KR101730036B1 - Method for Preparing Nanoparticle Cluster Using DNA Binding Protein - Google Patents
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Abstract
The present invention relates to a method of manufacturing magnetic nanoparticles (NPCs) capable of controlling size, and more particularly, to a method of manufacturing a magnetic nanoparticle cluster (NPCs) capable of controlling the size of a zinc finger protein, ≪ / RTI > to form a zinc finger-DNA complex; And (b) combining the zinc finger-DNA complex with the nanoparticles modified with the second binding material to form a nanoparticle cluster having the zinc finger-complex bound to the nanoparticle, wherein the zinc finger- And a nanoparticle cluster produced by the method.
According to the present invention, since a zinc finger protein can be used instead of a chemical method in the production of nanoparticle clusters, the nanoparticle clusters can be applied to various nanoparticles and can be applied to various fields including diseases, diagnostic treatments and imaging .
Description
The present invention relates to a method of manufacturing magnetic nanoparticles (NPCs) capable of controlling size, and more particularly, to a method of manufacturing a magnetic nanoparticle cluster (NPCs) capable of controlling the size of a zinc finger protein, ≪ / RTI > to form a zinc finger-DNA complex; And (b) combining the zinc finger-DNA complex with the nanoparticles modified with the second binding material to form a nanoparticle cluster having the zinc finger-complex bound to the nanoparticle, wherein the zinc finger- And a nanoparticle cluster produced by the method.
Cluster structures composed of nanoparticles, such as magnetic nanoparticles, gold nanoparticles, and quantum dots, are attracting attention in that they have unique collective characteristics that are different from single nanoparticles. Nanoparticle clusters specifically exhibit optical or physical properties such as coupled-plasmon absorption, inter-particle energy transfer, electron transport and conductivity. Changes in these properties can be applied to nanoelectronic or nanoplasmonic devices. In addition, aggregates of one-dimensional or related nanoparticles may exhibit unusual mechanical properties such as viscosity reduction and form supra-crystals such as ionic solids or ionic liquids. In the case of biological sensors or biomedical imaging, there are examples of using clustering of gold nanoparticles and magnetic nanoparticles. When gold nanoparticles are clustered, the plasmon absorbance associated with the particles migrates to a lower energy level than that of a single nanoparticle. The magnetic particles significantly reduce the transverse relaxation time of the proton of adjacent water molecules in the presence of an external magnetic field. Since the transverse relaxation speed is proportional to the cross-sectional area of the magnetic material, clusters such as multimers and self-assemblies of magnetic nanoparticles having a large effective cross-sectional area have a much larger transverse relaxation speed, The degree of shortening is remarkably shortened. There are studies to analyze the presence or amount of the substance to be sensed or to use the magnetic nanoparticle cluster as a more advanced MRI contrast agent by using the phenomenon change occurring when the particle exists in a single particle state or when forming a cluster.
Nanoparticle clusters with a wide range of applicability due to various characteristic changes are required to have a clear and well-regulated composition in their fabrication to better represent their properties. Many attempts have been made to control the size and shape of nanoparticle clusters ( Nat. Commun. 2010 , 1, 87; Acc. Chem. Res. 2014 , 47, 1881-1890; ACS Nano 2014 , 8 , 3272-3284; Langmuir 2014 , 30, 7313-7318). Particularly, nanoparticle clusters made using DNA are receiving great attention. Methods for this include electrostatic bonding to a largely negative DNA phosphate backbone, binding to chemically modified DNA, and base complementation to single stranded DNA. However, these methods require chemical reduction of the metal ion or modification of the DNA structure, and it is difficult to modify the surface of the nanoparticles required for additional modification such as attaching the molecule.
On the other hand, zinc finger protein is a kind of protein that binds to DNA. It has two cysteines in structure and a zinc ion at histidine residue, and has a sequence specifically recognizing it. Zinc fingers are generally composed of tandem arrays of two or more fingers, and one finger recognizes and binds three base pairs of double stranded DNA. The sequence specificity and binding force of the zinc finger can be easily controlled by changing the finger number of the zinc finger and optimizing the linker between the finger. It is expected that the fabrication of nanoparticle clusters using the characteristics of zinc finger can be a more adaptable manufacturing method by a more biocompatible method.
The present inventors have made extensive efforts to develop a method for producing nanoparticle clusters capable of controlling size. As a result, they have found that a template DNA designed to have a plurality of zinc finger binding sites using zinc finger protein- It has been confirmed that nanoparticle clusters capable of size control can be prepared when binding nanoparticles to a zinc finger-DNA complex in which zinc finger aggregates are combined, thereby completing the present invention.
It is an object of the present invention to provide a method for producing nanoparticle clusters which are scalable.
Another object of the present invention is to provide a nanoparticle cluster which can be manufactured by the above method and which can be resized.
It is another object of the present invention to provide a contrast agent composition containing the nanoparticle clusters.
It is still another object of the present invention to provide a cell marking composition containing the nanoparticle cluster.
In order to achieve the above object, the present invention provides a method for producing a zinc finger-DNA complex, comprising: (a) binding a zinc finger protein having a first binding substance modified thereto to a DNA template containing a sequence specifically binding to the zinc finger protein, ; And (b) combining the zinc finger-DNA complex with the nanoparticles modified with the second binding material to form a nanoparticle cluster having the zinc finger-complex bound to the nanoparticle, wherein the zinc finger- Lt; RTI ID = 0.0 > nanoparticle < / RTI >
The present invention also provides a nanoparticle cluster comprising a sequence that specifically binds to a zinc finger protein and nanoparticles bound to a zinc finger-DNA complex to which a DNA template and a zinc finger protein are bound.
