CN109646677B - Magnetic nano particle and method for preparing ocular hypertension animal model by using same - Google Patents
Magnetic nano particle and method for preparing ocular hypertension animal model by using same Download PDFInfo
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Abstract
The invention provides a magnetic nanoparticle and a method for preparing an ocular hypertension animal model by using the same. The invention is based on a magnetic nano particle with adjustable size, which has a multistage assembly structure and can be prepared by an emulsion polymerization method; when the animal model with high intraocular pressure is prepared, the intraocular pressure is introduced by anterior chamber injection, self-crosslinking or de-crosslinking can be adjusted according to the specific microstructure structure of the aqueous humor circulation path-trabecular meshwork/Schlemm's canal, a stable aqueous humor barrier layer is formed, the aqueous humor running channel is blocked in an omnibearing and multi-level manner, the aqueous humor reflux circulation is limited, the aqueous humor outflow resistance is increased, and the high intraocular pressure is maintained for a long time. The preparation method of the intraocular pressure animal model is simple, convenient, safe and long-acting.
Description
Technical Field
The invention relates to a magnetic nanoparticle and a method for preparing an ocular hypertension animal model by using the same, and belongs to the technical field of nano materials.
Background
The existing methods for preparing the glaucoma ocular hypertension animal model comprise peribulbar vein ligation method, dexamethasone method, anterior chamber gel injection method and anterior chamber magnetic bead injection method, and the methods can prepare the ocular hypertension animal model but have various problems. The general characteristics are poor intraocular pressure stability and low success rate of operation. The peribulbar vein ligation method can stimulate the blood vessels to compensate hyperplasia, bypass the ligated blood vessels, realize vein reflux and cause the failure of molding; dexamethasone can cause long-term biotoxicity of various tissues in eyes, increase complexity of problems and be not beneficial to accurate investigation of single problems; the gel method has short time for maintaining the ocular hypertension, and has limited deep investigation effect on long-term ocular hypertension diseases; the particle size of the magnetic beads selected by the anterior chamber magnetic bead injection method is about 3 mu m, the magnetic beads can be only enriched at the position of the small beam network of the chamber angle and cannot penetrate into the internal tissue of the aqueous humor operating channel, the basic properties of the used magnetic beads have no adjustability and cannot be well adapted to a specific microstructure, the stability and the durability of the intraocular pressure are not ideal, and the success rate of the intraocular pressure of an animal model is only less than one third. Therefore, the preparation of an ocular hypertension animal model capable of stably maintaining ocular hypertension for a long time has become a first priority for research related to ocular hypertension pathogenesis and drug screening.
Disclosure of Invention
Aiming at the problems, the invention provides a magnetic nanoparticle and a method for preparing an ocular hypertension animal model by using the magnetic nanoparticle. The invention is based on a size-adjustable magnetic nanoparticle which has a multistage assembly structure, and is characterized in that a magnetic nanoparticle dispersion liquid is introduced into eyes in an anterior chamber injection mode, under the guidance of a magnet, the magnetic nanoparticle deeply circulates to each stage of tissue of an aqueous humor operation channel along with aqueous humor, the particle size of the magnetic nanoparticle is adjusted according to the specific microstructure structure of the aqueous humor circulation channel, namely trabecular meshwork/Schlemm's canal, the aqueous humor circulation channel is blocked in an omnibearing and multilevel manner, the aqueous humor outflow resistance is increased, and the ocular hypertension is durably maintained. The particle size of the magnetic nanoparticles can be adjusted by cross-linking and de-cross-linking among particles, the dissociated nanoparticles move to a new position to be re-cross-linked with other particles, and the cross-linking and de-cross-linking processes among particles dynamically occur and finally tend to balance.
