of 99% 8-arm-PEG-NHS(17mg), iron oxide nanoparticles(5mg), 4-(benzyl aminoacyl) phenylboronic pinacol ester(1mg), RGD(1mg) into 2ml PBS buffer solution, slowly drop into the prepared solution(50ml n-hexane, 50ml liquid paraffin, 2ml span80), magnetic stirrer 8000rpm stirring for half an hour, the solution is placed for 1h, the magnetic microspheres are separated by magnet, then, washed with PBS buffer solution for 5 times, get magnetic micro robot.

[0067] Figure 5 The optical microscope of the above magnetic micro robot is shown in the figure. Figure 5 It can be seen that the magnetic particles of the micro robot are arranged as multiple chains distributed in the microspheres.

[0068] Comparative example 1

[0069] A method for preparing a micro robot, the steps are as follows:

[0070] Weigh 8-arm-PEG-NH2 (25 mg) with a purity of 96.8%, 8-arm-PEG-NHS (17 mg) with a purity of 99%, iron oxide nanoparticles (5 mg), 4-(benzylaminoacyl)phenylboronic acid pinacol ester (1 mg), and RGD (1 mg) and add them to 2 mL of PBS buffer. Slowly drip them into a pre-prepared solution (50 ml of n-hexane, 50 ml of liquid paraffin, 2 ml of span80). Stir at 5000 rpm with a non-magnetic stirrer for half an hour. Let the solution stand for 1 hour. Use a magnet to separate the magnetic microspheres. Then, wash them 5 times with PBS buffer to obtain a microrobot.

[0071] Figure 6 This is an optical microscope image of the microrobot. Figure 6 It can be seen that the magnetic particles of the microrobot are concentrated at the bottom of the microsphere due to the effect of gravity.

[0072] Experimental Example 1

[0073] Taking the magnetic microrobot prepared in Example 2 as an example, its motion behavior under a magnetic field was studied. 100 microrobots were dispersed in 1 mL of PBS, and 500 μl of the dispersion was placed in a cube-shaped transparent mold. The mold was manipulated under a magnetic control device. The results are shown in FIG. Figure 7 shown.

[0074] The movement speed of the magnetic microrobot prepared in Example 2 of the present invention is 180 μm / s. Figure 7 It can be seen that the magnetic microrobot has controllable movement behavior under the action of a magnetic field, which is conducive to achieving precise targeted delivery of therapeutic drugs or cells in the body.

[0075] Experimental Example 2

[0076] Taking the magnetic microrobot prepared in Example 1 as an example, its ability to remove ROS was studied. The microrobot was co-cultured with stem cells and hydrogen peroxide was used to induce a ROS environment. Figure 8 shown.

[0077] Depend on Figure 8 It can be seen that the magnetic microrobot prepared in Example 1 of the present invention can effectively reduce the fluorescence intensity of the ROS fluorescent probe (DCFH-DA). After stimulation with 100 μM hydrogen peroxide, intracellular ROS is significantly enhanced, and the addition of the magnetic microrobot can effectively eliminate ROS.

[0078] Experimental Example 3

[0079] Taking the magnetic microrobot prepared in Example 1 as an example, its effect on erectile dysfunction was studied. The cell-loaded microrobot was injected into rats with erectile dysfunction. The ICP was evaluated one month later. The results were as follows:Figure 9 The results show that, after one month, the ICP value of the group of injecting micro-robot and stem cells is obviously improved compared with the group of injecting pure stem cells, and can be maintained for a certain period of time.

[0080] The results show that, after one month, the ICP value of the group of injecting micro-robot and stem cells is obviously improved compared with the group of injecting pure stem cells, and can be maintained for a certain period of time. Figure 9 ) This shows that the magnetic micro-robot of the application has a certain treatment effect on penile erectile dysfunction.

[0081] The above only describes the preferred embodiments of the present application, and it should be pointed out that, for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a magnetic micro-robot with ROS response and shape adaptability, characterized in that, It comprises the following steps: (1) mixing eight-arm polyethylene diamine with eight-arm succinimidyl carboxymethyl ester, iron oxide nanoparticles, antioxidant material, cell adhesion molecule and PBS buffer to obtain a mixture; (2) mixing the mixture with n-hexane, liquid paraffin and span 80 and performing magnetic stirring, and then standing to obtain a magnetic micro-robot mixture; (3) separating the magnetic micro-robot mixture by a permanent magnet to obtain magnetic gel microspheres, i.e. magnetic micro-robots; In step (1), the mixing ratio of the eight-arm polyethylene diamine, eight-arm succinimidyl carboxymethyl ester, iron oxide nanoparticles, antioxidant material, cell adhesion molecule and PBS buffer is 12-25 mg:16-18 mg:4-6 mg:0.5-1.5 mg:0.5-1.5 mg:1-3 mL; In step (1), the diameter of the iron oxide nanoparticles is 10-50 nm, the antioxidant material is 4-(benzyl aminoacyl) phenylboronic pinacol ester, and the cell adhesion molecule is RGD; In step (2), the volume ratio of the mixture, n-hexane, liquid paraffin and span 80 is 1-3:48-52:48-52:1-3, the stirring speed is 3000-8000 rpm, the stirring time is 25-35 min, and the standing time is 0.5-2 h.

2. The method for preparing a magnetic microrobot with ROS response and shape adaptability according to claim 1, characterized in that: In step (1), the purity of the eight-arm polyethylene diamine is 96-97%, and the purity of the eight-arm succinimidyl carboxymethyl ester is 98.5-99.5%.

3. The method of claim 1, wherein the ROS-responsive and shape-adaptable magnetic microrobot is prepared by the steps of: In step (3), the permanent magnet is a neodymium-iron-boron magnet, and the diameter of the magnetic gel microspheres is 50-500 μm.

4. The magnetic micro-robot with ROS response and shape self-adaptability prepared by the preparation method of any one of claims 1-3.

5. The application of the magnetic micro-robot with ROS response and shape self-adaptability of claim 4 in the preparation of a drug for treating male erectile dysfunction.

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