The present invention also provides a contrast agent composition containing the nanoparticle cluster.
The present invention also provides a cell labeled nanoparticle cluster characterized in that the cell target material is modified in the nanoparticle cluster.
The present invention also provides a cell marking composition containing cell marking nanoparticle clusters.
The present invention also provides a cancer-detecting nanoparticle cluster characterized in that the nanoparticle cluster is modified with a cancer cell target material.
The present invention also provides a cancer diagnostic composition containing the above-described cancer diagnostic nanoparticle cluster.
The present invention also provides a nanoparticle cluster for cancer treatment, characterized in that the nanoparticle cluster is modified with a cancer cell targeting therapeutic substance.
The present invention also provides a composition for treating cancer comprising the above-mentioned nanoparticle clusters for cancer treatment.
According to the present invention, since a zinc finger protein can be used instead of a chemical method in the production of nanoparticle clusters, the nanoparticle clusters can be applied to various nanoparticles and can be applied to various fields including diseases, diagnostic treatments and imaging .
FIG. 1 is a schematic diagram showing the formation of nanoparticle clusters using DNA-binding zinc finger proteins and DNA template design by three lengths. FIG.
Figure 2 is an electron micrograph and DLS analysis graph showing the purification of NPCs using the glycerol density gradient method of the present invention.
FIG. 3 is an electron transmission microscope photograph and a graph showing the formation of magnetic nanoparticle clusters according to an embodiment of the present invention.
FIG. 4 is a graph showing a T2 relaxation ratio comparison between a magnetic nanoparticle cluster and a feridex according to the present invention, and a T2-weighted image.
In the present invention, a zinc finger-DNA complex capable of controlling the length of a DNA template binding to a zinc finger is prepared by using the ability of a zinc finger protein to bind to DNA of a specific sequence, and the zinc finger- Resulting in nanoparticle clusters that are scalable.
Accordingly, in one aspect, the present invention relates to a method for producing a zinc finger protein, comprising: (a) binding a DNA template comprising a sequence specifically binding to the zinc finger protein to a zinc finger protein to which the first binding substance is modified, Forming a complex; And (b) combining the zinc finger-DNA complex with the nanoparticles modified with the second binding material to form a nanoparticle cluster having the zinc finger-complex bound to the nanoparticle, wherein the zinc finger- To a method for producing nanoparticle clusters to which a complex is bound.
In the present invention, the DNA template may include at least one zinc finger binding sequence, and at least two DNA blocks containing at least one zinc finger binding sequence may be linked to a linker having a restriction enzyme recognition site have.
The zinc finger protein used in the present invention may be any zinc finger protein having DNA binding ability. Examples of zinc finger proteins that can be used include Cys2His2 including QNK-QNK-RHR, Gag knuckle, Treble clef, zinc ribbon, Zn2 / Cys6, TAZ2 domain like (TAZ2 domain like).
In one embodiment of the present invention, QNK-QNK-RHR is used as a zinc finger protein and the DNA sequence specifically binding to the zinc finger protein QNK-QNK-RHR is "GAGGCAGAA ".
In the present invention, the DNA template may be a DNA structure including various types of DNA origami.
In the present invention, the nanoparticles may be nanoparticles doped with magnetic particles selected from the group consisting of Fe 2 O 3, Fe 3 O 4 ,
In the present invention, the first binding substance and the second binding substance may be DNA, RNA, protein, peptide, fat, or carbohydrate capable of binding to each other.
In the present invention, the first binding substance is biotin and the second binding substance is selected from the group consisting of streptavidin, avidin, neutravidin and captavidin. Wherein the first binding substance is selected from the group consisting of streptavidin, avidin, neutravidin and captavidin, and the second binding substance is selected from the group consisting of biotin . ≪ / RTI >
In another aspect, the present invention relates to a nanoparticle cluster in which nanoparticles are bound to a zinc finger-DNA complex in which a DNA template containing a sequence specifically binding to a zinc finger protein and a zinc finger protein are bound.
An embodiment of the method for producing magnetic NPCs using the DNA binding zinc finger protein of the present invention is shown in FIG. In FIG. 1 and FIG. 1, the first biomolecule is biotin, the second biomolecule is neutravidin, and the zinc finger is a zinc finger consisting of three fingers F1-F2-F3 (finger 1-finger 2-finger 3) It corresponds to QNK-QNK-RHR. The neutravidin of the magnetic nanoparticles forms a magnetic nanoparticle cluster by specific binding with the biotin formulated at the N-terminal of the zinc finger. In FIG. 1, the designed DNA template having three different lengths is composed of 2, 4, and 8 blocks, respectively. There are 7 zinc finger binding sites in one block. Therefore, the size of the magnetic nanoparticle clusters made according to the length of the DNA template can be controlled by the specific binding between the zinc finger and the DNA.
In the following Examples and FIG. 1, QNK-QNK-RHR, which is one of the zinc finger proteins, was modeled as a mediator of magnetic nanoparticle cluster formation. However, the present invention is not limited thereto, and all kinds of zinc finger proteins The same principle can be used. Accordingly, any structure or form including any sequence or sequence may be used as long as DNA sequence or form can be specifically bound to a specific zinc finger, but it is of course not limited thereto.
The first biomolecule is bound to the second biomolecule by a specific binding Any DNA, RNA, protein, peptide, fat or carbohydrate may be used as long as it can form clusters of magnetic nanoparticles. When the first biomolecule is DNA / RNA, the second biomolecule may be DNA / RNA having a sequence complementary to the first biomolecule or a fragment thereof. Likewise, if the first biomolecule is a protein, a peptide, a fat, or a carbohydrate, a ligand capable of specifically binding thereto can be used as the second biomolecule. For example, if the first biomolecule is biotin as in the embodiment of the present invention, the second biomolecule may be any biomolecule capable of binding specifically to biotin. Examples of the biomolecule include streptavidin, avidin, But are not limited to, neutravidin or captavidin.