The invention firstly provides magnetic nanoparticles, wherein the particle size range of the magnetic nanoparticles is 100-1200 nm, and the magnetic nanoparticles are formed by assembling and crosslinking small-size magnetic nanoparticles;
the small-size magnetic nanoparticles have the particle size range of 10-100 nm and have a core-shell structure, the core is ferroferric oxide magnetic nanoparticles, the shell is a mesoporous silica nanolayer, and the mesoporous silica nanolayer is coated on the surface of the ferroferric oxide magnetic nanoparticles to protect the ferroferric oxide magnetic nanoparticles and provide better material biocompatibility; a mesoporous silica intermediate layer containing a fluorescent probe is arranged between the ferroferric oxide magnetic nanoparticles and the mesoporous silica nano layer, so that the in-vivo operation and tissue distribution of the magnetic nanoparticles can be observed and tracked through nuclear magnetic resonance or a fluorescence microscope in the following process; the magnetic nanoparticles have multi-modal tracking and detection characteristics, are beneficial to systematically evaluating the preparation method of the intraocular pressure animal model and are also beneficial to other related research works based on the intraocular pressure animal model;
the surface of the mesoporous silica nano layer is also provided with a polymer molecule modification layer, and small-size magnetic nanoparticles are assembled into magnetic nanoparticles through cross-linking reaction among polymer molecules; the polymer molecule comprises a siloxane group at one end and a sulfhydryl group at the other end; the polymer molecules are grafted to the surface of the mesoporous silica nano-layer through siloxane groups, intermolecular crosslinking is realized through a mercapto group at the other end, and then the assembly crosslinking of the small-size magnetic nano-particles is realized through intermolecular crosslinking of the polymer. The small molecule sulfhydryl reagent can generate oxidation reaction with the sulfhydryl group of the polymer to form disulfide bond, which can prevent the crosslinking reaction between the nano particles in a disulfide bond mode.
Preferably, the particle size range of the magnetic nanoparticles is 300-1000 nm, and the particle size range of the small-size magnetic nanoparticles is 20-80 nm. More preferably, the particle size range of the magnetic nanoparticles is 500-800 nm, and the particle size range of the small-size magnetic nanoparticles is 40-60 nm.
The polymer molecule has good biological safety and comprises a non-degradable polymer and a degradable polymer, wherein the non-degradable polymer comprises any one or any combination of polyethylene, polypropylene, polyacrylate, aromatic polyester, polysiloxane and polyformaldehyde, or any copolymerization of monomers or chain segments thereof; the degradable polymer comprises any one or any combination of collagen, gelatin, silk fibroin, chitosan, chitin, cellulose, polyamino acid, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer and polycaprolactone, or any copolymerization of monomers or chain segments thereof; preferably, the polymer is any one of polysiloxane, polyethylene glycol, polyamino acid, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer and polycaprolactone or any combination thereof, or any copolymerization of monomers or chain segments thereof; the molecular weight of the polymer is between 2000 and 20000.
The invention also provides a method for preparing an ocular hypertension animal model based on the magnetic nanoparticles, which comprises the following steps:
s1: preparation of small-sized magnetic nanoparticles
S11: from Fe3O4Nanoparticles to Fe3O4@SiO2(FITC)
Diluting the magnet powder-n-hexane dispersion in cyclohexane solution of n-octanol and polyethylene glycol octyl phenyl ether, adding ammonia water to form stable reverse microemulsion, adding ethyl silicate (TEOS) under continuous mechanical stirring for reaction to form magnetic nanoparticle dispersion coated with silicon dioxide, namely Fe3O4@SiO2A sample; in the above Fe3O4@SiO2Adding Fluorescein Isothiocyanate (FITC) fluorescent probe mother solution into the dispersion solution, stirring in dark condition, purifying by magnetic separation, washing with ethanol, and vacuum drying to obtain Fe3O4@SiO2(FITC) magnetic nanoparticles;
s12: from Fe3O4@SiO2(FITC) to Fe3O4@SiO2(FITC)@mSiO2
Fe3O4@SiO2Dispersing (FITC) magnetic nanoparticles in a mixed solution of Cetyl Trimethyl Ammonium Bromide (CTAB), deionized water and n-hexane, performing ultrasonic treatment, and uniformly dispersing; dropwise adding an ammonia water solution and TEOS into the mixed solution under continuous stirring, stirring under a dark condition, collecting a product through magnetic separation, and repeatedly washing with ethanol; refluxing the obtained sample in ethanol solution to remove CTAB template reagent, further washing with ethanol, and magnetically separating to obtain Fe3O4@SiO2(FITC)@mSiO2Magnetic nanoparticles;
s13: from Fe3O4@SiO2(FITC)@mSiO2To Fe3O4@SiO2(FITC)@mSiO2-
(P)-SH
mercapto-Polymer (P) -propyltrimethoxysilane added to Fe3O4@SiO2(FITC)@mSiO2Reacting in ethanol dispersion of magnetic nanoparticles, separating the- (P) -SH modified nanoparticles by magnetic separation method, and repeatedly washing and purifying with ethanol to obtain Fe3O4@SiO2(FITC)@mSiO2-(P)-SH；
