CN110755608A

CN110755608A - Application of specific antibody, implanted medical instrument and preparation method thereof

Info

Publication number: CN110755608A
Application number: CN201810847146.8A
Authority: CN
Inventors: 王玄; 周婷; 李俊菲; 姚瑶
Original assignee: Shanghai Minimally Invasive Medical Devices (group) Co Ltd
Current assignee: Shanghai Minimally Invasive Medical Devices (group) Co Ltd; Shanghai Microport Medical Group Co Ltd
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2020-02-07
Also published as: WO2020020090A1

Abstract

The invention belongs to application of a specific antibody, an implanted medical instrument and a preparation method thereof. The application of the specific antibody of the hematopoietic stem cell marker in preparing the medicine for promoting endothelial repair. The surface of the implanted medical device is loaded with a specific antibody of the hematopoietic stem cell marker. The method for producing the hematopoietic stem cell-labeled antibody comprises a step of supporting an antibody specific to the hematopoietic stem cell-labeled antibody on at least a part of the surface of the implantable medical device and a step of supporting an antibody specific to the hematopoietic stem cell-labeled antibody in a hole or a groove of the implantable medical device. The technical scheme provided by the invention creatively uses the specific antibody of the hematopoietic stem cell marker to realize the effect of promoting endothelial repair of the medical apparatus and instruments, compared with the existing endothelial repair mode, the method captures the hematopoietic stem cells of the human body and induces the generated cells to realize targeted endothelial repair, and the mode has small side effect of medicines, can not bring other adverse effects, and has more ideal comprehensive curative effect.

Description

Application of specific antibody, implanted medical instrument and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a specific antibody, an implanted medical instrument and a preparation method thereof.

Background

YIn et al 1997 have demonstrated that AC133 is a marker of hematopoietic stem cells. In 2000, AC133 was formally named CD133 at the 7 th international conference on human leukocyte differentiation antigens held by Harrogate, uk. The CD133 antigen can be recognized by 3 CD133 antibodies: clones AC133, 293C3 and AC 141. Clone AC133 binds directly to a CD133/1 glycosylated epitope and can be used to analyze and sort CD133 positive cells from peripheral blood, bone marrow, cord blood and other tissues. The monoclonal antibodies 293C3 and AC141 recognize the epitope CD133/2 and are used primarily for fluorescent staining of CD133+ cells after MACS sorting.

The left atrial appendage is an ear-like pouch extending from the left atrium, and is part of the left atrium, which is the main component of the left atrium. The left atrial appendage occlusion is researched and developed by boston science, is approved to be marketed in europe in 2006, is officially approved to be marketed in 12-31 months in 2013 by the national food and drug administration bureau of supervision and management in china, and is officially marketed in china in 3 months in 2014. The left atrial appendage, which is the root site for the occurrence of thrombus in patients with atrial fibrillation, is closed through the interventional operation, so that the risk of stroke in patients with atrial fibrillation can be reduced. The surface of the occluder is covered by an expandable polytetrafluoroethylene membrane, and the same general implanted medical device is also required to be loaded with a drug for repairing damaged tissues so as to prevent adverse effects caused by operation.

At present, the medical apparatus for promoting endothelial repair is generally realized by loading a small molecular compound or a composition on the surface of the medical apparatus, and is based on a mechanism for promoting cell growth, but the specificity is poor, so that the growth of other cells including smooth muscle, fiber cells and the like is promoted while the endothelial growth is promoted, adverse effects such as vascular restenosis, thrombus and the like are caused, and the mode has great side effect on medicines and can bring other adverse effects.

Disclosure of Invention

The invention provides an application of a specific antibody, an implanted medical instrument and a preparation method thereof, and aims to provide a brand-new method for promoting endothelial repair.

The invention provides an application of a specific antibody, wherein the specific antibody is a specific antibody of a hematopoietic stem cell marker, and the application is an application in preparing a medicament for promoting endothelial repair. Hematopoietic stem cells are adult stem cells in the blood system, and have long-term self-renewal capacity and the potential to differentiate into various types of mature blood cells. The aim of promoting endothelial repair can be achieved by inducing the hematopoietic stem cells to differentiate into required cell types in situ according to requirements.

Optionally, the antibody specific for the hematopoietic stem cell marker is an AC133 antibody. In most cases, CD133/1 and CD133/2 recognized the same cell but with differences in expression intensity, but CD133/1 and CD133/2 were found to be expressed differently or with a disturbed normal expression intensity in myelodysplastic syndrome and some acute myelogenous leukemias, and AC133 was considered to be a primitive hematopoietic cell population marker.

