KR20110115660A - Fluorescence nanoporous silica nanopaticle coated with lipid bilayer and method of preparing the same - Google Patents

Abstract

The present invention relates to a fluorescent porous silica nanoparticles coated with a lipid bimolecular film and a method of manufacturing the same, and more particularly, to form a ionic polymer thin film on the surface of the porous silica nanoparticles, and then to the lipid vapor The present invention relates to a fluorescently porous silica nanoparticle with increased biocompatibility prepared by forming a lipid bimolecular film using a lipid vesicle, and a method of manufacturing the same. Fluorescent porous silica nanoparticles coated with a lipid bimolecular film according to the present invention can be provided for bioanalysis such as bioimaging, drug delivery. In particular, it is very useful because it can be used in the production of high sensitivity biosensors for detecting or analyzing various phenomena between biological materials by using excellent biocompatibility with the optical detection method.

Description

Fluorescence nanoporous Silica Nanopaticle Coated with Lipid Bilayer and Method of Preparing the Same}

The present invention relates to a fluorescent porous silica nanoparticles coated with a lipid bimolecular film and a method of manufacturing the same, and more particularly, to form a ionic polymer thin film on the surface of the porous silica nanoparticles, and then to a lipid vapor containing fluorescent lipid particles. The present invention relates to a fluorescently porous silica nanoparticle with increased biocompatibility prepared by forming a lipid bimolecular film using a lipid vesicle, and a method of manufacturing the same.

Nanoporous silica colloidal particles are well-defined nanostructure systems that self-assemble nanoscale devices into larger hierarchical structures, very complex shapes commonly found in living systems. Since the study of the first ordered mesoporous silica was reported in 1992, much research has been conducted on its synthesis, analysis and application. Nanoparticles with regularly ordered pore structure, large surface area and pore volume are known to be very useful materials for biotechnology such as delivery of drugs, enzymes, DNA, etc. The mesoporous silica nanoparticles are expected to be used as multifunctional materials such as light emission, magnetic force, cell display, and therapeutic function.

Colloidal nanoparticles, on the other hand, have been widely studied as probes in biomedical imaging because of their unique sized electronic, optical, and magnetic properties. For example, magnetic nanoparticles have been used as T2 MRI contrast enhancers, semiconductor nanoparticles (quantum dots) have been used as luminescent probes in peso display, tracking, and imaging, and gold nanoparticles have been used for cDNA sensing. In addition, the possibility of using mesoporous silica with a large pore volume as a drug delivery medium has been studied.

Recently, the Prasad group reported on a multifunctional nanoprobe that simultaneously implants quantum dots and magnetic nanoparticles into a silica shell to image cancer cells. They also made magnetic nanoparticles functionalized with dyes bio-friendly and adjusted to magnetic properties to show cell markings with optical traces.

The research team at Seoul National University synthesized multifunctional nanomaterials with magnetic nanocrystals (Fe ₃ O ₄ -MSN) on the surface of silica nanoparticles doped with uniform mesoporous dyes and applied them to MRI, luminescence imaging, and drug delivery media. . The formation of many magnetic nanocrystals on the silica surface enhances the magnetic properties that enhance the MR signal, and the small dye molecules in the silica serve as optical imaging. In addition, drugs to treat cancer can be loaded into porous silica nanoparticles and used for drug delivery.

By attaching other types of nanostructures to porous silica nanoparticles in a similar way, they can be applied elsewhere, and the present nanocomposites provide multifunctional nanostructures.

Fluorescent organic dyes, for example, can be physically encapsulated in self-sealing nanochannels at micron sizes of up to 30 nm. Nanoscale-constrained fluorescent organic dyes in silica substrates can produce silica particles that emit very bright fluorescence. It can emit up to 260 times brighter than the brightest possible colloidal agglomeration of quantum dots of the same size. By combining with other molecules, it is also possible to create microdevices / microparticles that can respond complexly to external stimuli.

