KR102008622B1 - A linker material for introducing biological materials and a magnetic nanoparticle attached the said linker - Google Patents

Abstract

The present invention, in a method for measuring biomaterials such as DNA, RNA, antibodies in vivo using magnetic nanoparticles, relates to magnetic nanoparticles in which a magnetic nanoparticle is bound to a linker that specifically binds to a biomaterial in order to efficiently detect the biomaterials and the like when the magnetic nanoparticles are injected in vivo. Magnetic nanoparticles surface-treated with the linker compound provided in the present invention can specifically bind to biomaterials such as DNA, RNA, antibodies, proteins, etc., thereby simplifying the biomaterial measurement process. In addition, the magnetic nanoparticles may specifically bind to a desired biomaterial, thereby improving the specificity and sensitivity of the magnetic label.

Description

Linker material for introducing biological materials and a magnetic nanoparticle attached the said linker}

The present invention provides a method for measuring biomaterials such as DNA, RNA, and antibodies (Ab) in vivo using magnetic nanoparticles, wherein the magnetic nanoparticles efficiently infuse the biomaterials when the magnetic nanoparticles are injected in vivo. The present invention relates to a magnetic nanoparticle having a linker that specifically binds a biomaterial to a magnetic nanoparticle for detection.

Generally, a variety of nanoparticles can be used to measure the activity and activity effects of genetic materials such as DNA and RNA, and proteins such as antibodies. As nanoparticles, gold nanoparticles, semiconductor nanoparticles, magnetic nanoparticles and the like are mainly used. The present invention relates to magnetic nanoparticles among the nanoparticles, and a linker compound capable of specifically binding a biological material to magnetic nanoparticles and a magnetic compound into which the linker compound is introduced so as to easily bind the biological material such as the genetic material or the protein. It relates to nanoparticles.

Fluorescent and magnetic polymeric particles are known for use as markers and indicators in various biomedical assays. Among them, the markers most commonly used for cell sorting are, for example, immunoconjugates or immunological labels comprising immuno-fluorescent and immuno-magnetic labels. Immuno-fluorescent labels generally include, for example, fluorescent molecules bound to the antibody. Immuno-magnetic labels generally include strong paramagnetic particles that are bound to, for example, primary or secondary antibodies. Cell labeling can be performed, for example, by attaching the antibody to a subject marker (eg, a receptor site) on the surface of the cell, ie, a cell surface marker. However, the chemical and physical structure of cell surface markers and the density of immunological labels attached to the cell surface are generally difficult to accurately measure.

Magnetic particles, such as known magnetically active substances, may, for example, be bound or attached to antibodies, such as monoclonal antibodies, specificity for a particular cell type, antigen, or other target. The resulting magnetic-antibody can then be mixed with a large population of many individual cell types such as, for example, crude tissue samples, cells grown in a reactor, and the like. The magnetic-antibody is therefore attached to a pre-selected target cell type, forming a magnetic-antibody-cell conjugate. This conjugate can then be separated from the rest of the cell population using a magnetic field. The cell or other biological target measurement object is for example by binding a specific paramagnetic compound to a specific hapten on the cell, or the paramagnetic metal or metal complex directly or specifically specific to a cell, such as a metal binding microorganism. The disadvantage of magnetic particles is the lack of specificity of the magnetic label, as paramagnetic will be imparted by a number of individual pathways that can confuse the analytical and diagnostic information produced by or by methods such as phagocytosis. . Other problems faced by magnetic particles used in detection and diagnostics include, for example, the difficulty in obtaining highly accurate quantification of magnetic susceptibility in cell populations.

Therefore, it is very important to attach magnetic nanoparticles selectively to the biological material to be measured.