Alternatively, the AC133 antibody is Clone AC133(AC133 monoclonal antibody). Clone AC133 was purchased from Meitian and whirlpool Biotech, Inc., Germany.

The invention also provides an implanted medical appliance, and the surface of the implanted medical appliance is loaded with the specific antibody of the hematopoietic stem cell marker. The specific antibody of the hematopoietic stem cell marker can capture hematopoietic stem cells in blood, further induce the differentiation of the hematopoietic stem cells to endothelial cells, and improve the treatment and tissue repair effects of the apparatus.

Optionally, the antibody specific for the hematopoietic stem cell marker is an AC133 antibody.

Optionally, the AC133 antibody is Clone AC 133.

Optionally, an antibody specific for the hematopoietic stem cell marker is loaded on at least one face of the implanted medical device.

Optionally, the implanted medical device is a vascular prosthesis, a membrane of a prosthetic valve, a prosthetic valve stent, a left atrial appendage occluder membrane, a left atrial appendage occluder stent, a heart occluder membrane, a heart occluder stent or a prosthetic patch.

Optionally, the concentration range of the antibody specific for the hematopoietic stem cell marker per unit area on the surface of the implantable medical device is: 10 to 1000. mu.g/mm². The concentration is too low, the antibody is dissolved in blood quickly, the concentration reaches a certain degree, the antibody binding site is saturated, and the effect is not increased any more.

The present invention also provides a method for producing the above-mentioned implantable medical device, comprising a step of supporting an antibody specific to the hematopoietic stem cell marker on at least a part of the surface of the implantable medical device and a step of supporting an antibody specific to the hematopoietic stem cell marker in a hole or a groove of the implantable medical device.

Alternatively, the antibody specific to the hematopoietic stem cell marker is loaded on the surface of the implantable medical device by a direct coating method, a chemical grafting method, and/or an electrostatic adsorption method.

The direct coating method is to directly coat the specific antibody solution of the hematopoietic stem cell marker on the surface of the implanted medical appliance, and the coating method is spray coating, dip coating or spin coating.

The chemical grafting method can be to chemically modify the antibody molecule to chemically link with the surface of the device or the surface of the device coating. It is also possible to bind the antibody molecules to the surface of the device by using antibody linkers or cross-linkers.

In the electrostatic adsorption method, a coating with opposite charges is coated on the surface of the device, so that the antibody molecules are coated on the surface of the device through electrostatic adsorption force.

The technical scheme provided by the invention creatively uses the specific antibody of the hematopoietic stem cell marker to realize the effect of promoting endothelial repair of the medical apparatus and instruments, compared with the existing endothelial repair mode, the method captures the hematopoietic stem cells of the human body and induces the generated cells to realize targeted endothelial repair, and the mode has small side effect of medicines, can not bring other adverse effects, and has more ideal comprehensive curative effect.

Drawings

FIG. 1 is a fluorescence chart of a sample loaded with an antibody specific to a hematopoietic stem cell marker in an antibody loading experiment of example 5, wherein (1) to (4) correspond to the samples of examples 1 to 4 in this order, after staining with a secondary fluorogenic dye;

FIG. 2 is a fluorescence chart showing the cells in blood captured by a sample carrying a specific antibody against a hematopoietic stem cell marker in the cell capture experiment of example 5, and the cells are subjected to fluorescent staining with the specific antibody, wherein (1) to (4) correspond to the samples of examples 1 to 4 in this order;

FIG. 3 is a SEM representation of scaffolds loaded with AC133 antibody and drug scaffolds implanted into rabbit iliac artery 14 days after animal experiments of example 5, with the sample of the scaffold of example 1 on the left, the sample of the scaffold of example 2 in the middle, and the sample of the scaffold of the control group on the right;

figure 4 is an SEM representation of AC133 antibody loaded membranes sewn into the left atrial appendage occluder stent implanted for 14 days in the animal experiment of example 5, with the sample of example 3 on the left, the sample of example 4 in the middle, and the untreated membrane on the right.

Detailed Description

For ease of understanding, the use of antibodies specific for the hematopoietic stem cell markers is illustrated below in connection with examples, which are to be understood as merely illustrative of the present invention and not as limiting the scope of the present invention.

The raw materials and instruments used in the present invention are commercially available, unless otherwise specified.

