CN216824184U - Local bionic structure of target myocardial infarction - Google Patents
Local bionic structure of target myocardial infarction Download PDFInfo
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- CN216824184U CN216824184U CN202023327509.7U CN202023327509U CN216824184U CN 216824184 U CN216824184 U CN 216824184U CN 202023327509 U CN202023327509 U CN 202023327509U CN 216824184 U CN216824184 U CN 216824184U
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Abstract
The utility model provides a pair of local bionic structure of target myocardial infarction, its characterized in that: comprises a core unit and an outer membrane unit; wherein the outer membrane unit wraps the core unit; wherein, the core unit is a polydopamine nanoparticle; the outer membrane unit is a macrophage membrane. This bionical medicine compares traditional anti-oxidant medicine, and this bionical nanostructure can get into capillary through blood circulation, is absorbed with the mode of pinocytosis by the cell, has improved the bioavailability of medicine, can realize the effectual accumulation in infarction position after the modification of macrophage membrane, and the side effect is less, and the half-life is prolonged than traditional anti-oxidant medicine to the medicine elimination, and in vivo utilization is high.
Description
Technical Field
The utility model provides a bionical nano-material specifically relates to a local bionic structure of target myocardial infarction.
Background
Acute myocardial infarction is the leading cause of death worldwide, and the coronary artery of infarction is dredged in time through treatment means such as medicines and operations at present. After coronary recanalization, although the ischemic myocardium is restored to normal perfusion, the tissue damage is progressively aggravated, which limits the benefit of myocardial reperfusion therapy. This damage following coronary occlusion due to revascularization of the ischemic myocardium is known as myocardial ischemia reperfusion injury. How to reduce the malignant influence caused by myocardial ischemia reperfusion injury and improve the reperfusion treatment effect and prognosis of patients with myocardial infarction is a key and hot point problem for treating patients with myocardial infarction. The massive production of reactive oxygen radicals plays an important role in myocardial ischemia reperfusion injury.
A large number of animal experiments and in vitro researches prove that the antioxidant can effectively relieve ischemia reperfusion injury, effectively reduce the area of myocardial infarction and promote the recovery of myocardial function when used for treating myocardial infarction. Therefore, targeted scavenging of reactive oxygen species is a key approach to alleviating myocardial ischemia reperfusion injury. After myocardial infarction, macrophages can migrate rapidly to the infarcted site. The existing antioxidant drugs have low selectivity, low half-life, low in-vivo utilization rate and unobvious curative effect.
SUMMERY OF THE UTILITY MODEL
The utility model discloses a macrophage membrane parcel has polydopamine nanoparticle of strong anti-oxidant characteristic, combines together the directional migration ability of macrophage and polydopamine's antioxidant capacity, constructs the bionical nanometer medicine at target myocardial ischemia reperfusion injury position, realizes highly accumulating at myocardial ischemia reperfusion focus position, reaches the efficiency of target treatment myocardial ischemia reperfusion injury.
The utility model provides a pair of local bionic structure of target myocardial infarction, its characterized in that: comprises a core unit and an outer membrane unit;
wherein the outer membrane unit wraps the core unit;
wherein, the core unit is a polydopamine nanoparticle;
the outer membrane unit is a macrophage membrane.
Further, the utility model provides a pair of local bionic structure of target myocardial infarction, its characterized in that: the outer membrane unit has a double-layer structure.
Further, the utility model provides a pair of local bionic structure of target myocardial infarction, its characterized in that: the outer membrane unit consists of a first membrane layer and a second membrane layer;
the first film layer wraps the core unit;
the second film layer wraps the first film layer.
Further, the utility model provides a pair of local bionic structure of target myocardial infarction, its characterized in that: the outer membrane unit also has a plurality of surface receptors thereon.
Further, the utility model provides a pair of local bionic structure of target myocardial infarction, its characterized in that: the surface receptor is selected from one or more of scavenger receptor, integrin and surface antigen.
The utility model provides a preparation method of local bionic structure of target myocardial infarction, its characterized in that: the polydopamine nanoparticle with strong antioxidation property is wrapped by a macrophage membrane.
Further, the utility model provides a pair of local bionic structure's of target myocardial infarction preparation method, its characterized in that includes the following step:
step 1, after macrophage membrane vesicles are collected, extruding the macrophage membrane vesicles through a liposome extruder back and forth, and repeating the extruding and the extruding for 20 to 100 times;
step 2, adding polydopamine solution, and repeatedly squeezing for 20-100 times;
and 3, collecting the extruded liquid, centrifugally collecting the precipitate, and then using a buffer solution for heavy suspension.