Efforts have been made to overcome these problems by modifying the magnetic nanoparticle surface with silica compounds. Silica is chemically stable, improves dispersibility of nanoparticles, and has a high concentration of silanol groups on its surface, which can be used for various surface bonding reactions and immobilization of other molecules. In a commonly used sol-gel process, it is easy to control the thickness of the silica layer formed on the outer surface of the magnetic nanoparticles by controlling ammonia and TEOS / H ₂ O ratio. The silica shell not only protects the magnetic core but also secures dispersibility and oxidation stability in aqueous solution. However, as the thickness of the nonmagnetic silica layer increases, the saturation magnetization of the magnetic nanoparticles decreases relatively rapidly.

When a large amount of magnetic nanoparticles are bound to the outer core of the silica core in the form of a cluster, the magnetic value (that is, the degree of saturation magnetization) and the size can be controlled by controlling the size of the silica core and the number of magnetite bound to the outer shell. Stoeva, J. Am. Chem. Soc., 127, 15362 (2005), a single silica core-clustered magnetite shell incorporating negatively-charged Fe ₃ O ₄ (15 nm) nanoparticles onto an amine-functionalized silica core surface by electrostatic interaction. The complex was formed. Nagao prepared positively-charged magnetic nanoparticles (8 nm) in Langmuir, 24, 98049808 (2008), and then mixed them with silica nanoparticles (negatively-charged) prepared by the Stutter method. The nanocomposite was electrostatically bonded to the surface. And again through the coating process of sodium silicate to form a thin silica layer on the outer shell to improve the stability of the magnetic nanoparticles. Salgueirino-Maceria is available from Adv. Func. Mater., 15, 1036-1040 (2005) combines cobalt / cobalt oxide nanoparticles on the surface of silica nanoparticles using layer-by-layer (LbL) technology to form a clustered outer layer and then coated with silica layer again It was. The manufacturing method of the magnetic nanocomposite composed of such a single silica core-magnetite cluster shell is complicated to fix, and it is difficult to recover a large amount of magnetic nanoparticles not bound to the silica core shell, and the oxidation stability of the magnetic nanoparticles bonded to the surface is difficult. And there is a problem that it is necessary to form a secondary protective layer for the surface functionalization.

The present inventors have developed a linker compound that can specifically bind to a biological material while maintaining the magnetization of the magnetic nanoparticles, and measure the genetic material, protein, etc. in vivo by binding the linker compound to the magnetic nanoparticles. The present invention has been completed by developing magnetic nanoparticles having high bonding efficiency, excellent sensitivity and affinity, and greatly simplifying the process of producing charitable nanoparticles.

Korean Patent Publication No. 10-2011-0103009 Korean Patent Publication No. 10-2011-0035010

The present invention is a magnetic label by modifying the surface of the magnetic nanoparticles by using a linker compound that allows the magnetic nanoparticles to specifically bind to the desired biomaterials when measuring the biological material using the magnetic nanoparticles To improve the specificity and sensitivity of the solution.

In addition, the conventional technology has a problem that the process is complicated because the measurement experiments must be carried out by independently applying the magnetic nanoparticles, the antibody and the surfactant, etc. In the present invention, the biological material by applying the surface-modified magnetic nanoparticles The advantage of simplifying the measurement is that

In order to solve the above problems, the present inventors have developed a linker compound represented by the following Chemical Formulas 1 and 2, which enables specific binding to biological materials such as DNA, RNA, proteins, antibodies, and the like, to magnetic nanoparticles The above problem could be solved by binding to the particles.

[Formula A]

In said formula, m is an integer of 1-20, Preferably it is an integer of 1-10, More preferably, it is an integer of 1-5.

R ₁ is an alkyl silane or alkoxy silane having 1 to 5 carbon atoms.

[Formula B]

N is an integer of 1-20, Preferably it is an integer of 1-10, More preferably, it is an integer of 1-5.

R ₂ is an alkyl group having 1 to 5 carbon atoms or an alkoxy group.

