CN112516110A - Method for coating nano enzyme on cell membrane - Google Patents

Method for coating nano enzyme on cell membrane Download PDF

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Publication number
CN112516110A
CN112516110A CN202011093134.4A CN202011093134A CN112516110A CN 112516110 A CN112516110 A CN 112516110A CN 202011093134 A CN202011093134 A CN 202011093134A CN 112516110 A CN112516110 A CN 112516110A
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cell membrane
cell
membrane
double
buffer solution
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CN112516110B (en
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刘惠玉
刘爽
潘雪婷
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Beijing University of Chemical Technology
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Beijing University of Chemical Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses a method for coating a nano enzyme on a cell membrane. The cell membranes of different cells are cracked and purified, and the cells are coated on the surface of the double-layer hollow manganese silicate with catalase activity, so that the targeting property of the nano enzyme in vivo is enhanced, the circulation time in vivo is prolonged, the nano enzyme is effectively enriched at a tumor part, hydrogen peroxide is catalyzed to generate oxygen, and tumor hypoxia is relieved. The nano enzyme coated by the cell membrane has good biocompatibility, provides an idea for precise treatment and has a great clinical application prospect.

Description

Method for coating nano enzyme on cell membrane
The technical field is as follows:
the invention belongs to the technical field of nano materials, and relates to a method for coating a nano enzyme on a cell membrane.
Background art:
hypoxia is a common feature of most solid tumors, mainly due to the rapid proliferation of cancer cells that consume oxygen in large quantities and the inefficient uptake of oxygen by the microvasculature in the tumor. Hypoxia not only decreases the efficacy of anticancer therapies such as chemotherapy, photodynamic therapy and radiotherapy, but also increases tumor infiltration and metastasis, leading to death of cancer patients. In addition, hypoxic conditions promote the polarization of macrophages from M1, which has pathogen clearance and anti-tumorigenic properties, to M2, which has pro-angiogenic and pro-tumorigenic properties, leading to a reduced attack of the immune system on tumor cells. Therefore, it is important to alleviate tumor hypoxia.
Traditional medical approaches have attempted to increase tumor oxygen levels by hyperbaric oxygen inhalation. However, the inefficient microvasculature at the tumor site prevents oxygenation of hyperbaric blood, and the potential toxicity of excessive inhaled oxygen also prevents clinical application of hyperbaric oxygen inhalation. It has been reported that some nanoenzymes can promote tumor oxygenation by catalyzing hydrogen peroxide at the tumor site to generate oxygen, such as manganese dioxide. Compared with natural enzymes, the nano-enzyme has higher stability, adjustable enzyme activity and lower production cost, and shows good application prospect in the aspect of biomedical use.
However, the synthetic nanoenzymes, which act as foreign substances and are rapidly eliminated upon injection into the body, shorten the circulation time and limit the therapeutic effect, cause various degrees of immune response although they can be improved by surface modification with polymers. In addition, the nanoenzyme can be passively targeted to the tumor site through the EPR effect, but the delivery efficiency is only 0.7%. Therefore, it is important to realize immune escape of the nano material, prolong the half-life of the nano material and enhance the tumor targeting property of the nano material. Functional protein exists on the surface of the cell membrane, and the blood circulation half-life period of the nano particles can be prolonged by utilizing the cell membrane coating, the nano particles are prevented from being identified by an immune system, and the active targeting property of nano enzyme is enhanced.
The invention content is as follows:
