CN114796157B - Fluorinated nanocapsules, preparation method and application thereof - Google Patents
Fluorinated nanocapsules, preparation method and application thereof Download PDFInfo
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- CN114796157B CN114796157B CN202210514404.7A CN202210514404A CN114796157B CN 114796157 B CN114796157 B CN 114796157B CN 202210514404 A CN202210514404 A CN 202210514404A CN 114796157 B CN114796157 B CN 114796157B
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- 238000002360 preparation method Methods 0.000 title abstract description 6
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Classifications
-
- 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
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Optics & Photonics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Physics & Mathematics (AREA)
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- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
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Abstract
The invention relates to a fluorinated nanocapsule, a preparation method and application thereof. The fluorinated nanocapsule is of a core-shell structure, wherein an inner core in the core-shell structure is a nucleic acid medicine, and an outer shell in the core-shell structure is an acrylic ester/acrylamide high polymer with a cross-linked structure and containing fluorocarbon alkyl chain blocks. The fluorinated nanocapsule is prepared by the following method: (1) Fully mixing the nucleic acid medicine, the polymerization reaction monomer and the perfluorinated acrylate to enable the polymerization reaction monomer and the nucleic acid medicine to be aggregated; (2) And (3) adding ammonium persulfate and N, N, N ', N' -tetramethyl ethylenediamine into the step (1) to initiate free radical polymerization, so as to obtain the fluorinated nanocapsule. The obtained fluorinated nanocapsules can provide good gastrointestinal stability for oral delivery of nucleic acids, and have excellent effects of crossing a mucus layer and promoting absorption of intestinal epithelial cells.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a fluorinated nanocapsule, a preparation method and application thereof.
Background
With the rapid development of biotechnology, many highly advantageous nucleic acid drugs have been widely used in major diseases such as tumor, inflammation, cardiovascular diseases, etc. Compared with the conventional medicines, the medicines have high specificity and less adverse reaction, but also have a plurality of limitations. Nucleic acid drugs have poor stability in vivo circulation and are easily inactivated by the influence of acid and alkali in vivo and enzyme, so that most of clinical injections are mainly used. The frequent administration for a long time causes huge body injury and mental confusion to patients, so the research of the non-injection administration mode of the medicine has profound significance. In clinical experience, oral administration is the most favored mode of administration because of its simple operation and good patient compliance. In oral delivery, the abundant mucus on the small intestine and small intestine epithelial cells are two large physiological barriers through which drugs are required to be absorbed, and most of nucleic acid drugs are low in fat solubility and large in molecular weight, and are difficult to enter the blood circulation through the small intestine epithelial cells, so that the clinical development of the drugs is severely restricted.
Nano-carriers such as chitosan (Advanced Healthcare Materials,2018: 1800285), hydrogel (Biomacromolecules, 2015,16 (3): 962-72), liposome (Nature reviews. Drug discovery,2007,6 (3): 231-48) and the like have been demonstrated to enhance the oral absorption efficiency of macromolecular drugs such as nucleic acids, and among these carriers, the endocytosis of small intestinal epithelial cells to drugs has been enhanced by means of interactions between ligands (Science Translational Medicine,2013,5 (213): 213-167), electrostatic interactions between cations and cell membranes (Chemical Society Reviews,2011,40 (1): 233-45) and the like. However, for oral delivery systems, the mucus layer barrier above the intestinal cells should also be an important consideration, as positively charged carriers tend to adsorb negatively charged mucins in the mucus layer causing agglomeration, which results in poor drug contact with the intestinal epithelial cells. The use of polyglycol modification to enhance carrier hydrophilicity has become an important means of enhancing mucus layer penetration (ACS Nano,2015,9 (9): 9217-27). Patent application CN 107335059 discloses a polymer named DSPE-PEG as an oral absorption enhancer, and polyethylene glycol polymer chains contained in the polymer can effectively avoid mucin adsorption and promote the permeation of a mucus layer of a carrier. Nevertheless, the addition of polyethylene glycol can hinder the interaction between the carrier and the cell membrane, reducing the endocytic capacity of the intestinal epithelial cells.