The present invention can simplify the biomaterial measurement process using the magnetic nanoparticles by introducing a linker that can specifically bind to biological materials such as DNA, RNA, antibodies, proteins, etc. on the surface of the magnetic nanoparticles. Since the particles can specifically bind to the desired biomaterial, there is an advantage in that the specificity and sensitivity of the magnetic label can be improved. In addition, since the magnetic nanoparticles have excellent antibody selectivity and binding degree, high concentrations of the magnetic nanoparticles and the antibody conjugate can be obtained, and thus the reliability of the measurement results is remarkably increased compared to the prior art.

1 is a comparison of the amount of adhesion of the rabbit IgG of the magnetic nanoparticles with the linker compounds A and B provided in the present invention and the conventional magnetic nanoparticles.
Figure 2 compares the amount of adhesion of the mouse IgG of the magnetic nanoparticles with the linker compounds A and B provided in the present invention and the conventional magnetic nanoparticles.

The present inventors have completed the present invention by developing a linker compound represented by the following formulas (A) and (B).

[Formula A]

In said formula, m is an integer of 1-20, Preferably it is an integer of 1-10, More preferably, it is an integer of 1-5.

R ₁ is an alkyl silane or alkoxy silane having 1 to 5 carbon atoms.

[Formula B]

N is an integer of 1-20, Preferably it is an integer of 1-10, More preferably, it is an integer of 1-5.

R ₂ is an alkyl group having 1 to 5 carbon atoms or an alkoxy group.

Hereinafter, a method for preparing the compound represented by Chemical Formulas A and B will be described in detail.

The present invention relates to the synthesis of linkers for the introduction of biomaterials such as DNA / Protein and Ab to the magnetic nanoparticles, as a specific embodiment of the synthesis of the linker introduced to the optical magnetic nanoparticles represented by the formula (1) and (2) It is about:

Hereinafter, the present invention will be described in more detail through examples and comparative experiments. However, the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

First, experimental apparatus, analytical equipment, and reagents used in Examples and Comparative Experiments will be described.

Instruments for FT-NMR spectroscopy were measured using Avance 300 and 500 from Bruker and LC / MSD (G-1956B) from Agilent for LC / MS.

Column chromatography for the separation and purification of organic compounds used Merck's kieselgel 60 (230-400 mesh) as a silica gel in the normal phase, silica gel 60 GF254 for thin layer chromatography (TLC) (0.25 mm, Merck) was applied, and the compound identification on TLC used ultraviolet rays of 254 nm and 365 nm, or Ceric Ammonoum Molybdate (CAM) or KMnO ₄ was used as a coloring agent.

Example 1 Preparation of a Compound of Formula 1

[Formula 1]

In the above formula, TMS represents trimethylsilyl ((CH ₃ ) ₃ Si).

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are not intended to limit the scope of the present invention as long as it illustrates the present invention.

(1) Synthesis of Compound a (I-0561)

Tetraethylene glycol (20 g, 0.10 mol, 1 eq, TCI) and succinic anhydride (51.5 g, 0.51 mol, 5 eq, TCI) are placed in a round flask and dissolved in 500 ml of chlorform. Then pyridine (41.6 ml, 0.51 mmol, 5 eq, Deoksan) was added and stirred at 60 ° C. for 5 hours. Then, the solvent was dried by distillation under reduced pressure, and 500 ml of 5% aqueous sodium bicarbonate solution (NaHCO ₃ ) was added thereto. To increase the acidity, 6M hydrochloric acid (HCl) solution was adjusted to pH 1, extracted with dichloromethane and acid solution, and the dichloromethane layer was dried over magnesium sulfate, filtered, and distilled under reduced pressure. Yellow liquid particles were obtained (40.1 g, 98.8%).