The "medical device" referred to in the present invention may be a device implanted in the body. The device may be used temporarily for a short period of time, or permanently implanted for a long period of time. In certain embodiments, suitable devices are those typically used to provide medical treatment and/or diagnosis for heart rhythm disorders, heart failure, valvular diseases, vascular diseases, diabetes, neurological diseases and disorders, orthopedic surgery, neurosurgery, oncology, ophthalmology, and ENT procedures. Medical devices to which the present invention relates include, but are not limited to, the following: stents, stent grafts, anastomotic connectors, synthetic patches, leads, electrodes, needles, guidewires, catheters, sensors, surgical instruments, angioplasty balloons, wound drains, shunt tubes (shunts), tubes, infusion sleeves (infusion sleeves), urethral cannulas, pellets, implants, blood oxygenators, pumps, vascular grafts, embedded intervention cartridges (vascalves), heart valves, annuloplasty rings, sutures, surgical clips, surgical staples, pacemakers, implantable defibrillators, neurostimulators, orthopedic instruments, cerebrospinal fluid shunts, implantable drug pumps, vertebral cages, artificial intervertebral discs, nucleus pulposus replacement instruments, ear tubes, intraocular lenses, and any tube used in interventional procedures. Preferably, the medical device to which the invention relates is in particular: a stent, balloon, occluder, valve or vascular prosthesis.

Example 1

Mu.g of the AC133 monoclonal antibody Clone AC133 (available from America, whirlwind, Biotech, Ltd., Germany) was dissolved in 10mL of the antibody dilution to prepare an antibody solution having a concentration of 10. mu.g/mL. 1mg/mL of polyelectrolyte sodium Hyaluronate (HA) is dissolved in 0.1% NaCl solution, 1mg/mL of polyelectrolyte Chitosan (CS) is dissolved in 0.1% NaCl solution, and 5mg/mL of base coat polyelectrolyte Polyimide (PEI) is dissolved in 0.1% NaCl solution.

Using a vascular stent H₂SO₄:H₂O₂The solution (30%, v/v) ═ 3:1 was soaked for 30 minutes, rinsed 3 times with purified water, and blown dry with nitrogen. Firstly, soaking the substrate in a Polyethyleneimine (PEI) solution to obtain a PEI layer; soaking the PEI-treated stent in a sodium Hyaluronate (HA) solution to obtain an HA outer layer; the stent was then immersed in a Chitosan (CS) solution to obtain a CS outer layer. And repeating the HA/CS electrostatic assembly process for 7 times to obtain the sodium hyaluronate/chitosan self-assembled multilayer film which is marked as PEI (HA/CS) 7. Soaking the support in the antibody solution for 30min, and taking out N₂And (5) drying. AC133 antibody-loaded scaffold preparation was completed. The concentration of the AC133 antibody per unit area of the surface of the scaffold was 19. mu.g/mm²。

Example 2

Mu.g of the AC133 monoclonal antibody Clone AC133 (available from America, whirlwind, Biotech, Ltd., Germany) was dissolved in 1mL of the antibody dilution to prepare an antibody solution having a concentration of 100. mu.g/mL. Using a vascular stent H₂SO₄:H₂O₂Soak in 3:1 solution for 30 minutes, rinse 3 times with purified water, and blow dry with nitrogen. Dipping in waterStanding in antibody solution for 30min, taking out, air drying, and storing at 4 deg.C. AC133 antibody-loaded scaffold preparation was completed. The AC133 antibody concentration per unit area of the surface of the scaffold was finally determined to be 71. mu.g/mm by weight calculation²。

Example 3

1mg of the AC133 monoclonal antibody Clone AC133 (purchased from America, Edison, Germany) was dissolved in 1mL of an antibody dilution to prepare an antibody solution having a concentration of 1 mg/mL. And (3) spraying the antibody solution on the surface of the left atrial appendage occluder membrane by using an ultrasonic spraying instrument, naturally airing, and storing at 4 ℃. AC133 antibody-loaded scaffold preparation was completed. The AC133 antibody concentration per unit area of the membrane surface was finally determined to be 690. mu.g/mm by weight calculation²。

Example 4

1mg of the AC133 monoclonal antibody Clone AC133 (purchased from America, Edison, Germany) was dissolved in 1mL of an antibody dilution to prepare an antibody solution having a concentration of 1 mg/mL. 1mg/mL of polyelectrolyte sodium Hyaluronate (HA) is dissolved in 0.1% NaCl solution, 1mg/mL of polyelectrolyte Chitosan (CS) is dissolved in 0.1% NaCl solution, and 5mg/mL of base coat polyelectrolyte Polyimide (PEI) is dissolved in 0.1% NaCl solution. Antibody linker NHS (100mM)/EDC (400mM) was dissolved in 0.1% NaCl solution.