The magnetic nanoparticles may be Fe 2 O 3 or Fe 3 O 4 , which is known as a magnetic material having a size ranging from several nanometers to several tens of nanometers, or any of the later developed magnetic materials. The nanoparticles may be not only magnetic nanoparticles but also gold nanoparticles, silver nanoparticles, quantum dots, and the like, provided that they are of the same nanoscale size.
Since the NPCs are formed by the specific binding of the first biomolecules located at the end of the zinc finger N and the second biomolecules on the surface of the magnetic nanoparticles, the probability of cross-linking of the NPCs due to the plurality of second biomolecules Is high. In order to prevent this, the binding ratio of the magnetic nanoparticles to the DNA-zinc finger complex was set to 200 when NPCs were formed in the examples of the present invention. Cross-linking between clusters was not observed when reacting 200-fold excess of magnetic nanoparticles, which was demonstrated by the nanoparticle analyzer (DLS) (Figure 2). The results show that it is necessary to react at least 200 times the magnetic nanoparticles to make the NPCs effective without cross-linking.
The sedimentation velocity of NPCs during the purification process of NPCs using density gradient will depend on the type of nanoparticles. It is important to obtain high-purity NPCs because the purity of the NPCs will greatly affect the subsequent application. In the examples of the present invention, conditions optimized for high-purity purification of magnetic nanoparticle clusters were established, which were verified by an electron transmission microscope (TEM) and a nanoparticle analyzer (FIG. 2). The average number of nanoparticles or the size and shape of the NPCs in the optimal NPCs depending on the application required will depend on the type, size and composition of the nanoparticles used, and the shape and size of the DNA template. It will be readily apparent to those skilled in the art to select optimal conditions for this.
In another aspect, the present invention relates to a contrast agent composition containing the nanoparticle clusters.
The magnetic nanoclusters produced by the magnetic nanoparticle cluster manufacturing method of the present invention can further be used for application to T2-weighted image contrast agent of MRI and specific intracellular delivery and imaging. The T2 relaxation rate (r2) comparison with Feridex, a conventional MRI contrast agent, was performed using the NPCs prepared in the following examples (FIG. 4). R2 of the NPCs manufactured in accordance with the present invention has shown a value corresponding to about three times the contrast effect of Feridex (108.2mM -1 s -1) as 315.1mM -1 s -1.
In another aspect, the present invention relates to a cell marking nanoparticle cluster characterized in that a cell target substance is modified in the nanoparticle cluster, and a cell marking composition containing the cell marking nanoparticle cluster.
In the present invention, the cell target material may be any cell target material for recognizing a specific cell. Any ligand of a specific cell receptor may be used as an example.
In one embodiment of the present invention, NPCs exposed to folate are loaded with a dye that is intercalated into DNA, and thus it is possible to specifically transfer the folate to HeLa cells, which are overexpressed follicular receptors. (Fig. 5). When treated with NPCs that were not treated with any of the NPCs and no NPCs exposed to folate, no fluorescence signal was seen in the cells. However, when treated with NPCs exposed to folic acid, the green fluorescence signal of the DNA-injecting dye appeared in the cells.
In another aspect, the present invention relates to a cancer-detecting nanoparticle cluster characterized by the fact that the cancer cell target substance is modified in the nanoparticle cluster and a cancer diagnostic composition containing the cancer-detecting nanoparticle cluster.
In the present invention, the cancer cell target material may be a substance capable of binding to a receptor specifically expressed in cancer cells, and a target substance used for cancer diagnosis may be used. Antibodies, antisense, PNA, And the like can be used.
According to another aspect of the present invention, there is provided a cancer therapeutic nanoparticle cluster characterized in that the nanoparticle cluster is modified with a cancer cell targeting substance and the cancer therapeutic agent is bound to the nanoparticle cluster, ≪ / RTI >
In the present invention, the cancer cell therapeutic material may be a substance that specifically binds to and can be treated in a cancer cell, and a target cell therapeutic material used in cancer therapy may be used.
