CN113244189A - Preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes - Google Patents
Preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes Download PDFInfo
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- CN113244189A CN113244189A CN202110199479.6A CN202110199479A CN113244189A CN 113244189 A CN113244189 A CN 113244189A CN 202110199479 A CN202110199479 A CN 202110199479A CN 113244189 A CN113244189 A CN 113244189A
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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
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
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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
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
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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
- A61K9/5073—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 having two or more different coatings optionally including drug-containing subcoatings
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention discloses a preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes, which comprises the following steps: step 1: preparing a PLGA acetone solution; step 2: quickly adding a PLGA acetone solution into precooled Tris-HCl deionized water; and step 3: stirring is kept until the acetone is completely volatilized, and PLGA nano-particles are obtained; and 4, step 4: obtaining erythrocyte membranes, and mixing the erythrocyte membranes with PLGA nano-particles to obtain a mixed solution; and 5: and carrying out water bath ultrasound on the mixed solution to obtain the bionic nano-particles wrapped by the erythrocyte membranes. Compared with the prior art, the invention has smaller specification, higher biological barrier penetration effect and wider application potential, and has higher synthesis efficiency.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes.
Background
In recent years, the biomimetic nano-medical technology is widely applied in the fields of drug delivery, biological detoxification, vaccine preparation and the like, wherein the biomimetic nano-material coated on the basis of erythrocyte membranes is one of the most researched and most widely applied, which is mainly due to the fact that erythrocytes come from an organism, so the biomimetic nano-material coated on the erythrocyte membranes has the characteristics of excellent biocompatibility, low immunogenicity and the like, can effectively avoid the capture of a mononuclear-macrophage system and a reticuloendothelial system, and furthest prolongs the blood circulation time of nano-particles in vivo. Based on this, the erythrocyte membrane-coated particles can deliver various drugs to the target organs/tissues.
In addition, research reports that the biological behavior of the bionic nano material prepared based on the erythrocyte membrane in vivo is closely related to the particle size of the bionic nano material.
The red blood cells are derived from the body, and have extremely high biocompatibility and biological safety. It has been reported that coating the cell membrane of erythrocyte on the surface of nanoparticle can reduce the clearance of nanoparticle by immune cell and increase the half-life of blood, and at the same time, it can not induce immune reaction. Based on this, the erythrocyte membrane-coated particles can deliver various drugs to the target organs/tissues.
In addition, polylactic-co-glycolic acid (PLGA) is a medical polymer material approved by the Food and Drug Administration (FDA) for clinical use.
The invention patent with the application number of CN202010542343.6 discloses a ring gamma-polyglutamic acid modified hydrogel loaded nano sponge detoxification system and a preparation method thereof, and the description [0014] discloses a method for preparing nano particles with the size of about 60nm by using PLGA, wherein the size of the nano particles is about 80nm after the nano particles are coated with erythrocyte membranes, and the particles with the size cannot effectively penetrate most biological barriers (such as blood brain barriers, blood-fetus barriers and the like), so that symptoms related to the barriers cannot achieve the treatment effect. And the preparation method also discloses that a nanosponge is obtained by mixing 1mg PLGA nanoparticles with 1ml of erythrocyte membrane vesicles prepared from whole blood and extruding 14 times in a 100nm polycarbonate porous membrane using an Avanti micro extruder. The scheme utilizes a mechanical extrusion mode to wrap erythrocyte membrane vesicles and PLGA, the manufacturing mode is low in efficiency, and a large amount of product residues are difficult to obtain.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of an ultra-small bionic nanoparticle based on an erythrocyte membrane, which has smaller specification and higher biological barrier penetration effect compared with the prior art, and has higher synthesis efficiency compared with the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes comprises the following steps:
step 1: preparing a PLGA acetone solution;
step 2: quickly adding a PLGA acetone solution into precooled Tris-HCl deionized water;
and step 3: stirring is kept until the acetone is completely volatilized, and PLGA nano-particles are obtained;
and 4, step 4: obtaining erythrocyte membranes, and mixing the erythrocyte membranes with PLGA nano-particles to obtain a mixed solution;
and 5: and carrying out water bath ultrasound on the mixed solution to obtain the bionic nano-particles wrapped by the erythrocyte membranes.