On the other hand, silica nanoparticles having a functionalized surface can transfer various DNA and chemicals to cells or skin of animals. However, in plants, the transfer of matter is restricted by the cell walls. To solve this problem, Torney et al., Iowa State University, conducted research to deliver DNA and chemicals to plant cells using honeycomb porous silica nanoparticles with a pore diameter of 3 nm. The team filled the dielectric and chemical derivatives with MSN and surfaced them with gold (Au) nanoparticles to prevent them from escaping. These porous silica nanoparticles can vary the size of the pores and can deliver chemicals of various functions, making them new possibilities for target site transport technologies such as proteins, nucleotides, and chemicals, which are needed for plant biotechnology. Is opening up. In addition, the combination with other porous silica nanoparticles and materials having different nanostructures is expected to be developed as a multifunctional nanomedical structure capable of simultaneously imaging or diagnosing and treating various materials.

Therefore, in order to use biologically and medically a wide range of porous silica nanoparticles which are expected to be used for such various purposes, the increase of biocompatibility and the change of the activity of the biomolecule bound by the surface may reduce the "substrate effect". There has been a need to develop a porous silica nanoparticle composite having a new structure.

Accordingly, the present inventors have made diligent efforts to develop a multifunctional nanostructure that can be used for imaging or diagnosis. As a result, the polymer compound is repeatedly coated on the surface of the porous silica nanoparticles to form a polymer thin film layer, and a lipid molecule including a fluorescent lipid molecule. The nanostructures of the new structure are prepared by self-assembly, and these nanostructures are not only excellent in biocompatibility but also a new structure that can reduce the "substrate effect" and confirm the fluorescence with excellent intensity. The invention has been completed.

An object of the present invention is to provide a nanostructure of a novel structure using porous silica nanoparticles that can be used in various fields such as imaging or drug delivery.

In order to achieve the above object, the present invention provides a method for producing fluorescent porous silica nanoparticles coated with a lipid bimolecular film comprising the following steps:

(a) preparing an ionic polymer thin film layer on the surface of the porous silica nanoparticles; And

(b) forming a lipid bimolecular film on the surface of the porous silica nanoparticles coated with the ionic polymer thin film layer with a lipid vesicle including fluorescent lipid particles.

The present invention also provides fluorescent porous silica nanoparticles coated with a lipid bimolecular film prepared by the above method.

The present invention also provides a method for labeling a biomaterial using fluorescent porous silica nanoparticles coated with the lipid bimolecular film.

The present invention has the effect of providing a fluorescent porous silica nanoparticles coated with a lipid bimolecular film and a preparation thereof. Fluorescent porous silica nanoparticles coated with a lipid bimolecular film according to the present invention can be provided for bioanalysis, such as bioimaging, drug delivery by fixing the fluorescent lipid molecules to fluoresce, various phenomena in the biological material It is very useful because it can be used to manufacture a biosensor for optically detecting or analyzing optically.

1 is a schematic diagram of an ionic polymer thin film layer and a lipid bimolecular film coated on porous silica nanoparticles according to the present invention.
Figure 2 is a graph showing the results of measuring the surface charge of the porous silica nanoparticles coated with an ionic polymer thin film layer through a particle size analyzer.
FIG. 3 is a photograph showing an image of the surface of the porous silica nanoparticles coated with the ionic polymer thin film layer of FIG. 2 observed with a transmission electron microscope (TEM).
4 is a photograph showing an optical microscope image of a lipid vesicle prepared to form a lipid bimolecular film on porous silica nanoparticles coated with an ionic polymer thin film layer.
Figure 5 is a photograph showing the image observed by optical microscopy of the fluorescent porous silica nanoparticles coated with a lipid bimolecular film.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

As used herein, "porous silica nanoparticles" are silica nanostructures having fine pore sizes of several nanometers to several micrometers, and have a well-defined regularity of pore arrays and material properties (pore size, ratio). Surface area, surface characteristics) can be adjusted, it is also called mesoporous silica.

As used herein, "biomaterial" refers to a biologically derived material such as proteins, enzymes, antibodies, peptides, lipids, DNA, RNA and PNA.