Disclosure of Invention
The invention provides an oral fluorinated nanocapsule for nucleic acid drugs, and a preparation method and application thereof, in order to solve the technical problem that the performance of promoting mucus layer permeation and small intestine epithelial cell absorption cannot be simultaneously considered. The invention provides a fluorine-containing nano-capsule for oral delivery, which is constructed by initiating free polymerization on the surface of a biomacromolecule drug (nucleic acid), and the obtained nano-gel can provide good gastrointestinal stability for the oral delivery of the nucleic acid and has excellent effects of crossing a mucus layer and promoting absorption of intestinal epithelial cells.
The first object of the present invention is to provide a fluorinated nanocapsule, which has a core-shell structure, wherein the inner core of the core-shell structure is a nucleic acid drug, and the outer shell of the core-shell structure is an acrylic ester/acrylamide polymer having a cross-linked structure and containing a fluorocarbon alkyl chain block.
The second object of the present invention is to provide a method for preparing the fluorinated nanocapsules, comprising the steps of:
(1) Fully mixing the nucleic acid medicine, the polymerization reaction monomer and the perfluorinated acrylic acid to enable the polymerization reaction monomer and the nucleic acid medicine to be aggregated;
(2) And (3) adding ammonium persulfate and N, N, N ', N' -tetramethyl ethylenediamine into the step (1) to initiate free radical polymerization, so as to obtain the fluorinated nanocapsule.
In one embodiment of the present invention, in step (1), the nucleic acid-based drug may include one or more of siRNA, miRNA or DNA.
In one embodiment of the invention, the nucleic acid agent is selected from the group consisting of siRNA.
In one embodiment of the present invention, in the step (1), the concentration of the nucleic acid drug is 1 to 30. Mu. Mol/L.
In one embodiment of the present invention, in step (1), the polymerization monomer is selected from one or more of N- (3-aminopropyl) methacrylamide, N' -bis (acryl) cystamine and perfluoroacrylic acid.
In one embodiment of the present invention, in step (1), the perfluorinated acrylate is selected from one or a combination of compounds as follows:
in one embodiment of the present invention, in step (1), the molar ratio of the nucleic acid drug to the crosslinking agent is 1: (100-3000); the molar ratio of the N- (3-aminopropyl) methacrylamide to the perfluorinated acrylic acid is (1-4): (2-8).
In one embodiment of the present invention, in the step (2), the ammonium persulfate concentration is 0.1 to 10mg/mL; the concentration of the N, N, N ', N' -tetramethyl ethylenediamine is 0.1-10 mg/mL.
In one embodiment of the invention, in step (2), the mixing reaction time is 0.5 to 3 hours and the reaction temperature is 0 to 10 ℃.
In one embodiment of the present invention, in step (2), the pH of the reaction mixture solution is 8 to 10.
Compared with the prior art, the technical scheme of the invention has the following advantages:
1. the nanocapsule is prepared by in-situ polymerization after the monomer is aggregated on the surface of the drug, and the polymer shell-shaped structure formed on the surface of the nanocapsule can keep the drug stable when the drug passes through the gastrointestinal tract.
2. Compared with a gel system obtained by conventional polymerization, the nanocapsule has smaller particle size, is easier to penetrate through a mucin network structure in a mucin layer, and the perfluorocarbon chain on the surface of the nanocapsule can reduce non-specific adsorption between a carrier and mucin, so that the capability of the carrier to penetrate through the mucin layer is greatly improved under the advantages of small particle size and non-adsorption.
3. The nanocapsule surface has certain density of electropositivity and is modified with a perfluorinated carbon chain which can help to penetrate cell membranes, so that the nanocapsule has good endocytosis capability, and the transport effect of the drug in the epithelium of the small intestine is effectively improved; the reversible crosslinking structure of the nanocapsule has good biological responsiveness, and can enable the medicine to be smoothly released in cells, so that the medicine can successfully act on target cells.
4. The overall preparation process is simple and efficient, and has wide application prospect.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 shows the particle size and potential of nanocapsules in examples and comparative examples of the present invention.
FIG. 2 is an electron micrograph of nanocapsules in examples and comparative examples of the present invention.