The bionic nano-particles prepared based on the method have the characteristics of small particles and high wrapping efficiency.
The PLGA nanoparticles obtained by step 2 and step 3 are much smaller than the 60nm specification in the prior art. Through steps 4 and 5, the red cell membrane can be directly smashed and wrapped with PLGA nano particles by adopting a water bath ultrasonic mode, and compared with a mechanical extrusion mode in the prior art, the method has the characteristics of higher wrapping efficiency and small collection loss, and can accelerate the production efficiency. Meanwhile, the bionic nano-particles coated by the erythrocyte membrane have higher biological barrier penetration effect and can penetrate more biological barriers so as to treat the biological barrier related pathological positions which cannot be directly reached in the prior art.
Preferably, Tris-HCl deionized water has a pH of 8. The weakly alkaline deionized water can provide a mild reaction environment, so that the reaction is more stable.
Preferably, the pre-cooling temperature of the Tris-HCl deionized water is 4 ℃.
Preferably, the stirring speed in step 3 is 600 rpm/min.
Preferably, the time for performing water bath ultrasound on the mixed solution is 5 min.
Preferably, the ratio of PLGA nanoparticles to erythrocyte membrane in step 4 is 1: 2 (w/w).
Preferably, the PLGA acetone solution is prepared by the following method: acetone was used to dissolve 50: 50 viscosity, carboxy-terminal modified PLGA (0.67dL/g) to 7 mg/mL.
Preferably, the PLGA nanoparticles and the erythrocyte membrane are proportioned, and then the volume is adjusted to 1.5mL, and the mixture is added into a 20mL glass bottle.
Preferably, the ratio of the PLGA acetone solution to the Tris-HCl deionized water in the step 2 is 1: 3.
Preferably, the erythrocyte membrane in step 4 is prepared by the following method:
step 1: obtaining blood, and mixing the blood with 10mM EDTA PBS solution according to the volume ratio of 1: 1 to obtain mixed solution;
step 2: centrifuging the mixed solution at 3500rpm for 15min, and collecting erythrocyte layer to obtain erythrocyte;
and step 3: adding the red blood cells into deionized water according to the volume ratio of 1: 6, fully and uniformly mixing, and incubating in an ice bath for 30 min;
and 4, step 4: adding 20XPBS to adjust the erythrocyte membrane solution to 1X PBS, then centrifuging at 13800rpm for 10min, and removing hemoglobin in the supernatant;
and 5: repeating steps 3 and 4 until no hemoglobin is detected in the centrifuged supernatant;
step 6: the supernatant was removed to obtain red cell membranes.
In the preparation method, the efficiency of removing the hemoglobin in the red blood cells is higher, and the efficiency of preparing the red blood cell membrane is higher.
The invention has the beneficial effects that:
1. the prepared PLGA nano-particles are about 30nm and far smaller than 60nm in the prior art, can penetrate more biological membranes, and the bionic nano-particles prepared after wrapping the erythrocyte membrane can penetrate more biological barriers so as to treat more diseases;
2. when the bionic nano-particles are prepared, the erythrocyte membrane and the prepared PLGA nano-particles are mixed, then the water bath ultrasound is adopted to break the erythrocyte membrane and wrap the PLGA nano-particles at one time, and the two steps of mechanically breaking the erythrocyte membrane and wrapping the PLGA nano-particles and the erythrocyte membrane in the prior art are not needed;
3. the reaction environment is mild, and the reaction is more stable.
Drawings
FIG. 1 is a TEM image of PLGA nanoparticles of the present invention;
FIG. 2 is a schematic representation of PLGA nanoparticles of the present invention;
FIG. 3 is a photograph of the red blood cells of the present invention before and after reaching the red blood cell membrane;
FIG. 4 is a TEM image of a biomimetic nanoparticle coated with erythrocyte membrane according to the present invention;
FIG. 5 is a schematic diagram of the characterization in vitro stability assay of the biomimetic nanoparticles coated with erythrocyte membranes of the present invention.