S2: assembling and crosslinking small-size magnetic nanoparticles into magnetic nanoparticles
S21：Fe3O4@SiO2(FITC)@mSiO2the-SH groups which are abundantly present on the surface of the- (P) -SH magnetic nanoparticles form-S-S-through oxidation so as to realize chemical crosslinking among polymers and chemical crosslinking among nanoparticles to form the magnetic nanoparticles;
s22: the crosslinking reaction in the step S21 is carried out in a limited environment of emulsion polymerization, and the magnetic nanoparticles obtained by crosslinking have uniform size to form stable magnetic nanoparticle dispersion liquid;
s3: intraocular pressure animal model prepared from magnetic nanoparticles
The magnetic nanoparticles are injected into the anterior chamber of the eye in the form of aqueous dispersion, the magnetic nanoparticles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, so that the magnetic nanoparticles are enriched in tissues at all levels of the aqueous humor operation channel to prevent the aqueous humor from flowing back to systemic circulation, thereby causing ocular hypertension.
Furthermore, in the method for preparing the ocular hypertension animal model by using the magnetic nanoparticles, the crosslinking reaction capability of the small-size magnetic nanoparticles can be adjusted by adjusting the thiol density at the molecular chain end of the polymer on the surface of the small-size magnetic nanoparticles by using a small-molecule thiol reagent, so that the particle size and the particle size distribution of the small-size magnetic nanoparticles and the stable dispersion in the dispersion liquid can be accurately adjusted and controlled under the dual control of the crosslinking action and the magnetic mutual repulsion action of the surface polymer molecules of the small-size magnetic nanoparticles. Preparing magnetic nano particles with uniform particle size by an emulsion polymerization method, terminating a crosslinking reaction by a small molecule sulfhydryl reagent, centrifugally separating, and re-dispersing to form a stable magnetic nano particle dispersion liquid.
Further, in the method for preparing the intraocular pressure animal model by using the magnetic nanoparticles, after the magnetic nanoparticles are enriched in the trabecular meshwork, the local concentration of the magnetic nanoparticles is increased, and the particles can further generate a crosslinking reaction to form the magnetic nanoparticles with larger particle size or a complete blocking layer; part of the magnetic nano particles can be subjected to crosslinking release to release a large amount of small-size magnetic nano particles, the small-size magnetic nano particles circulate to Schlemm's canal or venous capillary along with aqueous humor and are enriched at a proper position, and the enriched small-size magnetic nano particles can be subjected to cross linking among particles again due to the increase of local density to form an omnibearing, multi-layer and relatively stable-structure nano particle barrier film for maintaining ocular hypertension for a long time.
Further, in the method for preparing an animal model of ocular hypertension by using the magnetic nanoparticles, the dispersion liquid of the magnetic nanoparticles can be used for preparing the animal model of ocular hypertension alone; can also be used as a carrier material to further load a drug capable of promoting the increase of intraocular pressure so as to prepare an intraocular pressure animal model with higher intraocular pressure.
The preparation method of the intraocular pressure animal model has the advantages of simplicity, convenience, safety, long-acting effect and the like:
(1) the preparation of the ocular hypertension animal model by the anterior chamber injection method is a universal method for preparing the ocular hypertension animal model at present, and the method has the advantages that as the nano particles have the characteristic of adjustable particle size, the prepared aqueous humor outflow resistance is durable and stable, the target can be realized by one-time injection without multiple injections;
(2) the method is used for preparing an ocular hypertension animal model based on magnetic nanoparticles with adjustable particle sizes, can adjust the self-crosslinking degree and the particle size according to a specific aqueous humor operating environment, can realize aggregation in multi-stage tissues such as trabecular meshwork, Schlemm's canal, venous blood vessels and the like, and can plug aqueous humor circulation channels in an omnibearing and multi-level manner, and the plugging mechanism system is complete;
(3) in the ocular hypertension animal model prepared by the method, the resistance positions of the magnetic nanoparticles for blocking the aqueous humor are not in the range of the anterior chamber, so that the visual field and the tissue structure in the anterior chamber are not influenced, and various subsequent experiments based on the ocular hypertension animal model are not influenced;
(4) the magnetic nano particles adopted by the method can rapidly enter a preset position under the guidance of the annular magnet to establish functions; or can be rapidly de-crosslinked and de-functionalized under the action and guidance of a reducing agent and an external magnetic field.