The left atrial appendage occluder membrane is cleaned. Firstly, soaking in a PEI solution to obtain a PEI layer; soaking the support treated by the PEI in a sodium hyaluronate solution to obtain an HA outer layer; the stent was then immersed in a chitosan solution to obtain a CS outer layer. And repeating the HA/CS electrostatic assembly process for 7 times to obtain the sodium hyaluronate/chitosan self-assembled multilayer film which is marked as PEI (HA/CS) 7. The membrane was soaked in antibody linker NHS (100mM)/EDC (400mM), taken out into the antibody solution, taken out N2 and blown dry. The AC133 antibody-loaded membrane preparation was completed. The concentration of the AC133 antibody per unit area of the membrane surface is finally measured to be 956 by weighing calculation

μg/mm²。

Example 5

In this example, in vivo and in vitro functional evaluation of the antibody-supporting samples of examples 1, 2, 3 and 4 was performed.

5.1 evaluation of antibody Loading results

And (3) carrying out fluorescence staining on the AC133 antibody load sample by using a TRITC labeled anti-mouse IgG secondary antibody, and identifying whether the antibody is successfully loaded. As shown in fig. 1, the sample may be stained with a fluorescent color after loading. Indicating that the antibody was successfully loaded onto the sample surface.

5.2 cell Capture assay

Collecting 5mL of rabbit venous blood, diluting the rabbit venous blood by 1:1 with PBS and uniformly mixing; slowly spreading the diluted blood on the surface of the Ficoll lymphocyte separation solution, wherein the volume ratio is 1: 1; centrifuging at 2500g speed for 30 minutes; sucking the middle cloud layer into a clean centrifuge tube, adding 5mL PBS, blowing and beating the mixture by a suction tube, and centrifuging the mixture for 10 minutes at the speed of 2000 g; and (3) absorbing the supernatant, adding 5mL of PBS, blowing and stirring by a suction pipe, centrifuging at the speed of 2000g for 10 minutes, discarding the supernatant to obtain bottom cells, covering a centrifugal pipe cover, and transferring to a sterile operating platform. The centrifuge tube cap is opened, 6mL of EGM-2 medium is added to the bottom of the bottle, and the mixture is blown and beaten uniformly. The scaffolds loaded with the AC133 antibody of examples 1, 2, 3, and 4 were placed in different 6-well plates, respectively, using a 6-well plate with fibronectin. The cells were mixed well and 2mL of cell suspension was added to each well. Placing the six-hole plate at 37 ℃ and CO₂The culture was carried out in a 5% concentration incubator by shaking. After 2 hours, the 6-well plate was removed, the cells were fixed, and the cells were stained with an antibody dye (AC133-PE) grafted with phycoerythrin fluorophore to identify cell expression.

The cells captured by AC133 must express AC 133. Therefore, the capture cells can be fluorescently labeled with the fluorescent dye AC133-PE of the antibody corresponding to AC 133. As shown in FIG. 2, cells captured on the surface of the sample can be stained with phycoerythrin fluorescent dye to give a fluorescent color, indicating that the antibody-loaded sample can be successfully captured into hematopoietic stem cells.

5.3 animal experiments

(one) scaffold preparation

AC133 antibody-loaded scaffolds or membranes were prepared as in examples 1, 2, 3, 4. The tools and reagents used in the preparation process need to be sterilized in advance, and the whole preparation process is completed in an aseptic environment. The obtained scaffold is pressed, held, blow-molded, packaged in a sterile environment, and stored at 4 ℃ for later use. The obtained membrane is sewn on the surface of the left auricle occluder bracket, sterilized, packaged and stored at the temperature of 4 ℃ for standby.

(II) Stent Implantation

9 New Zealand white rabbits (2.2-2.6 kg) were prepared, and the animal weights were matched with the age. The experimental groups were two different cell-capturing scaffolds of examples 1 and 2, and the control group was a rapamycin drug scaffold, with 3 animals per group. Each animal was implanted with 1 stent in each of the left and right iliac arteries as a replicate. The device is implanted according to the ratio of the target stent to the artery size of 1.10: 1.0-1.20: 1.0. The target vessel will be evaluated by angiography prior to stent implantation. All target vessels will also be again evaluated angiographically after stent implantation.