R _f = 0.25 (Silicagel, dichloromethane / methanol 10: 1 v / v)

LC / MS, calculated C ₁₆ H ₂₆ O ₁₁ 394.37, found 417.3 (+ Na ⁺ )

(2) Synthesis of Compound b (I-0691)

(Bn in the above formula represents a benzene ring)

To a round flask, Compound a (10 g, 0.025 mol, 1 eq) and 100 ml of Dichloromethane were added, and triethylamine (5.29 ml, 0.038 mol, 1.5 eq, TCI) was added at -20 ° C. Add Benzyl bromide (3.92 ml, 0.032 mol, 1.3 eq, TCI) and 100 ml of dichloromethane to the dropping funnel, and slowly add and stir for about 2 hours. Thereafter, the mixture was stirred at room temperature for 16 hours. After extracting with dichloromethane and 1M aqueous hydrochloric acid solution, the dichloromethane layer was dehydrated with magnesium sulfate and filtered to obtain yellow liquid particles through distillation under reduced pressure, and the developing solution was mixed with dichloromethane and methanol in a ratio of 10: 1. Purification by silica gel normal chromatography gave pure compound b (2.08 g, 16.9%).

R _f = 0.15 (Silicagel, dichloromethane / methanol 10: 1 v / v)

LC / MS, calcd C ₂₃ H ₃₂ 0 ₁₁ 484.50, found 523.2 (+ K ⁺ )

(3) Synthesis of Compound c (I-0712)

After adding compound b (2 g, 4.12 mmol, 1 eq) and 100 ml of chloromethane (Dichloromethane), the solution was placed in a round flask, and EDCI (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) ( 1.02 g, 5.36 mmol, 1.3 eq, TCI) and 4-dimethylaminopyridine (DMAP) (50 mg, 0.41 mmol, 0.1 eq, TCI), followed by Trimethylsilyl ethanol (0.76 ml , 5.36 mmol, 1.3 eq, TCI) was added slowly and stirred for about 10 minutes. Thereafter, the mixture was stirred at room temperature for 16 hours. After extracting ethyl acetate (Ethyl acetate) with 5% citric acid, saturated aqueous sodium bicarbonate (NaHCO ₃ ) and saturated aqueous sodium chloride solution (Sodium Chloride, Brine), the dichloromethane layer was extracted with magnesium sulfate. After removing water with filtration, and dried by distillation under reduced pressure, and purified by silica gel normal chromatography with a developing solution in which the ratio of ethyl acetate and hexane was mixed 1: 2 to obtain a pure compound c (1.77 g, 73.4%).

R _f = 0.4 (Silicagel, ethyl acetate / hexane 1: 2 v / v)

LC / MS, calculated C ₂₈ H ₄₄ O ₁₁ Si 584.73, found 623.2 (+ K ⁺ )

(3) Synthesis of Compound (Compound A) of Formula 1 (I-0713)

Compound c (1.74 g, 2.97 mmol, 1 eq) and 10 wt% Palladium hydroxide (Pd (OH) ₂ / C) (0.41 g, 0.59 mmol, 0.2 eq) were placed in a round flask, nitrogen was replaced in vacuo. 10 ml of ethyl acetate anhydride was injected into the syringe, and then hydrogen-substituted in a vacuum state and stirred for 4.5 hours. Thereafter, the mixture was filtered through celite, and the filtrate was dried through vacuum distillation to obtain pure Compound A. (0.96 g, 65.3%)

R _f = 0.2 (Silicagel, ethyl acetate / hexane 1: 2 v / v)

LC / MS, calculated C ₂₁ H ₃₈ 0 ₁₁ Si 494.61, found 517.0 (+ Na ⁺ )

¹ H NMR (500 MHz, CDCl ₃ ): δ 7.60 (s, 1H), 7.58 (d, 1H, J = 8.32 Hz), 7.32 (d, 1H, J = 7.99 Hz), 2.08 (s, 3H), 1.06 (s, 6H)

Example 2 Preparation of a Compound of Formula 2

[Formula 2]

In the above formula, EtO represents an ethoxy group.

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are not intended to limit the scope of the present invention as long as it illustrates the present invention.