Drawings
FIG. 1: preparation of small-sized magnetic nanoparticles and surface modification schematic (the polymer molecule is polyethylene glycol)
FIG. 2: SEM and TEM photographs of small-sized magnetic nanoparticles (a), and magnetic hysteresis loop (b)
FIG. 3: schematic diagram of aggregation and crosslinking of small-size magnetic nanoparticles into magnetic nanoparticles
FIG. 4: schematic diagram of multi-level (trabecular meshwork, Schlemm's canal and venous capillary) blocked aqueous humor passage by magnetic nanoparticles under guidance of magnet
FIG. 5: distribution of magnetic nanoparticles in tissues such as trabecular meshwork, Schlemm's canal and venous capillary
Abbreviations and abbreviations in the figures:
TEOS: tetra ethyl ortho silicate: tetraethoxysilane
APTS 3-Aminopropylriethoxysilane: 3-aminopropyltriethoxysilane
FITC: fluoroescein isothiocyant: fluorescein isothiocyanate
Octanol: octanol (I)
Trixton X-100: polyethylene glycol octyl phenyl ether
NH3H2O: aqueous ammonia
CTAB: cetylltriethynylaminomnonium Bromide: cetyl trimethyl ammonium Bromide
n-hexane: n-hexane
Remove CTAB: removal of cetyl trimethyl ammonium bromide
Fe3O4magnetic nanoparticles: ferroferric oxide magnetic nano particle
Fe3O4@SiO2(FITC): ferroferric oxide @ silicon dioxide (FITC fluorescent probe) nano particle
Fe3O4@SiO2(FITC)@mSiO2: ferroferric oxide @ silicon dioxide (FITC fluorescent probe) @ mesoporous silicon dioxide nano particle
Fe3O4@SiO2(FITC)@mSiO2- (P) -SH: ferroferric oxide @ silicon dioxide (FITC fluorescent probe) @ mesoporous silicon dioxide-polymer (P) -sulfydryl nano particle
Fe3O4@SiO2(FITC)@mSiO2-PEG-SH: ferroferric oxide @ silicon dioxide (FITC fluorescent probe) @ mesoporous silicon dioxide-polyethylene glycol-sulfydryl nano particle
TM Trbecular Meshwork: beam net
SC Schlemm's Canal: schlemm's canal
CB: Ciliary Body: ciliary body
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
Example 1
The preparation of the magnetic nano-particles comprises the following steps:
s1: preparation of small-sized magnetic nanoparticles
5mL of the magnet powder-n-hexane dispersion (20mg/mL) was diluted in 10mL of n-octanol and 10mL of polyethylene glycol octylphenyl ether (texton X-100) in cyclohexane. To the above solution was added 0.5mL of ammonia (28 wt%) to form a stable reverse microemulsion. Then, 0.04mL of ethyl silicate (TEOS) was added under continuous mechanical stirring and the reaction was allowed to proceed for 24 hours to form a silica-coated magnetic nanoparticle dispersion, i.e., Fe3O4@SiO2And (3) sampling. Then, in the above Fe3O4@SiO2To the dispersion solution was added a mother solution of a fluorescent probe (0.02g of 3-Aminopropyltriethoxysilane (APTS), 0.01g of Fluorescein Isothiocyanate (FITC), 0.02mL of TEOS, 5mL of ethanol). After further stirring for 12 hours in the dark, the product was purified by magnetic separation, washed three times with ethanol and dried under vacuum at 30 ℃ overnight to yield Fe3O4@SiO2(FITC) magnetic nanoparticles;
s12: from Fe3O4@SiO2(FITC) to Fe3O4@SiO2(FITC)@mSiO2
Fe3O4@SiO2The (FITC) magnetic nanoparticles are dispersed in a mixed solution of cetyltrimethylammonium bromide (CTAB, 0.2g), deionized water (40mL) and n-hexane (3mL) and uniformly dispersed by ultrasonic treatment. An aqueous ammonia solution (0.4mL, 28 wt%) and 0.2mL TEOS were added dropwise to the above mixed solution with continuous stirring, stirring was continued for 12 hours under dark conditions, and the product was collected by magnetic separation and washed repeatedly with ethanol. Refluxing the obtained sample in 80 deg.C ethanol solution for 12 hr to remove CTAB template reagent, further washing with ethanol, and magnetically separating to obtain Fe3O4@SiO2(FITC)@mSiO2Magnetic nanoparticles;
s13: from Fe3O4@SiO2(FITC)@mSiO2To Fe3O4@SiO2(FITC)@mSiO2-(P)-SH
0.05g of mercapto-polyethylene glycol (P) -propyltrimethoxysilane was added to Fe3O4@SiO2(FITC)@mSiO2Ethanol dispersion of magnetic nanoparticles (20mL, 0.6 wt%). After the reaction is carried out for 12 hours, the nano particles modified by the- (P) -SH are separated by a magnetic separation method, and are repeatedly washed and purified by ethanol to obtain Fe3O4@SiO2(FITC)@mSiO2- (P) -SH; the polymer here was polyethylene glycol (PEG) with a molecular weight of 5000.
S2: assembly crosslinking of small-sized magnetic nanoparticles