Further, the utility model provides a pair of local biomimetic structure's of target myocardial infarction preparation method, its characterized in that: the volume ratio of the macrophage membrane vesicle to the polydopamine solution is 1: 0.8-1.2.
Further, the utility model provides a pair of local biomimetic structure's of target myocardial infarction preparation method, its characterized in that: the mass concentration ratio of the macrophage membrane vesicle to the polydopamine solution is 1: 0.8-1.2.
Further, the utility model provides a pair of local biomimetic structure's of target myocardial infarction preparation method, its characterized in that the mass ratio of protein volume in the macrophage membrane vesicle and polydopamine in polydopamine solution is 1: 0.8-1.5.
Further, the utility model provides a pair of local biomimetic structure's of target myocardial infarction preparation method, its characterized in that: the preparation method of the polydopamine solution is as follows:
step 1, dissolving dopamine hydrochloride in deionized water;
step two, after vigorous stirring at 40-60 ℃, adding alkali liquor into the dopamine acid solution;
step three, carrying out polymerization reaction for 2-8 hours, and dispersing the nano particles in deionized water after centrifugally recovering the nano particles;
step four, removing ions in the solution by using an ultrafiltration tube, and centrifuging to collect the accumulated dopamine;
and step five, dissolving the polydopamine obtained by centrifugation into a PBS solution.
Further, the utility model provides a pair of local biomimetic structure's of target myocardial infarction preparation method, its characterized in that: the mass ratio of the dopamine to the alkali is 4-8: 1.
further, the utility model provides a pair of local biomimetic structure's of target myocardial infarction preparation method, its characterized in that: the extraction method of the macrophage membrane is as follows:
step 1, collecting macrophages, and adding a buffer solution for resuspension;
step 2, after grinding the cells, adding a sucrose solution and mixing with the cell homogenate to ensure that the final concentration of sucrose is 0.01-0.5 mol/L;
step 3, centrifuging for 5-20min, and taking supernatant;
step 4, centrifuging for 20-40min, and removing supernatant;
step 5, adding a buffer solution containing sucrose for resuspension;
and 6, centrifuging for 20-40min, and removing supernatant to obtain the tube bottom precipitate as the macrophage membrane.
Further, the utility model provides a local bionic structure of target myocardial infarction which characterized in that has at least one of following usage:
A. for use as a medicament for targeting the local part of a myocardial infarction;
B. for use as a medicament for relieving the degree of fibrosis in myocardial infarction;
C. a medicament for relieving symptoms of myocardial infarction;
D. drugs for endocytosis to be absorbed by cells, entering the capillaries via blood circulation;
E. a drug for achieving effective accumulation at a myocardial infarction site.
The utility model discloses an effect:
1) compared with the traditional antioxidant drugs, the bionic nano-object carrier can enter the capillary vessel through blood circulation and be absorbed by cells in a pinocytosis mode, so that the bioavailability of the drugs is improved;
2) the effective accumulation of infarcted parts can be realized after the modification of macrophage membranes, and the side effect is small;
3) the half life of the medicine is prolonged compared with the traditional antioxidant medicine, and the in vivo utilization rate is high.
Drawings
Fig. 1 is a schematic structural diagram of a biomimetic nanostructure in the present embodiment.
Fig. 2 is a schematic diagram of a synthetic route of the biomimetic nano structure of the present embodiment.
FIG. 3 is a graph of particle size of Polydopamine (PDA) and nano-biomimetic structures (MPDANPS) of the present example.
FIG. 4 is a transmission electron micrograph of polydopamine (Beforee) and biomimetic nanostructures (After).
FIG. 5 is the measurement of myocardial infarction markers of SD rats 1 day after myocardial infarction;
wherein, FIG. 5a is a CK-MB comparison graph;
FIG. 5b is a graph comparing LDH;
control as Control group, IR as ischemia reperfusion group, PDA as poly dopamine group, PDA @ RAW as nano bionic drug group;
statistical analysis: n is 6, p <0.05, p <0.01, and p < 0.001.
FIG. 6 is a photograph showing the comparison of sirius red staining of myocardial tissue sections of SD rats 30 days after myocardial infarction;
wherein, Control is a Control group, IR is an ischemia reperfusion group, PDA is a poly dopamine group, PDA @ RAW is a nano bionic drug group.