(1) Synthesis of Compound d (I-0707)

10-Undecenoic acid (10 g, 0.54 mol, 1 eq, TCI) and Potassium carbonate (15 g, 1.08 mol, 2 eq, Ducksan) are placed in a round flask and 50 ml of dimethylformamide. After dissolving, benzyl bromide (7 ml, 0.54 mmol, 1.11 eq, TCI) was added thereto, followed by stirring at 80 ° C. for 16 hours. After cooling to room temperature, the mixture was extracted with diethyl ether and distilled water, and then the diethyl ether layer was dried over magnesium sulfate, filtered, and filtered to obtain yellow liquid particles through distillation under reduced pressure (14.8 g, 100%).

R _f = 0.55 (Silicagel, hexane)

LC / MS, calculated C ₁₈ H ₂₆ O ₂ 274.40, found 278.1

(2) Synthesis of Compound e (I-0708)

To a round flask, add compound d (2 g, 7.28 mmol, 1 eq) and 19-21.5 wt% Karstedt'catalyst (0.96 g, 10 mol%, TCI) and add triethoxysilane (2.69 ml, 14.57 mmol, 2 eq, Sigma aldrich) was added and stirred at 80 ° C. for 24 hours at room temperature. Purified by silica gel normal chromatography with a developing solution in which the ratio of ethyl acetate and hexane was 1: 9 to obtain a pure compound e (921 mg, 28.8%).

R _f = 0.35 (Silicagel, ethyl acetate / hexane 1: 9 v / v)

LC / MS, calculated C ₂₄ H ₄₂ O ₅ Si 438.68, found 517.0 (+ 2K ⁺ )

(3) Synthesis of Compound (Compound B) of Formula 2 (I-0709)

Compound e (870 mg, 1.98 mmol, 1 eq) and 10 wt% Palladium on carbon (Pd / C) (0.21 g, 0.19 mmol, 0.1 eq) were placed in a round flask, nitrogen-substituted in vacuo, and ethyl acetate 10 ml of anhydride was injected into the syringe, and then hydrogen-substituted in a vacuum and stirred for 4.5 hours. Thereafter, the mixture was filtered through celite, and the filtrate was dried through vacuum distillation to obtain pure Compound B (0.591 mg, 85.5%).

R _f = 0.1 (Silicagel, ethyl acetate / hexane 1: 9 v / v)

LC / MS, calculated C ₁₇ H ₃₆ O ₅ Si 348.56, found 389.2 (+ K ⁺ )

¹ H NMR (500 MHz, CDCl ₃ ): 3.83-3.78 (q, 4H, J = 3.81 Hz), 2.35-2.32 (t, 2H, J = 2.33 Hz), 1.65-1.59 (m, 2H, J = 1.62 Hz), 1.29-1.20 (m, 23H, J = 1.23 Hz), 0.53-0.50 (m, 1H, J = 0.61 Hz), 0.05-0.03 (d, 1H, J = 0.04 Hz)

Test Example 1 Labeling Test of Magnetic Nanoparticles

The linker compounds represented by Chemical Formulas 1 and 2 according to the present invention were attached, and a test for carboxylating the magnetic nanoparticles coated with silica compounds was performed.

end. Cover experiment

Magnetic nanoparticles ZnMnFe ₂ O ₄ @SiO ₂ Prepare 1mg each sample, add 300uL of mixed solvent EtOH, leave the magnet stand, remove the filtrate, and then put again 300uL of EtOH and wash the sample. After repeating this three times, prepare the final volume to fill 200uL. Next, 10 mg of the linker compound represented by Compound A and Compound B were weighed in a new tube, and 300 μL of EtOH was added thereto, mixed with Vortex, and then mixed by adding 200 μL of the prepared magnetic nanoparticles. In this process, the homogenous distribution of the linker compound and the magnetic nanoparticles were released in a water sonicator, followed by overnight reaction at room temperature in a multi-mixer (B2, 15; mode) in an incubator. In order to remove the remaining linker compounds A and B after the reaction, the mixture was left to stand in a magnetic stand and the remaining filtrate was removed, followed by mixing by adding 500 µL of EtOH. This process was repeated three times to remove residual residue.