S21：Fe3O4@SiO2(FITC)@mSiO2the-SH groups which are abundantly present on the surface of the- (P) -SH magnetic nanoparticles form-S-S-through oxidation so as to realize chemical crosslinking among polymers and chemical crosslinking among nanoparticles to form the magnetic nanoparticles;
s22: the crosslinking reaction in the step S21 is carried out in a limited environment of emulsion polymerization, and the magnetic nanoparticles obtained by crosslinking have uniform size to form stable magnetic nanoparticle dispersion liquid;
s3: intraocular pressure animal model prepared from magnetic nanoparticles
The magnetic nanoparticles are injected into the anterior chamber of the eye in the form of aqueous dispersion, the magnetic nanoparticles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, so that the magnetic nanoparticles are enriched in tissues at all levels of the aqueous humor operation channel to prevent the aqueous humor from flowing back to systemic circulation, thereby causing ocular hypertension.
The magnetic nanoparticles obtained by the method have the particle size of 800nm and are formed by assembling and crosslinking small-size magnetic nanoparticles with the particle size of 30 nm; the small-size magnetic nanoparticles have a core-shell structure, the core is ferroferric oxide magnetic nanoparticles, the shell is a mesoporous silica nanolayer, and the mesoporous silica nanolayer is coated on the surface of the ferroferric oxide magnetic nanoparticles; a mesoporous silica intermediate layer containing a FITC fluorescent probe is arranged between the ferroferric oxide magnetic nano particles and the mesoporous silica nano layer; the surface of the mesoporous silica nano layer is also provided with a polymer molecule modification layer, and the polymer molecule is polyethylene glycol; it has a molecular weight of 5000, and comprises siloxane group at one end and sulfhydryl group at the other end; the polymer molecules are grafted to the surface of the mesoporous silica nano-layer through siloxane groups, intermolecular crosslinking is realized through a mercapto group at the other end, and then the assembly crosslinking of the small-size magnetic nano-particles is realized through intermolecular crosslinking of the polymer.
An ocular hypertension animal model is prepared based on the magnetic nanoparticles, and the steps are injecting the magnetic nanoparticles into the anterior chamber of the eye in the form of aqueous dispersion with the dosage of 100 mu L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 10 weeks, and can be increased to 3 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 2
This example is substantially similar to example 1, except that: the polymer molecule is polyamino acid with molecular weight of 10000; the particle size of the small-size magnetic nanoparticles is 50nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 900 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 80 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 9 weeks, and can be increased to 4 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 3
This example is substantially similar to example 1, except that: the polymer molecule is polylactic acid, and the molecular weight is 8000; the particle size of the small-size magnetic nanoparticles is 70nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 600 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 60 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 8 weeks, and can be increased to 3 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 4
This example is substantially similar to example 1, except that: the polymer molecule is polylactic acid-glycolic acid copolymer, and the molecular weight is 15000; the particle size of the small-size magnetic nanoparticles is 80nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 1000 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 100 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 7 weeks, and can be increased to 5 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 5
This example is substantially similar to example 1, except that: the polymer molecule is polysiloxane, and the molecular weight is 6000; the particle size of the small-size magnetic nanoparticles is 10nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 100 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 80 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 4 times higher than normal intraocular pressure, and is stably kept in a certain intraocular pressure data interval for 4 weeks.