in order to solve the problems of poor targeting property and short half-life of the nano enzyme in vivo, the invention aims to provide a method for coating the nano enzyme double-layer hollow nano manganese silicate by a cell membrane. In order to realize the purpose, the invention firstly utilizes hypotonic solution or freeze-thaw cycle to crack cells and extract cell membranes, and then wraps the nano enzyme into the biological membrane in an extrusion mode, and finally can form uniform membrane-coated nano enzyme.
The technical scheme of the invention is as follows:
a method for coating nano enzyme by cell membrane comprises the following steps:
extracting macrophage cell membranes: resuspend RAW 264.7 macrophages in Tris-magnesium buffer (pH 7.4, Tris and MgCl)2The mass ratio of (1): 12.72) concentration of 2.5X 107one/mL and extruded through an Avestin liposome extruder without polycarbonate membrane to destroy cells. The cell homogenate was mixed with 1M sucrose to a final concentration of 0.25M sucrose and then centrifuged at 2000g and 4 ℃ for 10 minutes. The supernatant was collected and centrifuged further at 3000g and 4 ℃ for 30min to collect cell membranes. The cell membranes were washed with Tris-magnesium buffer at 4 ℃ and collected by centrifugation at 3000g at 4 ℃ for 30 min. Resuspended in phosphate buffer solution at a ratio of RAW 264.7 macrophages to phosphate buffer solution of 2.5X 107The method comprises the following steps: 1 mL. Mixing the obtained cell membrane with double-layer hollow manganese silicate, wherein the mass ratio of the cell membrane suspension volume to the double-layer hollow manganese silicate is 1mL:200 μ g, passing through a polycarbonate membrane with the aperture of 200nm for at least 25 times, centrifuging to collect the nano enzyme coated by the cell membrane, wherein the centrifugal force is 8950g, the centrifugation time is 10min, and the operation is carried out at room temperature.
The cell membrane coated nanoenzyme synthesized by the invention has the advantages of uniform appearance and size, good dispersibility, simple synthesis method and process, high repeatability, economy, good biocompatibility, higher targeting property, longer in-vivo circulation time and peroxidase-like activity, and has wide application prospect in the field of biomedicine.
Description of the drawings:
fig. 1 is a TEM image of the resulting double-layered hollow manganese silicate nanoparticle.
Fig. 2 is a TEM image of the prepared macrophage membrane-coated double-layered hollow manganese silicate nanoparticle.
FIG. 3 shows peroxidase-like activity at different concentrations of the double-layered hollow manganese silicate nanoparticles coated with the macrophage membrane prepared in the above method.
Detailed Description
Example 1
The transmission electron micrograph of the double-layer hollow manganese silicate nanoparticle is shown in FIG. 1, and the particle size is 150 nm.
Example 2
Resuspend RAW 264.7 macrophages in Tris-magnesium buffer (TM-buffer, pH 7.4, 0.01M Tris and 0.001M MgCl2) At a concentration of 2.5X 107one/mL, and by passing withoutAn Avestin liposome extruder of polycarbonate membrane extruded to destroy cells. Mixing the cell homogenate with sucrose solution with concentration of 342.3g/L at a volume ratio of 1:3, centrifuging for 10min at centrifugal force of 2000g and centrifugal temperature of 4 deg.C, collecting supernatant, centrifuging again at rotation speed of 3000g and temperature of 4 deg.C. Collecting the precipitate, washing with Tris-magnesium buffer solution at 4 deg.C, centrifuging at 3000g centrifugal force at 4 deg.C for 30min, and suspending in phosphate buffer solution at a ratio of RAW 264.7 macrophage to phosphate buffer solution of 2.5 × 107The method comprises the following steps: 1 mL. Mixing the obtained cell membrane with a double-layer hollow manganese silicate solution, wherein the mass ratio of the cell membrane suspension volume to the double-layer hollow manganese silicate is 1mL:200 mug, passing through a polycarbonate membrane with the aperture of 200nm, the centrifugal force is 8950g, the centrifugal time is 10min, and collecting double-layer hollow manganese silicate nanoparticles coated by a cell membrane at room temperature. The sample produced is shown in FIG. 2.
Example 3
0-400 mug mL of the double-layer hollow manganese silicate coated by the cell membrane is dispersed in 1mL of phosphate buffer solution, and the weight ratio of the double-layer hollow manganese silicate to the phosphate buffer solution is 1: 15, 3',5,5' -tetramethylbenzidine and hydrogen peroxide, and the ultraviolet absorption was measured after standing at room temperature for 10 min.