Examples of cancer therapeutic substances that may be used in various embodiments of the present invention, including the pharmaceutical compositions and dosage forms and kits of the invention include, but are not limited to, axicidin; Aclarubicin; Acodazole hydrochloride; Acronine; Adozelesin; Aldethsky; Altretamine; Ammomycin; Amethanthrone acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Aspelin; Azacytidine; Azeta; Azothomaine; Batimastat; Benzodepa; Bicalutamide; Bisanthrene hydrochloride; Bisnipid dimesylate; Bezelesin; Bleomycin sulfate; Sodium brexanal; Bropyrimine; Layout; Cactinomycin; Callus teron; Caracamide; Carbethymers; Carboplatin; Carmustine; Carvedicin hydrochloride; Carzelesin; Sedefingol; Chlorambucil; Sirole remiacein; Cisplatin; Cladribine; Chinatol mesylate; Cyclophosphamide; Cytarabine; Takabazin; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Dexomaplatin; Not dejected; Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Droloxifene; Droloxifen citrate; Dlromotranolone propionate; Durazomycin; Etrexate; To flunitin hydrochloride; Elasmitruscin; Enloflatatin; Enpromeate; Epipropidine; Epirubicin hydrochloride; Erburosol; Esorubicin hydrochloride; Estra mestin; Estramustine phosphate sodium; Ethanedisole; Etoposide; Etoposide phosphate; Etopren; Hydrazone hydrochloride; Fazarabine; Fenretinide; Phlox uridine; Fludarabine phosphate; Fluorouracil; Fluloxilin; Phosquidone; Postriasin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Rubicin hydrochloride; Iospasmide; Ilmofosin; Interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a; Interferon alpha-2b; Interferon alpha-n1; Interferon alpha-n3; Interferon beta-Ia; Interferon gamma-Ib; Iuproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate; Liarozole hydrochloride; Lometrexol sodium; Rosemastin; Rosoxanthrone hydrochloride; MASO PROCOL; Maitansin; Mechlorethamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaryl; Mercaptopurine; Methotrexate; Methotrexate sodium; methoprone; Metourethamine; Mitomodide; Mitocarcin; Mitochromin; Mitogyline; Mitochondria; Mitomycin; Mitosper; Mitotane; Mitoxanthrone hydrochloride; Mycophenolic acid; Nocodazole; Nogalamycin; Ormaflatin; Oxysulane; Paclitaxel; Gaspard; Peliomycin; Pentamustine; Perfromycin sulfate; Perpospamide; Pipobroman; Chopped sulp; Pyrroxanthrone hydrochloride; Plicamycin; Flomestane; Sodium formate; Porphyromycin; Fred Nimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazopurine; Riboprene; Roglutimide; Saffing bone; Saffigold hydrochloride; Taxostin; SimTragen; Sparosate sodium; sparsomalcine; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; streptonigin; Streptozocin; Sulfophenur; Talisomycin; Tetogalan sodium; Tegapur; Teloxanthrone hydrochloride; Temoporphine; Tenifocide; Tetracyclone; Testolactone; Thiamipine; Thioguanine; Thiotepa; Thiazopurine; Tyrapazamin; Toremifens citrate; Tristolone acetate; Tricyribine phosphate; Trimetrexate; Trimetrexate glucuronate; Tryptophan; Pyrazole hydrochloride; Uracil mustard; Uredepa; Bafreotide; Vertefopin; Vinblastine sulfate; Vincristine sulfate; Bindeseo; Vindesine sulphate; Vinepidine sulfate; Vin glycinate sulfate; Shin Sulfate as a pessimistic; Vinorelvin tartrate; Vincrosidine sulfate; Bin zolyidine sulfate; Borosal; Nippleatin, zinostatin; Lt; / RTI > hydrochloride. Other anti-cancer drugs include, but are not limited to, 20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; Aviratorone; Aclarubicin; Acylphenol; Adhephenol; Adozelesin; Aldethsky; ALL-TK antagonists; Altretamine; Amvamustine; Amidox; Amipostin; Aminolevulinic acid; Amrubicin; Amsacrine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitors; Antagonist D; Antagonist G; Antaralicious; Anti-spinal cord protein-1; Antiandrogen, prostate carcinoma; Antiestrogen; Enneoplaston; Antisense oligonucleotides; Ampicillin glycinate; Cell annihilation gene regulators; Apoptosis regulator; apurinic acid; Ara-CDP-DL-PTBA; Arginine deaminase; Asulacrine; Atamestane; Artimustine; Acycinastatin 1; Acuminastatin 2; Acuminastatin 3; Azacetone; Azotoxin; Azathiocin; A baccatin III derivative; Valanol; Batimastat; BCR / ABL antagonist; Benzochlorine; Benzoylstaurosporine; Betalactam derivatives; Beta-alletin; Betaclomycin B; Betulinic acid; bFGF inhibitors; Bicalutroamide; Arsenate; Bissaridinylspermine; Bisnaphid; Bistatten A; Bezelesin; Brake plate; Bropyrimine; Subordinate titanium; Butyronine sulfoximine; calcipotriol; Calpostatin C; Camptothecin derivatives; Canary fox IL-2; Capecitabine; Carboxamide-amino-triazole; Carboxyamidotriazole; CaRest M3; CARN 700; Cartilage tissue induced inhibitors; Carzelesin; Casein kinase inhibitor (ICOS); Castanospermine; Secropin B; Set Laurelrix; Clawruns; Chloroquinoxaline sulfonamide; Sika Frost; Cis-porphyrin; Cladribine; Clomiphene analogs; Clotrimazole; Colistin A; Collymycin B; Combretastatin A4; Combretastatin analogs; Conazenine; Chlambacidin 816; Chinatol; Cryptophycin 8; Cryptophycin A derivatives; Curacin A; Cyclopentanthraquinone; Cyclophotam; Cypermycin; Cytarabine oxophosphate; Cell lysis factors; Cytostatin; Map when clicked; Decitabine; Dehydrodimdinin B; Deslorin; Dexamethasone; Dexiposphamide; Dexlazoic acid; Dex verapamil; Diaziquone; Dimedinin B; Dodox; Diethylnonsper