Example 6
This example is substantially similar to example 1, except that: the polymer molecule is polycaprolactone and has a molecular weight of 12000; the particle size of the small-size magnetic nanoparticles is 20nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 300 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 60 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 5 weeks, and can be increased to 2 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 7
This example is substantially similar to example 1, except that: the polymer molecule is polyethylene glycol with molecular weight of 2000; the particle size of the small-size magnetic nanoparticles is 40nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 500 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 100 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 6 weeks, and can be increased to 5 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 8
This example is substantially similar to example 1, except that: the polymer molecule is polysiloxane, and the molecular weight is 15000; the particle size of the small-size magnetic nanoparticles is 60nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 750 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 80 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 9 weeks, and can be increased to 4 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Example 9
This example is substantially similar to example 1, except that: the polymer molecule is polyamino acid, and the molecular weight is 20000; the particle size of the small-size magnetic nanoparticles is 100nm, and the particle size of the magnetic nanoparticles formed by assembling and crosslinking the small-size nanoparticles through an emulsion polymerization method is 1200 nm; the magnetic nanoparticles were injected into the anterior chamber of the eye as an aqueous dispersion at a dose of 60 μ L. The magnetic nano particles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, and are enriched in all levels of tissues of the aqueous humor operating channel. The intraocular pressure can be kept for 8 weeks, and can be increased to 4 times of normal intraocular pressure and stably kept in a certain intraocular pressure data interval.
Claims (4)
1. A method for preparing an ocular hypertension animal model based on magnetic nanoparticles, wherein the particle size range of the magnetic nanoparticles is 100-1200 nm, and the magnetic nanoparticles are assembled and crosslinked to form the ocular hypertension animal model; the particle size range of the small-size magnetic nanoparticles is 10-100 nm, the small-size magnetic nanoparticles have a core-shell structure, the core is ferroferric oxide magnetic nanoparticles, the shell is a mesoporous silica nanolayer, and the mesoporous silica nanolayer is coated on the surface of the ferroferric oxide magnetic nanoparticles; a mesoporous silica intermediate layer containing a fluorescent probe is arranged between the ferroferric oxide magnetic nanoparticles and the mesoporous silica nano layer, so that the in-vivo operation and tissue distribution of the magnetic nanoparticles can be observed and tracked through nuclear magnetic resonance or a fluorescence microscope in the following process; the surface of the mesoporous silica nano layer is also provided with a polymer molecule modification layer, and small-size magnetic nanoparticles are assembled into magnetic nanoparticles through cross-linking reaction among polymer molecules; the polymer molecule comprises a siloxane group at one end and a sulfhydryl group at the other end; polymer molecules are grafted to the surface of the mesoporous silica nano-layer through siloxane groups, intermolecular crosslinking is realized through a mercapto group at the other end, and then assembly crosslinking of the small-size magnetic nano-particles is realized through intermolecular crosslinking of the polymer; the method is characterized by comprising the following steps:
s1: preparation of small-sized magnetic nanoparticles
S11: from Fe3O4To Fe3O4@SiO2-FITC
Diluting the magnet powder-n-hexane dispersion in cyclohexane solution of n-octanol and polyethylene glycol octyl phenyl ether, adding ammonia water to form stable reverse microemulsion, adding ethyl silicate TEOS under continuous mechanical stirring to react to form magnetic nanoparticle dispersion coated with silicon dioxide, namely Fe3O4@SiO2A sample; in the above Fe3O4@SiO2Adding fluorescein isothiocyanate FITC fluorescent probe mother solution into the dispersion solution,stirring the mixture under the condition of keeping out of the sun, and performing magnetic separation and purification, ethanol washing and vacuum drying on the product to obtain Fe3O4@SiO2-FITC magnetic nanoparticles;