Detailed Description
The invention will be further described in detail with reference to the following examples, which are given in the accompanying drawings.
As shown with reference to figures 1-5,
example one
Preparation and characterization of PLGA particles
Preparing PLGA particles by adopting a nano coprecipitation method: acetone was used to dissolve 50: 50 viscosity, carboxy-terminal modified PLGA (0.67dL/g) to 7mg/mL for use. The 1mL of PLGA acetone solution was quickly added to 4 ℃ pre-cooled 3mL ph 8.0Tris-HCl deionized water, followed by maintaining the stirring speed at 600rpm/min until the acetone was completely volatilized. And performing basic physical characterization on the prepared and collected PLGA nano particles, wherein the basic physical characterization comprises the steps of observing the particle morphology by a transmission electron microscope and detecting the hydraulic size and the surface potential of the particles by using a particle size potentiometer. The TEM results of fig. 1 show that PLGA nanoparticles are around 30nm in size; the particle size potentiometer detection result of fig. 2 shows that the size of the PLGA nanoparticles is about 35 nm in hydraulic dimension, and the surface potential is about-35 mV.
Example two
Extraction and purification of ICR mouse erythrocyte membrane
ICR mouse erythrocyte extraction: blood was drawn through the cheek and mixed 1: 1 by volume with 10mM EDTA in PBS. Centrifuging at 3500rpm for 15min, carefully removing serum and leukocyte layer, and collecting erythrocyte layer. Adding the red blood cells into deionized water according to the volume ratio of 1: 6, fully mixing and incubating in an ice bath for 30 min. Subsequently, a volume of 20XPBS was added to adjust the erythrocyte membrane solution to 1 XPBS, and then centrifuged at 13800rpm for 10min to remove hemoglobin from the supernatant. The above process was repeated 3 to 4 times until no hemoglobin could be detected in the centrifuged supernatant. Removing supernatant, resuspending erythrocyte membrane with deionized water, measuring membrane protein concentration by BCA method, and packaging at-80 deg.C. FIG. 3 shows a comparison of erythrocytes before and after removal of internal hemoglobin. The right graph shows that hemoglobin in erythrocytes can be efficiently removed by the above method, and erythrocyte membranes with high purity can be obtained.
EXAMPLE III
Preparation, characterization and in vitro stability determination of erythrocyte membrane-coated bionic nanoparticles (PLGA @ RBC)
PLGA: the volume of RBC is adjusted to 1.5mL according to 1: 2(w/w), then the mixture is added into a 20mL glass bottle, and water bath ultrasound is carried out for 5min to prepare the bionic nano-particles wrapped by erythrocyte membranes, and the bionic nano-particles are marked as PLGA @ RBC. And performing basic physical characterization on the prepared and collected PLGA @ RBC, wherein the basic physical characterization comprises the steps of observing the morphology of the particles by a transmission electron microscope and detecting the hydraulic size and the surface potential of the particles by using a particle size potentiometer. The TEM results of fig. 3 show that PLGA @ RBC exhibit a core-shell structure with a size of about 50 nm; the particle size potentiometer test result of FIG. 4 shows that the size of the PLGA @ RBC is about 60nm in hydraulic dimension and the surface potential is about-27 mV. Fig. 5 shows that PLGA @ RBC has very high stability in PBS compared to PLGA nanoparticles alone.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (10)
1. A preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes is characterized by comprising the following steps:
step 1: preparing a PLGA acetone solution;
step 2: quickly adding a PLGA acetone solution into precooled Tris-HCl deionized water;
and step 3: stirring is kept until the acetone is completely volatilized, and PLGA nano-particles are obtained;
and 4, step 4: obtaining erythrocyte membranes, and mixing the erythrocyte membranes with PLGA nano-particles to obtain a mixed solution;
and 5: and carrying out water bath ultrasound on the mixed solution to obtain the bionic nano-particles wrapped by the erythrocyte membranes.