Min; Dihydro-5-azacytidine; Dihydrotaxol, 9-; Dioxamycin; Diphenyl spiromustine; Docetaxel; Dococanol; Dolasitron; Doxifluridine; Droloxifene; Dronabinol; Duocamaxine SA; Epselene; Ecomustine; Edelosine; Ed recolor map; Fluorinitin; Elements; Emitepur; Epirubicin; Episteide; Estramustine homolog; Estrogen agonists; Estrogen antagonists; Ethanedisole; Etoposide phosphate; Exemestane; Fadrosol; Fazarabine; Fenretinide; Phil Grass Team; Pinastellite; Flavopyridone; Flaselastine; Fluastarone; Fludarabine; Fluorodanolunhyde hydrochloride; Porphnimex; Formestane; Post lysine; Potemustine; Gadolinium tetrapyrin; Gallium nitrate; Galoshita bin; Ganilelix; Gelatinase inhibitors; Gemcitabine; Glutathione inhibitors; Hept sulfam; Herlegolin; Hexamethylene bisacetamide; Hyperperishin; Ibandronic acid; dirubicin; Iodoxifene; Idraemantone; Ilmofosin; Start Roman; Imidazoacridone; Imiquimode; Immunostimulatory peptides; Insulin-like growth factor-1 receptor inhibitors; Interferon agonists; Interferon; Interleukin; Iobenguan; Iododoxirubicine; I-propanol, 4-; Iroplast; Irgoglidine; Isobenzazole; Isomerized halocondrine B; Itacetone; Jas flakinoid; Carbhalide F; Lamellarin-N triacetate; Lanthanide; Reinamycin; Reno Grass Team; Lentan sulfate; Leptolastatin; Letrozole; leukemia inhibitory factor; Leukocyte alpha interferon; Leuprolide + estrogen + progesterone; Leuprorelin; Levamisole; Riazolone; Linear polyamine homologs; A chimeric disaccharide peptide; A lipophilic platinum compound; Lysochlinamide 7; Roba flatin; Lombardin; Lonometrin; Ronnidamin; Rosantanone; Lovastatin; Rock sound blank; Rutotethene; Lutetium tetrapyrine; Lysophylline; Degradable peptides; Mytansine; Mannostatin A; Marimastat; MASO PROCOL; Town spin; Matrilysin inhibitors; Matrix metalloproteinase inhibitors; menogalil; Merbaron; Methelin; Methionine; Methocloframide; MIF inhibitors; Mifepristone; Miltefosine; Premoist team; Double stranded RNA mismatched; Mitoguazone; Mitolactol; Mitomycin analogs; Mitomapride; Mitotoxin fibroblast growth factor-serine; Mitoxantrone; Mofarotene; Morgol Ramos Team; Monoclonal antibody, human chorionic gonadotropin; Monophosphoryl lipid A + myobacterium cell wall sk; Fur damol; Multiple drug resistance gene inhibitors; Multiple tumor suppressor 1-based therapy; Mustard anticancer agent; My Cafe Rockside B; Mycobacterium cell wall extract; Pre-apron; N-acetyl dinalin; N-substituted benzamides; Napalelin; Nagres tips; Naloxone + pentazocine; Napa bin; Naphterpine; Nartograsi Team; Your dapplatin; Nemorubicin; Neridronic acid; Neutral endopeptidase; Nilutamide; Nisamycin; Nitric oxide modifiers; Nitroxides antioxidants; Nitrulline; O6-benzylguanine; Octreotide; Oxycenone; Oligonucleotides; Onafristone; Ondansetron; Ondansetron; Oracle; Oral cytokine inducers; Ormaflatin; Osaterone; Oxaliplatin; Oxathinomycin; Paclitaxel; Paclitaxel homolog; Paclitaxel derivatives; Palauamine; Palmytoiligin; Pamidronic acid; Paraxyltriol; Panomimpen; Paramartin; Pascel liptin; Pegasper; Peledin; Sodium pentosan polysulfate; Pentostatin; Pentrozole; Perfluorobutane; Perphosphamide; Peryl alcohol; Phenazinomycin; Phenylacetate; Phosphatase inhibitors; Fish barnil; Pilocarpine hydrochloride; Pyra rubicin; Pyrite tree core; Plasetin A; Plasetin B; Plasminogen activator inhibitors; Platinum complex; Platinum compounds; Platinum-triamine complex; Sodium formate; Porphyromycin; Prednisone; Propyl bis-acridone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae; Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; Furfurin; Pyrazoloacridine; Pyridoxylated hemoglobin polyoxyethylene conjugates; raf antagonists; Ralitriptycide; Ramosetron; rasparnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; Demethylated letelipidin; Rhenium Re 186 etidronate; Liqin; Ribozyme; RII retinamide; Roglutimide; Lohitkin; Rosemutide; Quinimex; Ruby Guinea B1; Leuco; Saffing bone; Tosin; SarCNU; Sarcopitol A; Sarragramos Team; Sdi 1-like substances; Taxostin; Aging induced inhibitor 1; Sense oligonucleotides; Signal transduction inhibitors; Signaling modulators; Single chain antigen binding protein; Xanthopyran; Bovine acid; Sodium borocapthalate; Sodium phenylacetate; Sorbrole; Somatomedin binding protein; Sonermin; Sparfolic acid; Spicamycin D; Spiromustine; Splenopentin; Spongistatin 1; Squalamine; Hepatocyte inhibitors; Liver-cell division inhibitors; Stipiamide; Stromelysin inhibitors; Sulfinosine; And active vasoactive intestinal peptide antagonists; Suradistar; Suramin; Surainsonin; Synthetic glycosaminoglycan; Talimustine; Tamoxifen methiodide; Tauromustine; Tazaroten; Tetogalan sodium; Tegapur; Telulapyrilium; Telomerase inhibitors; Temoporphine; Temozolomide; Tenifocide; Tetrachlorodecoxide; Tetrazomine; Talli blastin; Thiocolrine; Thrombopoietin; Thrombopoietin-like substances; Thalam fascin; A thymopoietin receptor agonist; Thymolitin; thyroid stimulating hormone; Tin ethyl thiopurfurin; Tyrapazamin; Titanocene chloride; Topcentin; Toremie pen; Tortifotent hepatocyte factor; Translational inhibitors; Tretinoin; Triacetylglycine; Trishyribine; Trimetrexate; Tryptophan; Trophis set ron; Truostearate; Tyrosine kinase inhibitors; Triphostin; UBC inhibitors; Yubenimex; Urogenital steal-derived growth inhibitory factor; urokinase receptor antagonist; Bafreotide; Barrydin B; Vector system, red blood cell gene therapy; Velarresol; Veramin; Berdins; Vertefopin; Vinorelbine; Vinxaline; Non-thaxin; Borosal; Zanoterone; Nipple latin; Zilas corv; And zinostatin stimemer. Preferred additional anti-cancer drugs are 5-fluorouracil and leucovorin.