(III) occluder implant

9 beagle dogs weighing about 30kg were prepared. Experimental group samples made from the membranes of examples 3 and 4, control group left atrial appendage occluders made from untreated membranes, and experimental and control groups of 3 animals each. The left atrial appendage membranes of the experimental group were loaded with antibodies, and the left atrial appendage membranes of the control group were untreated. After animal anesthesia, perform the interatrial puncture under the X-ray perspective environment, after the puncture succeeds, the propelling movement puncture sheath gets into the left atrium, withdraw from the pjncture needle, insert superhard seal wire along the expander, exchange left auricle occluder sheath pipe, send into 6F pigtail catheter to left auricle along the sheath pipe, carry out the radiography, observe left auricle shape and measure left auricle opening width and degree of depth, select suitable specification occluder to implant left auricle, the radiography is observed the shutoff effect and is tractive test shutoff steadiness. The plugging effect is good, and the plugging device is released when the plugging device is stable. Withdrawing the sheath tube, performing operation ending work, and completing the left atrial appendage occluder implantation operation.

(IV) results of the experiment

And after the stent is implanted for 14 days, taking out the implanted stent section, splitting the implanted stent section along the axial direction of the blood vessel, flushing the implanted stent section for 1 minute by using physiological salt containing heparin, transferring the blood vessel into 2.5 percent glutaraldehyde for fixation, taking out the blood vessel after 24 hours, dehydrating, drying and observing the endothelial coverage condition by using a scanning electron microscope. The stent samples are characterized by SEM 14 days after being implanted into the iliac arteries of the rabbit, the SEM characterization images of the examples 1 and 2 are sequentially shown in the figure 3 from left to right, and the SEM characterization image of the stent sample of the control group is shown at the rightmost side, so that the vascular endothelium is completely covered by endothelial tissues and the shape of endothelial cells is good after the stent samples of the examples 1 and 2 are 14 days. Compared with the inner wall graph of the blood vessel of the drug stent in the control group, the inner wall of the blood vessel is covered by a large amount of fibrin, and the endothelium is poor. The result shows that the prepared stem cell capturing bracket can achieve better effect of promoting endothelial repair.

After the occluder is implanted for 14 days, the animal is euthanized, the left atrial appendage occluder is dissected out and taken out, and the left atrial appendage occluder membrane is taken off from the support. After fixation and dehydration, the endothelialization of the membrane surface was observed by SEM. As shown in FIG. 4, the sample of example 3 is on the left, the sample of example 4 is in the middle, the untreated membrane is on the right, the left and middle samples are both completely covered by endothelial cells on the membrane surface, and a large amount of fibrin and blood cells are deposited on the untreated membrane surface on the right. It is demonstrated that endothelialization can be rapidly achieved with a left atrial appendage occluder using antibody loaded membranes.

Finally, it should be noted that: the above examples are only for illustrating the technical solutions of the present invention, and are not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features may be equivalently replaced, and such modifications or replacements may not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the specific antibody is characterized in that the specific antibody is the specific antibody of a hematopoietic stem cell marker, and the application is the application in preparing a medicament for promoting endothelial repair.

2. The use of a specific antibody according to claim 1, wherein the specific antibody for the hematopoietic stem cell marker is Clone AC 133.

3. An implantable medical device, wherein the surface of the implantable medical device is loaded with Clone AC 133.

4. The implantable medical device according to claim 4, wherein an antibody specific for said hematopoietic stem cell marker is loaded on at least one face of said implantable medical device.

5. The implantable medical device according to claim 4, wherein the implantable medical device is a vascular prosthesis, a membrane of a prosthetic valve, a prosthetic valve stent, a left atrial appendage occluder membrane, a left atrial appendage occluder stent, a cardiac occluder membrane, a cardiac occluder stent or a prosthetic patch.

6. The implantable medical device according to claim 5, wherein the implantable medical device is a left atrial appendage occluder membrane or a cardiac occluder membrane.

7. The implantable medical device according to any one of claims 3 to 6, wherein the concentration range of the antibody specific for the hematopoietic stem cell marker per unit area of the surface of the implantable medical device is: 10 to 1000. mu.g/mm²。

8. The method for producing an implantable medical device according to any one of claims 3 to 6, comprising a step of loading an antibody specific to the hematopoietic stem cell marker on at least a part of the surface of the implantable medical device or a step of loading an antibody specific to the hematopoietic stem cell marker in a hole or a groove of the implantable medical device.

9. The method for producing an implantable medical device according to claim 8, wherein the antibody specific to the hematopoietic stem cell marker is loaded on the surface of the implantable medical device by a direct coating method, a chemical grafting method and/or an electrostatic adsorption method.

10. The method for producing an implantable medical device according to claim 9, wherein the antibody specific to the hematopoietic stem cell marker is loaded on the surface of the implantable medical device by a method combining a chemical grafting method and an electrostatic self-assembly method.