FIG. 3 is a diagram showing transfection of nanocapsules in RAW264.7 cells according to test example 1 of the present invention.
FIG. 4 is a graph showing the multi-particle trace of nanocapsules in mucus in test example 2 of the present invention.
FIG. 5 is a graph showing percentage adsorption of nanocapsules to different mass fractions of mucin in test example 2 according to the present invention.
FIG. 6 is a graph showing the results of experiment on the single layer transport of nanocapsules in Caco-2 cells in experimental example 2 of the present invention.
FIG. 7 is a graph showing the results of experiments on the expression level of TNF-. Alpha.mRNA in liver tissue after completion of treatment in a liver injury model of a mouse according to test example 3 of the present invention.
FIG. 8 is a graph showing the experimental results of the content of glutamic oxaloacetic transaminase in serum after the treatment of the liver injury model of the mouse of experimental example 3 of the present invention.
FIG. 9 is a graph showing the experimental results of the glutamic pyruvic transaminase content in serum after the treatment of the liver injury model of the mouse of the experimental example 3.
FIG. 10 shows the survival rate of mice in test example 3 after the end of liver injury treatment.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Examples
1OD TNF-alpha siRNA (sense strand: 5'-GUCUCAGCCUCUUCUCAUUCCUGCT-3', antisense strand: 5 '-AGCAGGAAmUGmAAmGAGGmCUGAmGAACmAmU-3') lyophilized powder was dissolved to 20. Mu. Mol/L with DEPC water (125. Mu.L), then transferred to a two-necked flask, and the flask was placed in an ice-water bath. An aqueous solution of N- (3-aminopropyl) methacrylamide (3.56. Mu.L, 4. Mu. Mol) was then added. After mixing, the siRNA concentration in the solution was adjusted to 5. Mu. Mol/L with RNase-inactivated deionized water and the flask was evacuated. After 15 minutes, a solution of 1H, 2H-octyl perfluoroacrylate (25. Mu.L, 2. Mu. Mol) in tetrahydrofuran and a solution of N, N' -bis (acryl) cystamine (6.5. Mu.L, 2.5. Mu. Mol) in dimethyl sulfoxide were added thereto, and thoroughly mixed. Under the protection of nitrogen, 5 mu L of ammonium persulfate solution with the mass fraction of 1% and 2 mu L of N, N, N ', N' -tetramethyl ethylenediamine solution with the mass fraction of 5% are added into a bottle to initiate free radical polymerization. After magnetically stirring for 2 hours, the obtained solution is placed in a 10-kDa dialysis bag, dialyzed in phosphate buffer solution for 24 hours at 4 ℃ and unreacted monomers are removed, thus obtaining the fluorine-containing modified siRNA nanocapsules. The particle size and potential of the nanocapsules of the obtained examples are shown in FIG. 1, and the electron microscope chart is shown in FIG. 2.
Comparative example
1OD TNF-alpha siRNA (sense strand: 5'-GUCUCAGCCUCUUCUCAUUCCUGCT-3', antisense strand: 5 '-AGCAGGAAmUGmAAmGAGGmCUGAmGAACmAmU-3') lyophilized powder was dissolved to 20. Mu. Mol/L with DEPC water (125. Mu.L), then transferred to a two-necked flask, and the flask was placed in an ice-water bath. Then an aqueous solution of N- (3-aminopropyl) methacrylamide (8.9. Mu.L, 4. Mu. Mol) was added. After mixing, the siRNA concentration in the solution was adjusted to 5. Mu. Mol/L with RNase-inactivated deionized water and the flask was evacuated. After 15 minutes, a solution of N, N' -bis (acryl) cystamine (6.5. Mu.L, 2.5. Mu. Mol) in dimethyl sulfoxide was added thereto, and thoroughly mixed. Under the protection of nitrogen, 5 mu L of ammonium persulfate solution with the mass fraction of 1% and 2 mu L of N, N, N ', N' -tetramethyl ethylenediamine solution with the mass fraction of 5% are added into a bottle to initiate free radical polymerization. After magnetically stirring for 2 hours, the obtained solution is placed in a 10-kDa dialysis bag, dialyzed in phosphate buffer solution for 24 hours at 4 ℃ and unreacted monomers are removed, thus obtaining the siRNA nanocapsules without fluorine modification. The particle size and potential of the nanocapsules of the comparative example are shown in FIG. 1, and the electron microscope chart is shown in FIG. 2.