The suitability of the particular route of administration used for a particular active ingredient will depend on the active ingredient itself (e.g. whether it can be orally administered without degradation prior to introduction into the bloodstream) and the disease being treated. For example, treatment of tumors on the skin or on exposed mucosal tissues may be more effective than administration of one or both active ingredients via topical, transdermal or mucosal routes (e.g., by nasal, sublingual, ball, rectal or vaginal) It can be effective. Treatment of tumors in the body, or prevention of cancer that can spread from one part of the body to another, may be more effective when either one or two active ingredients are administered orally or parenterally. Similarly, parenteral administration may be desirable for acute treatment of disease, while transdermal or subcutaneous routes of administration may be used for chronic treatment or prevention of disease.
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.
Example 1: Preparation of magnetic nanoparticle clusters
1) Production of DNA template by length
For the production of three different lengths of DNA template, block DNA of about 100 bp in length was synthesized. Block DNA was designed to have seven zinc finger binding sites. Restriction enzyme treatment and ligation were used to efficiently produce DNAs of 251 bp, 437 bp and 809 bp of SEQ ID NOS: 1 to 3 having 2, 4 and 8 repeated block DNAs, respectively.
In addition, as shown in FIG. 1B, the light blue color indicates seven zinc finger binding sites (underlined, nine base sequences), and four random base sequences are distributed among the zinc finger binding sites. Red is NdeI restriction enzyme recognition sequence; Gray is the XhoI restriction enzyme recognition sequence; Yellow is SalI restriction enzyme recognition sequence; Blue represents the EcoRI restriction enzyme recognition sequence. Italic is an adapter sequence containing the restriction enzyme recognition sequence (bold) required for cloning.
More specifically, two DNA blocks with Nde I enzyme recognition sequences at the 5 'or 3' ends were synthesized by PCR and treated with Nde I enzyme. Two DNA blocks were ligated to each other through ligation. The bound DNA of the desired size was extracted from the gel after confirmation through agarose gel. The extracted DNA was subjected to PCR with Nco I at the 5 'end and BamH I recognition sequence at the 3' end, and then cloned into pBEL118N phagemid. Finally, a DNA template of 251 bp in length was obtained by PCR using the prepared phagemid as a template. Next, a DNA template of 437 bp in length was obtained by repeating the same procedure using 251 bp as a template. At this time, the Xho I recognition sequence was used for ligation. The DNA template of 809 bp in length was also subjected to the same procedure as above using 437 bp DNA as a template and the Sal I recognition sequence was introduced.
2) Expression and purification of zinc finger proteins with biotin-modified N-terminus
(SEQ ID NO: 5) of the zinc finger protein (SEQ ID NO: 4) named QNK-QNK-RHR was synthesized and PCR was carried out to introduce the Nde I recognition sequence at the 5 'end and the Xho I recognition sequence at the 3' Respectively. In particular, an amino acid linker containing cysteine was introduced at the N-terminus. The resulting gene was cloned into pET21a vector and transformed into BL21 (DE3) competent cells by heat shock method.
For the expression of zinc finger proteins, a single colony of transformed cells was inoculated in 10 mL of LB medium for 12 hours or more, and the cultured cells were inoculated again into a 1 L medium at a ratio of 1/100 to obtain 37 Lt; 0 > C for 3 hours. By the time the optical density at the 600 nm wavelength of the cells reached 0.6, IPTG and ZnSO4 were additionally added at 0.5 mM and 0.2 mM, respectively. Thereafter, the cells were further cultured at 18 ° C for 20 hours, and finally, the cells were obtained at a centrifugal force of 6000 g. The resulting cells were sonicated by dissolving in 30 mL lysis buffer (20 mM Tris-Cl, 0.2 mM ZnSO4, and 2 mM β-mercaptoethanol, pH 7.5). The supernatant containing the protein was separated by centrifugation, and a zinc finger protein with a histidine tag was purified by a nickel-fixed column (GE Healthcare, USA). The N-terminal biotin formulations were buffer exchanged with 50 mM HEPES buffer (150 mM NaCl, 0.2
Analysis of the finally obtained protein was performed by polyacrylamide gel electrophoresis (PAGE), and biotin binding assay (HABA; Sigma-Aldrich) confirmed binding of biotin molecules to proteins at a ratio of about 1: 1. The purified protein was stored at -70 ° C prior to use.
3) Preparation of magnetic nanoparticles modified neutravidin on the surface
For the synthesis of magnetic nanoparticles modified with Nutrabide, DSPE-PEG2000-methoxy and DSPE-PEG3400-biotin were coated at a ratio of 95: 5 at 15 nm. iron oxide magnetic nanoparticles were conjugated to neutra- vidin.
First, superparamagnetic iron oxide nanoparticles of 15 nm in size were synthesized according to the prior art (J. Cheon, Angew. Chem. Int. Ed. 2009 , 48, 1234-1238).
Three times of filtration using a centrifugal filter with a molecular weight cut-off of 100 kD was performed by adding 200 times neutravidin (Pierce Chemical) to magnetic nanoparticles in 50 mM borate buffer (pH 8.3) for 1 hour To remove unreacted neurotavidin. Calculated protein conjugated to magnetic nanoparticles by the Bradfordassay method showed that 200 neutravidin molecules were expected to be immobilized on the magnetic nanoparticles.
