KR102628114B1 - Novel calcium binding nanoprobe and contrast agent comprising the same - Google Patents

Abstract

The present invention relates to a novel calcium-binding nanoprobe and a contrast agent composition containing the same.
The compound represented by formula (I) according to the present invention may have selectivity and binding properties for calcium ions. Accordingly, it targets cells or tissues containing calcium ions due to its excellent affinity and emits near-infrared fluorescence for a long period of time in the body, thereby targeting bone tissue, calcific plaque, or osteogenic macrophages. It can be used as a contrast agent composition that can image the back.

Description

Novel calcium binding nanoprobe and contrast agent comprising the same}

The present invention relates to a novel calcium-binding nanoprobe and a contrast agent composition containing the same.

Bone, a dynamic tissue, undergoes modeling and remodeling through a balance between mineralization by osteoblasts and demineralization by osteoclasts. Additionally, osteoblast-like cells are observed in blood vessel walls, and vascular calcification represents a major problem in cardiovascular diseases. Importantly, hydroxyapatites (HAps) are the main inorganic minerals in bone tissue and calcified vascular cells produced by osteoblasts. Therefore, hydroxyapatite in cells and bone tissue is an attractive target for drug delivery and imaging.

Bisphosphonates bind strongly to hydroxyapatite through coordination of divalent cations with the oxygen atom of each phosphonate group. Their strong and selective affinity has led to their use as specific targeting ligands for bone tissue imaging. So far, bone-targeting ligands containing tetracycline derivatives or bisphosphonates have been used as bone-targeting imaging contrast agents that emit near-infrared fluorescence (NIRF), such as IRDye78, IRDye800CW, Alexa Fluor 647, ZW800-1, P700, and P800. It has been developed through chemical combination with NIR dye. Based on their chemical structure and properties, they are highly soluble in aqueous solutions due to the presence of sulfate groups in the near-infrared phosphor. Because near-infrared phosphors can minimize interference caused by autofluorescence from biological tissue and can minimize photon scattering from bone in the ultraviolet-visible region, near-infrared phosphor-based bone imaging contrast agents are It is the optimal fluorescent substance for in vivo bone imaging.

Recently, several researchers have combined alendronate with carbon quantum dots and fluorescein 5(6)-isothiocyanate (FITC) or rhodamine for bone target imaging and drug delivery. A nanoparticle-sized fluorescent probe was developed by chemically combining nanodiamonds labeled with rhodamine B isothiocyanate (RITC).

However, in their report, since the fluorescence wavelength of the nanoparticle probes for bone targeting is within the visible light spectrum range, these nanoparticle probes cannot avoid the interference of autofluorescence and photon scattering in the visible light region.

Meanwhile, vascular calcification (VC) occurs not only during the normal aging process but also pathologically in diabetes, arteriosclerosis, and chronic kidney disease. Two distinct patterns of vascular calcification have been identified, one of which, medial calcification, occurs in the media of blood vessels associated with phenotypic transformation of smooth muscle cells into osteoblast-like cells, while the other, One, atherogenesis, is associated with lipid-laden macrophages and intimal hyperplasia.

Intimal wall calcification can occur in relatively young patients with chronic renal failure and is common in patients with diabetes mellitus even in the absence of renal disease. The presence or absence of calcium within the intimal walls of the arteries distinguishes this type of vascular calcification from atherosclerosis-related vascular calcification. Atherosclerotic vascular calcification develops from atheromatous plaques along the intima layer of the artery. Calcification is usually most evident in large, well-advanced lesions and increases with age. The degree of arterial calcification in patients with atherosclerosis generally corresponds to the severity of the disease. Unlike intimal wall calcification, atherosclerotic vascular lesions invade the arterial lumen, regardless of whether they contain calcium, and disrupt blood flow. Localized deposition of calcium may occur within atherosclerotic plaques due to inflammation due to oxidized lipids and other oxidative stresses and infiltration by monocytes and macrophages.

Since there are no nanoprobes for diagnosing these diseases related to vascular calcification, their development is necessary.

Accordingly, the present inventors designed a new calcium-binding near-infrared nanoprobe and demonstrated binding ability to calcium-containing inorganic minerals, detection of calcium levels in vitro , targeting characteristics of bone tissue in vivo , and targeting characteristics of calcific plaques in blood vessels in vivo. was evaluated, and based on this, the present invention was completed.

Patent Document 1. Registered Patent Publication No. 10-1469156

The present invention was designed to solve the above-described conventional problems. The present invention detects intracellular calcium ions based on strong binding force with calcium ions, targets bone tissue in vivo , and targets calcified sclerotic plaques in blood vessels in vivo. The purpose is to provide a possible new calcium-binding near-infrared nanoprobe and a contrast agent composition containing the same.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

Hereinafter, embodiments of the present invention will be described in detail. However, this is presented as an example, and the present invention is not limited thereby, and the present invention is only defined by the scope of the claims to be described later.