2. The method of claim 1, wherein the PH of Tris-HCl deionized water is 8.
3. The method for preparing ultra-small biomimetic nanoparticles based on erythrocyte membranes as claimed in claim 1, wherein the pre-cooling temperature of Tris-HCl deionized water is 4 ℃.
4. The method for preparing ultra-small biomimetic nanoparticles based on erythrocyte membranes as claimed in claim 1, wherein the stirring speed in step 3 is 600 rpm/min.
5. The method for preparing ultra-small bionic nanoparticles based on erythrocyte membranes according to claim 1, wherein the time for performing water bath ultrasound on the mixed solution is 5 min.
6. The method of claim 1, wherein the ratio of the amount of PLGA nanoparticles to the amount of the erythrocyte membrane proteins in step 4 is 1: 2 (w/w).
7. The method for preparing ultra-small biomimetic nanoparticles based on erythrocyte membranes as claimed in claim 6, wherein PLGA acetone solution is prepared by the following method:
dissolving 50 parts by using acetone: 50 viscosity, carboxyl end modified PLGA (0.67dL/g) to 7 mg/mL.
8. The method for preparing ultra-small bionic nanoparticles based on erythrocyte membranes as claimed in claim 6, wherein the volume is 1.5mL after the PLGA nanoparticles are matched with the erythrocyte membranes, and the mixture is added into a 20mL glass bottle.
9. The method for preparing ultra-small biomimetic nanoparticles based on erythrocyte membranes as claimed in claim 1, wherein the ratio of PLGA acetone solution to Tris-HCl deionized water in step 2 is 1: 3.
10. The method for preparing ultra-small biomimetic nanoparticles based on erythrocyte membranes as claimed in claim 1, wherein the erythrocyte membranes in step 4 are prepared by the following method:
step 1: obtaining blood, and mixing the blood with 10mM EDTA PBS solution according to the volume ratio of 1: 1 to obtain mixed solution;
step 2: centrifuging the mixed solution at 3500rpm for 15min, and collecting erythrocyte layer to obtain erythrocyte;
and step 3: adding the red blood cells into deionized water according to the volume ratio of 1: 6, fully and uniformly mixing, and incubating in an ice bath for 30 min;
and 4, step 4: adding 20XPBS to adjust the erythrocyte membrane solution to 1X PBS, then centrifuging at 13800rpm for 10min, and removing hemoglobin in the supernatant;
and 5: repeating steps 3 and 4 until no hemoglobin is detected in the centrifuged supernatant;
step 6: the supernatant was removed to obtain red cell membranes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668772A (en) * | 2022-03-15 | 2022-06-28 | 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） | Composition loaded with amphotericin B bionic nano system, preparation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103857387A (en) * | 2011-06-02 | 2014-06-11 | 加利福尼亚大学董事会 | Membrane encapsulated nanoparticles and method of use |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103857387A (en) * | 2011-06-02 | 2014-06-11 | 加利福尼亚大学董事会 | Membrane encapsulated nanoparticles and method of use |
Non-Patent Citations (2)
| Title |
|---|
| ANDREAS REISCH ET AL.: "Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles", 《ACSNANO》 * |
| AUSTIN L. LAGROW ET AL.: "A Novel Biomimetic Nanosponge Protects the Retina from the Enterococcus faecalis Cytolysin", 《MSPHER》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668772A (en) * | 2022-03-15 | 2022-06-28 | 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） | Composition loaded with amphotericin B bionic nano system, preparation method and application |
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Inventor after: Chen Yijie Inventor after: Chen Mengchun Inventor after: Duan Ping Inventor after: Chen Sailing Inventor after: Wang Fang Inventor after: Yang Xuewei Inventor before: Chen Yijie Inventor before: Chen Mengchun Inventor before: Chen Sailing Inventor before: Wang Fang Inventor before: Yang Xuewei |