4) Manufacture of NPCs
To prepare magnetic NPCs, a DNA template at a concentration of 20 nM was reacted with a biotin-labeled zinc finger at a 10-fold excess of the number of zinc finger binding sites. BSA was added to the reaction to a final concentration of 0.01 mg / ml for stability of the zinc finger-DNA complex. After 30 minutes of reaction at room temperature, the zinc finger without complex was removed using a centrifugal filter with a molecular weight cut-off of 30 kD. The resulting complex was further conjugated with neutron-coated magnetic nanoparticles at a concentration of 200 nM at 4 DEG C for at least 12 hours.
Density gradient centrifugation was used to purify high purity magnetic nanoparticle clusters. More specifically, glycerol having a concentration range of 30 to 60% was prepared using a gradient maker, and magnetic NPCs having a concentration of 400 nM based on magnetic nanoparticles were loaded. The centrifugation was carried out at 5000 rpm for 12 hours with an ultracentrifuge (Beckman Coulter XL-100 ultracentrifuge, SW 55 Ti rotor). NPCs obtained in the form of pellet were resuspended in zinc finger storage buffer and glycerol in aqueous solution was removed by dialysis.
5) Confirmation of formation of scaled NPCs by DNA template length
For the analysis of the size-adjusted NPCs according to the DNA template lengths prepared in the above examples, DNA templates of three different lengths were used as in the above example. The prepared DNA templates were 251 bp, 437 bp and 809 bp in length, respectively. The image of the NPCs was observed using transmission electron microscopy (TEM, JEOL JEM-2100F).
Each solution of NPCs with DNA lengths was dropped on a 300-mesh carbon-coated grid and dried at room temperature for 60 minutes. The TEM image was obtained and the results are shown in FIG.
As can be seen in FIG. 3 (a), the formation of NPCs was clearly observed, and the size and shape of the NPCs were well controlled according to the length of the DNA template. 4 (b) is a histogram representing the size distribution by analyzing over 380 NPCs on a TEM image. As the length of the DNA template increased from 251 bp to 809 bp, the peak of the distribution shifted to the right. On average, the number of magnetic nanoparticles bound to the DNA template was 2.9 ± 0.9, 5.8 ± 1.3, and 11.6 ± 2.0, respectively, from the shortest length DNA template. This was well matched with the theoretically expected values of 3, 6 and 12, which proved that the method of manufacturing the NPCs can be manufactured in a size-controlled manner.
Example 2: Initial step application of fabricated NPCs
1) MR imaging effects of magnetic NPCs
In order to confirm the applicability of the NPCs prepared in the above examples to the T2-weighted MRI contrast agent, T2 relaxation rate of NPCs was measured and the results are shown in FIG. At this time, NPCs made of 809 bp DNA template were used for the measurement.
To measure the T2 relaxation time, MR imaging experiments were performed with a 3-T MRI (Siemens Magnetom Verio, Germany) of a 12-channel head matrix receiver coil using the following parameters to determine the T2 of the NPCs sample - T2-weighted MR images were measured; spin echo mode; section thickness = 10 mm; echo time (TE) = 7.8, 10, 15, 20, 30, 40, 50, 70, 100 ms; repetition time (TR) = 5,000 ms; number of average = 1.
In FIG. 4, Feridex is the result of measuring T2 relaxation as a control for comparison. As can be seen in FIG. 4, the T2 relaxation rate of NPCs showed a T2 relaxation rate three times higher than that of Feridex, indicating that the NPCs showed enhanced contrast enhancement properties than Feridex. This is also true for the T2-weighted image (the image of FIG. 4). When the Fe concentration was measured at 0.18 mM, NPCs showed darker images due to the enhanced contrast effect compared to Feridex.
2) Confirmation of cellular targeting and intracellular delivery of magnetic NPCs
Another application of NPCs has been cell experiments for intracellular targeting. For this purpose, 1% of DSPE-PEG5000-folate was added to the coating of magnetic nanoparticles used to make NPCs. Folate was used to target specific cells. In order to observe intracellular delivery of NPCs, YOYO-1 stain, a dye that interferes with the DNA template, was used.
10 4 HeLa cells overexpressing the folate receptor were cultured for 24 hours, and the cells were treated with 0.1 nM stain labeled magnetic NPCs for 4 hours. After treatment, the cells were washed 3 times with DPBS buffer and nuclear staining was performed with DAPI. Fixed cells were observed at 400x magnification with a LSM 780 confocal microscope system (Zeiss, Germany) microscope equipment (FIG. 5).