Detailed Description
The bionic structure for targeting a local part of myocardial infarction provided by the embodiment is composed of a core unit 100 and an outer membrane unit 200, as shown in fig. 1;
wherein the outer membrane unit 200 wraps the entire core unit 100;
wherein, the core unit 100 is a polydopamine nanoparticle;
the outer membrane unit 200 is a macrophage membrane, the outer membrane unit 200 having a phospholipid bilayer with several surface receptors thereon, such as: integrin (intergin a4)210, integrin (intergin b1)220, scavenger receptor 230, surface antigen (CD40)240, and the like.
The specific synthesis method of the biomimetic nanostructure targeting the local myocardial infarction provided by the embodiment is as follows (as shown in fig. 2):
step one, synthesizing polydopamine particles
Dissolving 180mg of dopamine hydrochloride in 135ml of deionized water;
② after vigorously stirring at 50 ℃, adding NAOH solution (790ul,1mol/L) into dopamine acid solution;
③ recovering NPs through centrifugation (20000rpm,10min) after 4h and dispersing in deionized water;
and fourthly, removing ions in the PDA solution by using a 3000da ultrafiltration tube, then centrifugally collecting again, and dissolving in the PBS solution.
Step two, extracting macrophage membrane
Macrophage was collected, resuspended by adding 4 ℃ TM buffer, and after grinding the cells, an appropriate amount of 1 mol. L-1 sucrose was added and mixed with the cell homogenate to give a final sucrose concentration of 0.25 mol. L-1. The following operations are then performed:
centrifuging at 4 ℃ for 10min at 3000g, and taking supernatant;
② centrifuging for 30min at the temperature of 4 ℃ and 4000g, and removing supernatant fluid;
③ adding 0.25 mol.L-1 sucrose TM buffer at 4 ℃ for heavy suspension, centrifuging for 30min at 4000g at 4 ℃, and discarding the supernatant. The sediment at the bottom of the tube is the macrophage membrane.
Step three, wrapping the nano particles by the macrophage membrane
After macrophage membrane vesicles (protein amount: 200ug/ml) are collected, the macrophage membrane vesicles are extruded back and forth through a liposome extruder to pass through a nano polycarbonate film, and the process is repeated for about 50 times;
adding polydopamine solution with the same volume of 200ug/ml, and repeatedly squeezing for about 50 times;
③ collecting the extruded liquid, centrifuging at 50000g, collecting the precipitate, and then using PBS for heavy suspension.
In connection with the above-described specific example,
in the process of preparing the nano particles, the mass ratio of the dopamine to the alkali can be 4-8: 1, is selected within the range of 1. The alkali solution may be an organic base or an inorganic base, and preferably an inorganic base is used.
In the process of preparing and extracting the macrophage membrane, the final concentration of the sucrose can be selected within the range of 0.01-0.5mol/L according to the requirement of actual medicines, and the centrifugation time can be adjusted for 5-40min according to the form condition after centrifugation;
in the process of preparing the macrophage membrane-wrapped nano-particles, the volume ratio of the macrophage membrane vesicles to the polydopamine solution can be 1: the mass concentration ratio of 0.8-1.2 is selected from the range of 1: the mass ratio of the protein amount in the macrophage membrane vesicle to the polydopamine in the polydopamine solution can be 1: selected within the range of 0.8-1.5. In the first step and the second step, the repeated extrusion times of the liposome extruder can be selected within the range of 20-100 times according to the state of the extruded product.
For the biomimetic nano-drug of the above embodiment, the following verification test is also performed on the performance of the biomimetic nano-drug in this embodiment:
performance verification test I, determination of myocardial infarction marker of SD rat 1 day after myocardial infarction.
A. The colorimetric detection of serum LDH comprises the following steps:
1. respectively adding 35 mu L, 25 mu L and 25 mu L of double distilled water into the blank tube, the standard tube and the control tube;
2. adding 10 mu L of standard solution into the standard tube, and respectively adding 10 mu L of samples to be detected into the measuring tube and the comparison tube; to each tube was added 125. mu.L of matrix buffer, and to each assay tube was added only 25. mu.L of coenzyme I application solution. Fully mixing the materials in a vortex mixer, and putting the mixture into a water bath at 37 ℃ for 15 minutes;
3. adding 125 mu L of 2, 4-dinitrophenylhydrazine into each tube, fully mixing the mixture by a vortex mixer, and placing the mixture into a water bath at 37 ℃ for 15 minutes;
4. adding 1.25ml of 0.4mol/L NaOH solution into each tube, fully mixing by using a vortex mixer, standing at room temperature for 3 minutes, measuring the absorbance value of each tube at 440nm and 1cm of light path, and adjusting the double distilled water to zero;
5. LDH activity (UI/L) was calculated for each tube.
As shown in fig. 5a, LDH in ischemia-reperfusion group was significantly elevated after myocardial ischemia-reperfusion injury occurred. After the nano bionic drug (PDA @ RAW) is injected, LDH is reduced, and the myocardial infarction is prompted to be relieved.