Test example 1
The nanocapsules containing TNF-alpha siRNA obtained in the examples and comparative examples were diluted with deionized water to a nanocapsule solution containing 25. Mu.g/mL siRNA. RAW264.7 cells were 1X 10 per well 4 The individual cells were inoculated into 96-well plates and cultured in DMEM medium containing 10% fbs for 24 hours. The siRNA nanocapsules described above were then added to the wells at a dose of 0.1. Mu.g siRNA per well and incubated at 37℃for 24 hours. After 4 hours of lipopolysaccharide stimulation at 7.5 ng/well, the cells were lysed to extract RNA and the gene expression levels of TNF- α in the cells were detected by reverse transcription kit and real-time PCR kit instructions. Knot(s)As shown in FIG. 3, the examples have better levels of gene silencing for TNF- α than the comparative examples.
Test example 2
The Brownian movement of the particles in the mucus is researched by a multiple particle tracking method, and the wider the movement track range is, the smaller the resistance of the particles in the mucus is, and the easier the particles penetrate through the mucus layer. FITC-labeled examples and comparative nanocapsules were added to the mucus, then transferred to a small petri dish and equilibrated for 30 minutes at 37 ℃. A confocal fluorescence microscope was used to obtain the motion profile of the nanocapsules moving in 1 second in mucus. Analysis was then performed using Imaris software and the time-average mean square displacement for each trace was calculated. The results are shown in fig. 4, and the mean square displacement of the movement track of the example in the mucus is larger than that of the comparative example in the same time, which shows that the addition of the fluorine alkane chain increases the migration rate of the nanocapsule in the mucus.
FITC-labeled examples and comparative nanocapsules were dispersed in 0.1%,0.3%,0.5% by mass of mucin solution, respectively, and vortexed and incubated at room temperature in a shaker. After 30 minutes, the mixture was centrifuged at 1500rpm for 2 minutes and the pellet was washed twice with PBS. 200 mu LNaOH (5 mol/L) was then added to the precipitate for treatment; finally, the fluorescence intensity is measured by a fluorescence spectrometer. The results are shown in FIG. 5, where the adsorption strength of the examples to mucin is smaller than that of the comparative examples while maintaining the same mass fraction of mucin. Further, the addition of the fluoroalkane chain helps to enhance the ability of the nanocapsule to adsorb anti-mucin.
In vitro modeling of small intestine epithelial cell transport, caco-2 cells were first cultured at 5×10 4 Cell/well density was seeded into the topside of the Transwell and then cultured for 17-21 days to form a monolayer of cells. Changing the culture medium on the top and outer sides every day until the transepithelial resistance (TEER) of the monolayer reaches 450-550 Ω/cm 2 And then the model establishment is completed. Before the beginning of the formal experiment, the medium on the top and bottom sides of the Transwell was replaced with Hanks balanced salt buffer. After the system was equilibrated for 0.5 hours, FITC-labeled examples and comparative nanocapsules were topped with an addition of 1. Mu.g siRNA per wellIncubation was performed for 4 hours. Repeating the above operation steps, adding 1% of mucin by mass into Hanks balanced salt buffer solution on the top side of the Transwell, adding FITC-labeled example and comparative example nanocapsules into the top test, and incubating for 4 hours. Finally, 50. Mu.L of the buffer solution in the bottom side was taken, the fluorescence intensity thereof was detected by an enzyme-labeled instrument, and the apparent permeation system (P app ) The formula is P app =q/Act. Where Q is the total amount of siRNA permeated (ng) and A is the diffusion area of the cell monolayer (cm 2 ) C is the initial concentration of top siRNA (ng/cm 3) and t is the time of penetration. The results are shown in FIG. 6, for P in nanocapsules of examples and comparative examples in the absence of mucin app The values were not significantly different. However, in the presence of mucin, example P app The values are significantly higher than for the comparative examples. In conclusion, the addition of the fluoroalkyl chain can improve the cell transport capacity of the nanocapsule in the presence of the mucus layer.