I. Analysis of Antibody Binding Ability of Magnetic Nanoparticles with Linker Compounds

Goat anti-Rabbit IgG (H + L) (31210, ThermoFisher Scientific, San Jose, CA, USA) to confirm that the terminal portions of the magnetic nanoparticles to which Compounds A and B are bound are modified with a carboxyl group (-COOH). Antibodies and Goat anti-Mouse IgG (H + L) (31160, ThermoFisher Scientific, San Jose, Calif., USA) antibodies were attached to bind the magnetic nanoparticle carboxyl group to the antibody. Analyzes were made with MagnaBind Carboxyl Derivatized Beads (21353, ThermoFisher Scientific, San Jose, Calif., USA) already carboxylated for comparison with magnetic nanoparticles prepared by the prior art. The magnetic nanoparticles prepared in this test example in which the silane linkers represented by Compounds A and B were introduced were dispensed into tubes each 1 mg and placed on a magnetic stand. The filtrate is then removed and 300 μL of 1 × PBS (pH 7.4) (70011044, ThermoFisher Scientific, San Jose, CA, USA) is added. Repeat this process three times, adjust the volume to the final 200 uL, mix with Vortex, and store in 1.5 mL Tube Rack.

The antibody is added to 200ug per mg of each magnetic nanoparticles and reacted for 1 to 2 hours in a 25 ℃ incubator. After completion of the reaction, each tube was allowed to stand for 30 min on a magnetic stand, and the filtrate was collected for each sample. In the collected samples, the magnetic nanoparticles were sufficiently removed to measure the concentration of the unattached antibody, the nanodrop was measured, and the antibody introduction ratio of the magnetic nanoparticles having the silane linkers represented by the compounds A and B was finally introduced. It confirmed and shown in FIG.

As shown in Table 1 below, the compound incorporating the linker according to the present invention was effectively bound to the magnetic nanoparticles. As a result of labeling each prepared antibody, it was confirmed that the antibody is bound, including the Control product (MagnaBind Carboxyl Derivatized Beads) and analyzed.

Table 1. Antibody Attachment Amount Rabbit IgG Attachment Average (ug) Control (Thermo) Compound A Compound B 6.083 109.667 105.333 Mouse IgG Attachment Average (ug) Control (Thermo) Compound A Compound B 36.500 59.500 63.333

As shown in the above table, compared to the conventional magnetic nanoparticles, the linker compound provided in the present invention is a magnetic nanoparticle whose surface is modified with a linker specific to a biological material such as an antibody on the surface of the magnetic nanoparticle in terms of antibody adhesion amount. It proved to be excellent.

The present invention is industrially available.

Claims (15)

A linker compound represented by the following Formula A which is introduced to the surface of the magnetic nanoparticles and specifically bound to a biological material
[Formula A]

Wherein m is an integer from 1 to 20, R ₁ is an alkyl silane or alkoxy silane having 1 to 5 carbon atoms.
According to claim 1, wherein in Formula A, m is a linker compound specifically bound to a biological material, characterized in that an integer of 1 to 5 The linker compound of claim 1, wherein the linker compound is represented by the following formula:

The linker compound according to any one of claims 1 to 3, wherein the biomaterial is any one selected from DNA, RNA, and antibody. Magnetic nanoparticles to which the linker compound of claim 1 is bound and specifically bound to a biological material Magnetic nanoparticles to which the linker compound of claim 3 is bound and specifically binds to a biological material The magnetic nanoparticle of claim 5 or 6, wherein the biomaterial is any one selected from DNA, RNA, and antibody. delete delete delete delete delete delete delete Method for measuring a biomaterial, characterized in that by introducing a linker compound represented by the formula A to the magnetic nanoparticles to measure any one biomaterial selected from DNA, RNA, antibodies
[Formula A]

Wherein m is an integer from 1 to 20, R ₁ is an alkyl silane or alkoxy silane having 1 to 5 carbon atoms.

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