B. The steps of detecting serum CKMB by enzyme-linked immunosorbent assay (ELISA) are as follows:
1. diluting the standard substance, and preparing a concentration gradient;
2. sample adding: and blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way), standard holes and sample holes to be detected are respectively arranged. Accurately adding 50 mul of standard sample on an enzyme-labeled coating plate, adding 40 mul of sample diluent in a sample hole to be detected, and then adding 10 mul of sample to be detected;
3. and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 30 minutes at 37 ℃;
4. washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, discarding, repeating the steps for 5 times, and patting dry;
5. adding an enzyme: adding 50 mul of enzyme-labeled reagent into each hole, and then incubating for 30 minutes at 7 ℃;
6. color development after 5 washes: adding 50 μ l of color-developing agent A into each well, adding 50 μ l of color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min;
7. and (4) terminating: the reaction was stopped by adding 50. mu.l of stop solution to each well (blue color immediately turned yellow).
8. And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with blank air conditioning of zero. CK-MB values were determined according to a standard curve.
As shown in fig. 5b, CK-MB was significantly elevated in the ischemia-reperfusion group after myocardial ischemia-reperfusion injury occurred. After the nano bionic drug (PDA @ RAW) is injected, CK-MB is reduced, and the myocardial infarction is prompted to be relieved.
Performance verification test two, SD rat myocardial tissue section sirius red staining 30 days after myocardial infarction (ruler: 100 μm, fibrosis staining is red).
The dyeing steps of sirius red are as follows:
1. fixing the myocardial tissue in 10% formalin fixing solution, and conventionally dehydrating and embedding;
2. slicing to 6 μm thickness, and dewaxing to water by conventional method;
3. dyeing the sirius red dyeing solution for 1 hour;
4. washing with running water slightly to remove dye liquor on the surface of the slice;
5. staining cell nuclei for 8-10 min by using Mayer hematoxylin staining solution;
6. cleaning for 10min, dehydrating, and sealing with neutral gum;
7. and (5) taking a picture by a microscope.
As shown in fig. 6, the fibrosis area of the ischemia-reperfusion group was significantly increased 30 days after the myocardial ischemia-reperfusion injury occurred, while the fibrosis degree was significantly alleviated after the injection of the nano-bionic drug (PDA @ RAW).
Claims (5)
1. A bionic structure for targeting myocardial infarction is characterized in that:
comprises a core unit and an outer membrane unit;
wherein the outer membrane unit wraps the core unit;
wherein, the core unit is a polydopamine nanoparticle;
the outer membrane unit is a macrophage membrane.
2. The biomimetic structure for targeting a local part of myocardial infarction of claim 1, wherein:
the outer membrane unit has a double-layer structure.
3. The biomimetic structure for targeting a local part of myocardial infarction of claim 1, wherein:
the outer membrane unit consists of a first membrane layer and a second membrane layer;
the first film layer wraps the core unit;
the second film layer wraps the first film layer.
4. The biomimetic structure for targeting a local part of myocardial infarction of claim 1, wherein:
the outer membrane unit also has a plurality of surface receptors thereon.
5. The biomimetic structure for targeting a local part of myocardial infarction of claim 4, wherein:
the surface receptor is selected from one or more of scavenger receptor, integrin and surface antigen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716913A (en) * | 2020-12-31 | 2021-04-30 | 上海市胸科医院 | Bionic nano-drug targeting myocardial infarction part and preparation method thereof |
| CN116270533A (en) * | 2023-04-26 | 2023-06-23 | 复旦大学附属中山医院 | Application of macrophage membrane-coated PCOD 585-containing nano-particles in myocardial ischemia reperfusion injury |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716913A (en) * | 2020-12-31 | 2021-04-30 | 上海市胸科医院 | Bionic nano-drug targeting myocardial infarction part and preparation method thereof |
| CN116270533A (en) * | 2023-04-26 | 2023-06-23 | 复旦大学附属中山医院 | Application of macrophage membrane-coated PCOD 585-containing nano-particles in myocardial ischemia reperfusion injury |
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