Claims (5)

1. A method for coating nano enzyme on cell membrane is characterized in that: mixing the cell membrane with double-layer hollow manganese silicate with peroxidase-like property in phosphate buffer solution, extruding through membrane, centrifuging and collecting double-layer hollow manganese silicate coated by cell membrane;
dispersing RAW 264.7 macrophage in Tris-magnesium buffer solution, adjusting RAW 264.7 macrophage number to 2.5 × 107Placing the cells/mL at 4 ℃ for 12h, and extruding the cells by an extruder for more than 20 times to destroy the cells to obtain cell homogenate; mixing the cell homogenate with a sucrose solution with a concentration of 342.3g/L at a volume ratio of 1:3, centrifuging for 10min at a centrifugal force of 2000g and a centrifugal temperature of 4 ℃, collecting the supernatant, centrifuging again at a rotating speed of 3000g and a temperature of 4 ℃; collecting the precipitate, washing with Tris-magnesium buffer solution at 4 deg.C, centrifuging at 3000g centrifugal force at 4 deg.C for 30min, and suspending in phosphate buffer solution, RAW 264.7 macrophage cell and phosphateThe ratio of the buffer solution was 2.5X 107The method comprises the following steps: 1 mL.
2. The method according to claim 1, characterized in that the synthesis of the double-layered hollow manganese silicate having peroxidase-like properties is carried out according to the following steps: the publication date is as follows: 20180306, publication No.: CN 107758676A.
3. The method of claim 1, wherein the mass ratio of the cell membrane suspension volume to the double layer hollow manganese silicate is 1mL:200 μ g.
4. The method of claim 1, wherein the polycarbonate membrane has a pore size of 200nm and is repeatedly pressed through the filter membrane more than 25 times.
5. The method of claim 1, wherein the centrifugation is performed at room temperature at a centrifugal force of 8950g for 10min to collect the cell membranes.
CN202011093134.4A 2020-10-13 2020-10-13 Method for coating nano enzyme on cell membrane Active CN112516110B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113750232A (en) * 2021-09-12 2021-12-07 重庆医科大学 Macrophage membrane coated arginine deiminase/catalase/IR 780 nanoparticle, and preparation method and application thereof
CN114306581A (en) * 2021-12-31 2022-04-12 重庆医科大学 Fusion cell membrane coated uricase/catalase lipid nanoparticle and preparation method thereof

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US20130336897A1 (en) * 2012-06-18 2013-12-19 Gerald L. Wolf Theranosis of macrophage-associated diseases with ultrasmall superparamagnetic iron oxide nanoparticles (uspio)
WO2019050998A1 (en) * 2017-09-05 2019-03-14 GLAdiator Biosciences, Inc. Method of targeting exosomes
CN110711249A (en) * 2019-09-19 2020-01-21 北京化工大学 Preparation method of lysosome membrane-coated nanoparticles
CN110876805A (en) * 2019-12-06 2020-03-13 郑州大学 Preparation method and application of macrophage membrane bionic bismuth triselenide nanoparticles
CN110974978A (en) * 2019-12-23 2020-04-10 暨南大学 Nano-catalyst for treating tumor and preparation method and application thereof

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US20130336897A1 (en) * 2012-06-18 2013-12-19 Gerald L. Wolf Theranosis of macrophage-associated diseases with ultrasmall superparamagnetic iron oxide nanoparticles (uspio)
WO2019050998A1 (en) * 2017-09-05 2019-03-14 GLAdiator Biosciences, Inc. Method of targeting exosomes
CN110711249A (en) * 2019-09-19 2020-01-21 北京化工大学 Preparation method of lysosome membrane-coated nanoparticles
CN110876805A (en) * 2019-12-06 2020-03-13 郑州大学 Preparation method and application of macrophage membrane bionic bismuth triselenide nanoparticles
CN110974978A (en) * 2019-12-23 2020-04-10 暨南大学 Nano-catalyst for treating tumor and preparation method and application thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113750232A (en) * 2021-09-12 2021-12-07 重庆医科大学 Macrophage membrane coated arginine deiminase/catalase/IR 780 nanoparticle, and preparation method and application thereof
CN114306581A (en) * 2021-12-31 2022-04-12 重庆医科大学 Fusion cell membrane coated uricase/catalase lipid nanoparticle and preparation method thereof
CN114306581B (en) * 2021-12-31 2023-06-02 重庆医科大学 Fusion cell membrane coated uricase/catalase lipid nanoparticle and preparation method thereof

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