In one aspect, the present invention relates to a fluorescent porous silica nanoparticles coated with a lipid bimolecular film, the method of manufacturing the method comprising the following steps:

(a) preparing an ionic polymer thin film layer on the surface of the porous silica nanoparticles; And

(b) forming a lipid bimolecular film on the surface of the porous silica nanoparticles coated with the ionic polymer thin film layer with a lipid vesicle including fluorescent lipid particles.

As shown in FIG. 1, the fluorescent porous silica nanoparticles according to the present invention are repeatedly coated alternately using a cationic polymer (PAH) and an anionic polymer (PSS) on the surface of the porous silica nanoparticle 110. After forming the polymer thin film layer 120, the fluorescent lipid molecules fluorescing may be prepared by forming the lipid bilayer 120 using a fixed lipid vesicle.

The present invention forms an ionic polymer thin film layer on the surface of the porous silica nanoparticles before forming the lipid bilayer on the porous silica nanoparticles, the ionic polymer thin film layer serves as a cushion (cushion) to facilitate the formation of the lipid bi-molecular film In addition, it may serve to reduce the "substrate effect" in which the activity of the biomolecule is changed by the surface and to maintain the activity of the loaded biomaterial.

At this time, in order to form the ionic polymer thin film layer, the surface of the porous silica nanoparticles can be coated with a cationic and anionic polymer compound in an aqueous solution by using a layer-by-layer (LbL) method, the coating of the two solutions The surface charges vary depending on the order (FIG. 2), and finally, the surface of the porous silica nanoparticles is negatively charged by coating with a polymer having a negative charge.

In addition, when repeatedly coating the cationic polymer compound and the anionic polymer compound alternately, it may be characterized in that it is preferably repeated 4 to 10 times. In case of repeating less than 4 times, the cushioning effect is reduced, and in case of repeating more than 10 times, when the drug or biomolecule is supported in the porous silica nanoparticles to release the drug or bind to the target biomolecule, Movement through the polymer membrane in and out can be difficult. However, more preferably it is characterized by repeating 4 to 6 times.

In this case, the cationic polymer compound for forming the ionic polymer thin film layer is not limited thereto, but polyarylamine hydrochloride (PAH), polydiarylammonium chloride (PDDA), polydiaryldimethylammonium chloride (PDADMAC) ), Poly-L-lysine (PLL), poly-L-arginine (PLA), polyethyleneimine (PEI) and chitosan (Citosan) can be used a cationic high molecular compound, preferably poly Arylamine hydrochloride (PAH) is used.

In addition, the anionic polymer compound for forming the ionic polymer thin film layer is not limited thereto, but polystyrene sulfonate (PSS), polyacrylic acid (PAA), polylactic acid (PLA), polyglutamic acid (PGA) ) And poly [ β- glucuronic acid- (1 → 3) -N- acetyl- β- galactosamine-6-sulfate- (1 → 4)] (PG) Compounds can be used, preferably polystyrene sulfonate (PSS).

Meanwhile, in the present invention, the porous silica nanoparticles coated with the ionic polymer thin film layer are again coated with a lipid bimolecular film, wherein the lipid bimolecular film is coated with a lipid vesicle containing fluorescent lipid particles with the ionic polymer thin film layer. After mixing with the porous silica nanoparticles, it may be characterized by forming by ultrasonication.

In the present invention, in order to form a bimolecular film on the porous silica nanoparticles, it is preferable to prepare lipid vesicle particles using lipid molecules. A step of preparing a lipid vesicle may be performed by mixing two or more kinds of lipid molecules. In the preparing of the lipid vesicle, a step of introducing lipid molecules having fluorescence characteristics may be performed.

At this time, the lipid molecule for forming the lipid vesicle (lipid vesicle) is not limited thereto, 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2- Diheptanoyl-sn-glycero-3-phosphocholine (1,2-diheptanoyl-sn-glycero-3-phosphocholine, DHPC), 1,2-dilauroyl-sn-glycero-3-phospho Choline (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC), 1,2-dipyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-distearoyl- sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-dilinoleyl-sn-glycero-3-phospho-L-serine ( 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 Stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, SOPS), 1-palmi Lipid particles selected from the group consisting of toyl-2-oleoyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and mixtures thereof Preferably, 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE) is used in combination.