Test example 3
Nanocapsules containing TNF-alpha siRNA as described in examples and comparative examples were orally gavaged to male C57BL/6 mice at a dose of 200. Mu.g/kg siRNA (6 mice per group), and non-dosed mice served as control groups. Twenty-four hours after administration, mice were induced to have acute liver injury by intraperitoneal injection of lipopolysaccharide (12.5 μg/kg) and galactosamine (1.25 g/kg). Five hours after induction, mice were harvested for blood from their eyeballs and serum levels of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were determined using commercial kits. Mice were then sacrificed, livers of the mice were removed, homogenized, RNA extracted, and TNF- α mRNA levels in tissue cells were detected by reverse transcription kit and real-time PCR kit instructions. The relative levels of TNF- α mRNA in liver tissue are shown in FIG. 7, respectively, and it can be seen that the examples have higher levels of silencing of the TNF- α gene than the comparative examples; as can be seen from FIGS. 8 and 9, the effect of the examples on the reduction of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase was superior to that of the comparative examples.
The nanocapsules containing TNF-. Alpha.siRNA described in examples and comparative examples were orally administered to male C57BL/6 mice at a dose of 200. Mu.g/kg siRNA (10 mice per group), and mice orally administered with phosphate were used as control groups. Mice were intraperitoneally injected with lipopolysaccharide (12.5. Mu.g/kg) and galactosamine (1.25 g/kg) to induce acute liver injury, and the non-induced group served as a blank group. Survival of the four groups of mice was then monitored in real time over 24 hours. As can be seen in fig. 10, the mice in the control group all died during the 24-hour observation period, and the survival rate of the mice in the group of oral example nanocapsules was 30% during 24 hours, which is higher than that of the group of oral comparative example nanocapsules. As can be seen from the above, the examples can inhibit the expression of inflammatory factor TNF-alpha in the acute liver injury model of mice more than the comparative examples, and can more effectively improve the survival rate of mice, and have better treatment efficacy on acute liver injury.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (7)
1. The fluorinated nanocapsule is characterized by being of a core-shell structure, wherein an inner core in the core-shell structure is a nucleic acid medicine, and an outer shell in the core-shell structure is an acrylic ester/acrylamide high-molecular polymer with a cross-linked structure and containing a fluorocarbon alkyl chain block;
the fluorinated nanocapsule is prepared by the following method:
(1) Fully mixing the nucleic acid medicine, the polymerization monomer, the cross-linking agent and the perfluorinated acrylate to enable the polymerization monomer and the nucleic acid medicine to be aggregated;
(2) Adding ammonium persulfate and N, N, N ', N' -tetramethyl ethylenediamine into the step (1) to initiate free radical polymerization, so as to obtain the fluorinated nanocapsule;
the polymerization monomer is selected from N- (3-aminopropyl) methacrylamide;
the cross-linking agent is N, N' -bis (propionyl) cystamine;
the perfluoroacrylate is selected from one or more of the following compounds:
。
2. the fluorinated nanocapsule of claim 1, wherein in step (1), the nucleic acid drug is selected from one or more of siRNA, miRNA, and DNA.
3. The fluorinated nanocapsule of claim 1, wherein in step (1), the concentration of the nucleic acid drug is 1-30 μmol/L.
4. The fluorinated nanocapsule of claim 1, wherein in step (1), the molar ratio of the nucleic acid drug to the crosslinking agent is 1: (100-3000); the molar ratio of the polymerization monomer to the perfluorinated acrylate is (1-4): (2-8).
5. The fluorinated nanocapsule of claim 1, wherein in step (2), the ammonium persulfate concentration is 0.1-10 mg/mL; the concentration of the N, N, N ', N' -tetramethyl ethylenediamine is 0.1-10 mg/mL.
6. The fluorinated nanocapsule of claim 1, wherein in step (2), the mixing reaction time is 0.5 to 3 hours and the reaction temperature is 0 to 10 ℃.
7. The fluorinated nanocapsule of claim 1, wherein in step (2), the pH of the reaction mixture solution is 8-10.
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