Observation revealed that NPCs exposed to folic acid enter well into HeLa cells. The green fluorescence of YOYO-1 was clearly visible in the cells. In contrast, the cells treated with control NPCs without folic acid showed no green fluorescence, suggesting that intracellular delivery was not achieved. Similarly, cells that did not undergo any treatment could not observe fluorescence at all. These results clearly demonstrate that receptor-mediated specific intracellular delivery of folic acid-conjugated NPCs is achieved. Especially, it is remarkable that it can be easily used by loading a large amount of dye that interferes with DNA rather than chemical bonding of existing dye. It also shows that both MRI and fluorescence imaging of cells using NPCs are possible at the same time.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> Korea Advanced Institute of Science and Technology <120> Method for Preparing Nanoparticle Cluster Using DNA Binding Protein <130> P14-B392 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 251 <212> DNA <213> Artificial Sequence <220> <223> block DNA <400> 1 atatccatgg ccattaccgt gagcacccga attcgaggca gaagttagag gcagaagccg 60 gaggcagaat atcgaggcag aaaaacgagg cagaaaactg aggcagaaca ctgaggcaga 120 acatatggag gcagaagtta gaggcagaag ccggaggcag aatatcgagg cagaaaaacg 180 aggcagaaaa ctgaggcaga acactgaggc agaagaattc cgtagcatta tttgcccgac 240 cggatccata t 251 <210> 2 <211> 437 <212> DNA <213> Artificial Sequence <220> <223> block DNA <400> 2 atatccatgg ccattaccgt gagcacccga attcgaggca gaagttagag gcagaagccg 60 gaggcagaat atcgaggcag aaaaacgagg cagaaaactg aggcagaaca ctgaggcaga 120 acatatggag gcagaagtta gaggcagaag ccggaggcag aatatcgagg cagaaaaacg 180 aggcagaaaa ctgaggcaga acactgaggc agaactcgag gaggcagaag ttagaggcag 240 aagccggagg cagaatatcg aggcagaaaa acgaggcaga aaactgaggc agaacactga 300 ggcagaacat atggaggcag aagttagagg cagaagccgg aggcagaata tcgaggcaga 360 aaaacgaggc agaaaactga ggcagaacac tgaggcagaa gaattccgta gcattatttg 420 cccgaccgga tccatat 437 <210> 3 <211> 809 <212> DNA <213> Artificial Sequence <220> <223> block DNA <400> 3 atatccatgg ccattaccgt gagcacccga attcgaggca gaagttagag gcagaagccg 60 gaggcagaat atcgaggcag aaaaacgagg cagaaaactg aggcagaaca ctgaggcaga 120 acatatggag gcagaagtta gaggcagaag ccggaggcag aatatcgagg cagaaaaacg 180 aggcagaaaa ctgaggcaga acactgaggc agaactcgag gaggcagaag ttagaggcag 240 aagccggagg cagaatatcg aggcagaaaa acgaggcaga aaactgaggc agaacactga 300 ggcagaacat atggaggcag aagttagagg cagaagccgg aggcagaata tcgaggcaga 360 aaaacgaggc agaaaactga ggcagaacac tgaggcagaa gtcgacgagg cagaagttag 420 aggcagaagc cggaggcaga atatcgaggc agaaaaacga ggcagaaaac tgaggcagaa 480 cactgaggca gaacatatgg aggcagaagt tagaggcaga agccggaggc agaatatcga 540 ggcagaaaaa cgaggcagaa aactgaggca gaacactgag gcagaactcg aggaggcaga 600 agttagaggc agaagccgga ggcagaatat cgaggcagaa aaacgaggca gaaaactgag 660 gcagaacact gaggcagaac atatggaggc agaagttaga ggcagaagcc ggaggcagaa 720 tatcgaggca gaaaaacgag gcagaaaact gaggcagaac actgaggcag aagaattccg 780 tagcattatt tgcccgaccg gatccatat 809 <210> 4 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> QNK-QNK-RHR <400> 4 Met Met Cys Lys Thr Gly Glu Lys Arg Pro Tyr Lys Cys Pro Gly Cys 1 5 10 15 Gly Lys Ser Phe Ser Gln Ser Ser Asn Leu Gln Lys His Gln Arg Thr 20 25 30 His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Gly Cys Gly Lys Ser Phe 35 40 45 Ser Gln Ser Ser Asn Leu Gln Lys His Gln Arg Thr His Thr Gly Glu 50 55 60 Lys Pro Tyr Lys Cys Pro Gly Cys Gly Lys Ser Phe Ser Arg Ser Asp 65 70 75 80 His Leu Ser Arg His Gln Arg Thr His Gln Asn Lys Leu Glu His His 85 90 95 His His His His 100 <210> 5 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> QNK-QNK-RHR <400> 5 atgatgtgca aaaccgggga gaaacgcccg tacaaatgtc caggttgcgg gaaaagcttt 60 agccagagtt ccaatctgca gaaacatcag cgaacccata ccggcgaaaa gccgtataaa 120 tgtcccggat gtggtaagag cttctcacag tctagtaacc tccagaagca tcagcgtact 180 cataccggtg aaaaaccgta taagtgccct ggatgcggta aatccttctc tcgcagtgat 240 cacctgagta gacaccaacg tacgcaccag aataaactcg agcaccacca ccaccaccac 300 300
Claims (28)
(a) forming a zinc finger-DNA complex by binding a DNA template containing at least one zinc finger binding sequence that specifically binds to the zinc finger protein to a zinc finger protein to which the first binding substance is modified; And
(b) combining the zinc finger-DNA complex with the nanoparticles modified with the second binding material to form a nanoparticle cluster having the zinc finger-complex bound to the nanoparticle;
Wherein the first binding material is biotin and the second binding material is selected from the group consisting of streptavidin, avidin, neutravidin and captavidin,
The first binding substance is selected from the group consisting of streptavidin, avidin, neutravidin and captavidin, and the second binding substance is biotin. ,
Wherein said zinc finger protein is QNK-QNK-RHR or Cys2His2,
The nanoparticles may be at least one selected from the group consisting of nanoparticles doped with magnetic particles, gold nanoparticles, silver nanoparticles, and quantum dots, or a combination thereof.
Here, the binding between the zinc finger-DNA complex and the nanoparticle may be characterized by an avidin-biotin bond,
Wherein said zinc finger protein is QNK-QNK-RHR or Cys2His2,
The nanoparticles may be at least one selected from the group consisting of nanoparticles doped with magnetic particles selected from the group consisting of Fe 2 O 3, Fe 3 O 4 , Zn 2+ and Mn 2+, gold nanoparticles, silver nanoparticles and quantum dots. Or a combination thereof.
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