On the other hand, it is possible to expect a biosensor effect for bioanalysis such as bioimaging and drug delivery by fixing fluorescent lipid molecules fluorescing between lipid vesicles prepared during formation of lipid bimolecular membranes. fluorescent lipid particles included in the lipid vesicle include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1, 3-benzosadiazol-4-yl (1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3-benzoxadiazol-4-yl, NBD PS) , 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (5-dimethylamino-1-naphthalenesulfonyl (1,2-dioleoyl-sn-glycero-3-phospho -L-serine-N- (5-dimethylamino-1-naphthalenesulfonyl, Dansyl PS), 1,2-dstearoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2- 1,3-benzosadiazol-4-yl (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1,3-benzoxadiazol-4-yl, NB D-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1,3-benzosadiazol-4-yl (1,2- Fluorescent lipid particles selected from the group consisting of dioleoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1,3-benzoxadiazol-4-yl, NBD-PE) and mixtures thereof can be used. , Preferably 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3-benzosadiazol-4-yl (1,2 -dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3-benzoxadiazol-4-yl, NBD PS) is used.

Fluorescent porous silica nanoparticles coated with a lipid bimolecular film according to the present invention can be used for labeling a biomaterial by immobilizing one or more functional groups capable of binding to the biomaterial to the biomaterial. Therefore, the present invention relates to a method for labeling a biomaterial using fluorescent porous silica nanoparticles coated with the lipid bimolecular film.

In addition, the fluorescent porous silica nanoparticles coated with a lipid bimolecular film according to the present invention can deliver various DNA and chemicals to animal cells or skin through functional groups, and can be used for drug delivery. It can be used as a probe in medical imaging.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Preparation of Porous Silica Nanoparticle Ionic Polymer Thin Film

In order to coat the surface of the porous silica nanoparticles with an ionic polymer, the cationic and anionic polymers were alternately coated using LbL (layer-by-laye) to form a polymer thin film.

That is, anionic polymer polystyrene sulfonate (PSS) and cationic polymer polyarylamine hydrochloride (PAH) are each prepared in an aqueous solution at a concentration of 1 mg / mL, and then the silica nanoparticles prepared in advance are dispersed in a PSS solution. After reacting for 10 minutes, the mixture was centrifuged and washed with water two or three times. This was again dispersed in the PAH solution, reacted for about 10 minutes, centrifuged and washed 2-3 times with water. This process was repeated 4-5 times and the outermost surface was treated with PSS to produce negatively charged nanoparticles.

As a result of confirming the surface charge by using a zeta potential measuring system (ELS-Z1, Otsuka Electronics, Japan) for each process, as shown in FIG. 2, the surface of the silica nanoparticles was repeatedly coated with a cationic and anionic polymer. It was confirmed that the polymer thin film was formed.

In addition, as a result of using a transmission electron microscope (TEM, JEOL LTD., Japan), as shown in Figure 3, it was confirmed that the polymer film was formed on the surface of the porous silica nanoparticles.

Lipid molecule  Fluorescence Vesicle  Produce

In order to form a bimolecular film structure on the surface of the porous silica nanoparticles coated with the polymer prepared in Example 1, a vesicle was prepared using lipid molecules.

That is, DOPC and DOPE were each prepared in a concentration of 10 mg / mL in chloroform, and then 100 μl of each was put in a vial and vortexed for 2 minutes. Chloroform was slowly evaporated using nitrogen gas so that the bilayer structure formed a film on the vial wall, and then dispersed in Tris buffer (5 mM, pH 7.4) and incubated in a thermostat (50 ° C.) for about 15 minutes. At this time, 2 mol% of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3- Benzoxadiazol-4-yl) (NBD PS) was added to the lipid solution dispersed in chloroform before evaporating the solvent.

After confirming the vesicle particles produced using an optical microscope, as shown in Figure 4, it was confirmed that the vesicle formed by the fluorescent lipid molecules exhibit a strong fluorescence.