s12: from Fe3O4@SiO2FITC to Fe3O4@SiO2-FITC@mSiO2
Fe3O4@SiO2Dispersing FITC magnetic nanoparticles in a mixed solution of Cetyl Trimethyl Ammonium Bromide (CTAB), deionized water and n-hexane, and performing ultrasonic treatment to disperse uniformly; under the condition of continuous stirring, dropwise adding an ammonia water solution and TEOS into the mixed solution, stirring under the condition of keeping out of the sun, collecting a product through magnetic separation, and repeatedly washing with ethanol; refluxing the obtained sample in ethanol solution to remove CTAB template reagent, further washing with ethanol, and magnetically separating to obtain Fe3O4@SiO2-FITC@mSiO2Magnetic nanoparticles;
s13: from Fe3O4@SiO2-FITC@mSiO2To Fe3O4@SiO2-FITC@mSiO2-P-SH
Adding mercapto-polymer P-propyltrimethoxysilane to Fe3O4@SiO2-FITC@mSiO2Reacting in ethanol dispersion of magnetic nanoparticles, separating-P-SH modified nanoparticles by magnetic separation method, and repeatedly washing and purifying with ethanol to obtain Fe3O4@SiO2-FITC@mSiO2-P-SH；
S2: assembling and crosslinking small-size magnetic nanoparticles into magnetic nanoparticles
S21：Fe3O4@SiO2-FITC@mSiO2the-SH groups which are abundantly present on the surface of the-P-SH magnetic nanoparticles form-S-S-through oxidation so as to realize chemical crosslinking among polymers and chemical crosslinking among nanoparticles to form the magnetic nanoparticles;
s22: the crosslinking reaction in the step S21 is carried out in a limited environment of emulsion polymerization, and the magnetic nanoparticles obtained by crosslinking have uniform size to form stable magnetic nanoparticle dispersion liquid;
s3: intraocular pressure animal model prepared from magnetic nanoparticles
The magnetic nanoparticles are injected into the anterior chamber of the eye in the form of aqueous dispersion, the magnetic nanoparticles are guided by the annular magnet to gather at the chamber angle, and gradually disperse in the trabecular meshwork, Schlemm's canal and venous capillary along with the circulation of aqueous humor, so that the magnetic nanoparticles are enriched in tissues at all levels of the aqueous humor operation channel to prevent the aqueous humor from flowing back to systemic circulation, thereby causing ocular hypertension.
2. The method for preparing an animal model with ocular hypertension from magnetic nanoparticles as claimed in claim 1, wherein the crosslinking reactivity of the small-sized magnetic nanoparticles can be adjusted by adjusting the thiol density at the molecular chain end of the polymer on the surface of the small-sized magnetic nanoparticles with a small-molecule thiol reagent, so that the particle size and particle size distribution of the small-sized magnetic nanoparticles can be precisely controlled under the dual control of the crosslinking action and the magnetic mutual repulsion action of the surface polymer molecules, and the small-sized magnetic nanoparticles can be stably dispersed in the dispersion liquid.
3. The method for preparing an animal model with ocular hypertension by using magnetic nanoparticles as claimed in claim 1, wherein after the magnetic nanoparticles are enriched in trabecular meshwork, the local concentration of the magnetic nanoparticles is increased, and further cross-linking reaction can occur between the particles to form magnetic nanoparticles with larger particle size or a complete blocking layer; part of the magnetic nano particles can be subjected to crosslinking release to release a large amount of small-size magnetic nano particles, the small-size magnetic nano particles circulate to Schlemm's canal or venous capillary along with aqueous humor and are enriched at a proper position, and the enriched small-size magnetic nano particles can be subjected to cross linking among particles again due to the increase of local density to form an omnibearing, multi-layer and relatively stable-structure nano particle barrier film for maintaining ocular hypertension for a long time.
4. The method for preparing an animal model of ocular hypertension using magnetic nanoparticles according to claim 1, wherein the dispersion of magnetic nanoparticles can be used alone for preparing an animal model of ocular hypertension; can also be used as a carrier material to further load a drug capable of promoting the increase of intraocular pressure so as to prepare an intraocular pressure animal model with higher intraocular pressure.
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