On silica nanoparticles coated with polymer Biparticulate  formation

On the surface of the silica nanoparticles coated with the polymer in Example 1, a bimolecular film structure was formed using a vesicle showing fluorescence prepared in Example 2.

That is, silica nanoparticles coated with the ionic polymer of Example 1 were added to the vesicle solution of Example 2, and ultrasonic treatment was performed to form a bimolecular film on the surface of the belocyclo-silica nanoparticles.

Then, confirming that the bimolecular film was formed on the surface of the nanoparticles by using an optical microscope, as shown in FIG. 5, the fluorescence image of the nanoparticles was confirmed, and thus, coated with the bimolecular film according to the present invention. It was found that silica fluorescent nanoparticles can be usefully used for bioanalyses such as bioimaging and drug delivery.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

110: porous silica nanoparticles 120: ionic polymer thin film layer
130: lipid bilayer

Claims (10)

A method for preparing fluorescent porous silica nanoparticles coated with a lipid bimolecular film comprising the following steps:
(a) preparing an ionic polymer thin film layer on the surface of the porous silica nanoparticles; And
(b) forming a lipid bimolecular film on the surface of the porous silica nanoparticles coated with the ionic polymer thin film layer with a lipid vesicle including fluorescent lipid particles.
The method of claim 1, wherein the step (a) comprises repeatedly forming the cationic polymer thin film and the anionic polymer thin film on the surface of the porous silica nanoparticles, and finally forming the anionic polymer thin film to form the ionic polymer thin film layer. Manufacturing method.
The method of claim 2, wherein the cationic polymer thin film and the anionic polymer thin film are alternately formed 4 to 10 times.
The method of claim 2, wherein the cationic polymer thin film is polyarylamine hydrochloride (PAH), polydiarylammonium chloride (PDDA), polydiaryldimethylammonium chloride (PDADMAC), poly-L-lysine (PLL) , Poly-L-arginine (PLA), polyethyleneimine (PEI) and chitosan (Chitosan) is characterized in that it is formed of a cationic polymer compound selected from the group consisting of.
The method of claim 2, wherein the anionic polymer thin film is polystyrene sulfonate (PSS), polyacrylic acid (PAA), polylactic acid (PLA), polyglutamic acid (PGA) and poly [ β- glucuronic Acid- (1 → 3) -N- acetyl- β- galactosamine-6-sulfate- (1 → 4)] (PG).
The lipid bimolecular film of step (b) is formed by mixing a lipid vesicle including fluorescent lipid particles with porous silica nanoparticles coated with the ionic polymer thin film layer and then ultrasonicating the same. Characterized in that.
According to claim 1, wherein the lipid vesicle (lipid vesicle) of step (b) is 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-diheptanonyl -sn-glycero-3-phosphocholine (1,2-diheptanoyl-sn-glycero-3-phosphocholine, DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (1 , 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC ), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-distearoyl-sn-glycer Rho-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-dilinoleyl-sn-glycero-3-phospho-L-serine (1,2 -dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1-stearoyl-2-oleoyl-sn-glycero-3- Phospho-L-serine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, SOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and mixtures thereof.
According to claim 1, wherein the fluorescent lipid particles contained in the lipid vesicle (lipid vesicle) of step (b) is 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3-benzosadiazol-4-yl (1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3- benzoxadiazol-4-yl, NBD PS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (5-dimethylamino-1-naphthalenesulfonyl (1,2- dioleoyl-sn-glycero-3-phospho-L-serine-N- (5-dimethylamino-1-naphthalenesulfonyl, Dansyl PS), 1,2-dstearoyl-sn-glycero-3-phosphoethanolamine- N- (7-nitro-2-1,3-benzosadiazol-4-yl (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1,3-benzoxadiazol- 4-yl, NBD-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1,3-benzosadiazol-4-yl ( 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2-1,3-benzoxadiazol-4-yl, NBD-PE) and mixtures thereof How to.
A fluorescent porous silica nanoparticle coated with a lipid bimolecular film prepared by the method of any one of claims 1 to 8.
A method for labeling a biomaterial using fluorescent porous silica nanoparticles coated with a lipid bimolecular film of claim 9.

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