In one embodiment of the present invention, a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof is provided:

[Formula I]

In formula I above

X is ,

, and

It is any one selected from the group consisting of;

a is an integer from 1 to 5;

b is an integer from 4 to 450; and

R ₁ , R ₂ and R ₃ are each independently -OH, -O ^- or -OR _c , and R _c is C ₁ to C ₃ alkyl.

The compound of Formula I is an amphipathic calcium-targeted near-infrared fluorescent compound in which alendronate, polyethylene glycol, and dibenzocyclooctyne labeled with a cyanine-based fluorescent substance are conjugated through click chemistry.

Alendronate is a member of the bisphosphonate series and binds strongly to the calcium of hydroxyapatite through coordination of the oxygen atom, thereby giving the compound of formula (I) the ability to target bone. That is, the bisphosphonate group of alendronate acts as a targeting ligand and has a strong binding ability to bone tissue. It may include being bonded in a covalent or non-covalent manner directly or indirectly through a conventional reaction, for example, it may include bonding through an ionic bond, an electrostatic bond, a hydrogen bond, a covalent bond, or a van der Waals bond. You can. Through this, the compound of Formula I can be applied as a novel calcium-binding near-infrared nanoprobe.

Hydrophilic polyethylene glycol serves to link alendronate and the fluorescent substance so that the compound of formula (I) can have structural stability. In addition, polyethylene glycol, which is biocompatible, plays a role in reducing the toxicity of nanoprobes and increasing circulation time in the body.

Dibenzocyclooctyne serves to link alendronate-polyethylene glycol and cyanine dye through click chemistry.

Near-infrared phosphor refers to a dye that fluoresces at near-infrared wavelengths. The near-infrared ray refers to a region with a short wavelength among electromagnetic waves in the infrared region, and is not limited thereto, but generally represents a wavelength region of about 670 nm to 900 nm. Since the rotational vibration spectrum of molecules generally appears within the above range, it is considered an important wavelength range for research on molecular structure. Near-infrared rays have better skin penetration compared to visible rays, so they can be applied to obtain molecular imaging images to view blood vessels or organs in the subcutaneous layer, or to guide sensitive parts such as nerves or blood vessels during surgical procedures. has.

Cyanine dye allows the compound of formula (I) to emit near-infrared (NIR) fluorescence.

In one specific embodiment, am.

In one embodiment, a may be 3.

In another embodiment, b may be 4 to 450.

In a preferred embodiment, b may be 4 to 350.

In a more preferred embodiment, b may be 4 to 250.

In a more preferred embodiment, b may be 4 to 150.

If b is less than 4, the hydrophilic groups present in the molecule are too small compared to the hydrophobic groups, so self-assembly cannot occur smoothly, and if b is more than 450, the size of the nanoprobe becomes too large and the hydrophilic groups present in the molecule are too small compared to the hydrophobic groups. If there are too many, problems such as self-assembly not being able to occur smoothly may occur.

In other embodiments, R ₁ , R ₂ and R ₃ are -OH, -O ^- or -OR _c and R _c is C ₁ to C ₃ alkyl.

In one preferred embodiment, R ₁ and R ₂ are each independently -OH or -O ^- and R ₃ is -OH.

The compound of Formula I is amphipathic due to hydrophobic Cy dye-labeled dibenzocyclooctyne and hydrophilic alendronate and polyethylene glycol, and thus self-assembles to form nanoparticles.

The size of the nanoparticles is preferably 50 to 500 nm.

In another embodiment, the size of the nanoparticles may be 50 to 400 nm.

In a preferred embodiment, the size of the nanoparticles may be 100 to 350 nm.

In a more preferred embodiment, the size of the nanoparticles may be 200 to 300 nm.

If the size of the nanoparticle is less than 50 nm, the strength of self-assembly is weak, so there may be difficulties in maintaining the physical form of the nanoparticle, and if the size of the nanoparticle exceeds 500 nm, the nanoparticle may not reach the target. Because there are difficulties in imaging bone using nanoparticles, difficulties may arise.

In the present invention, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, for example, inorganic ionic salts made of calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, etc. Inorganic acid salts made from iodic acid, perchloric acid, sulfuric acid, etc.; Acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid. Organic acid salts made from acids, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc.; Sulfonic acid salts made from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; Amino acid salts made from glycine, arginine, lysine, etc.; and amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts meant in the present invention are not limited by these salts listed.

In another embodiment of the present invention, a contrast medium composition containing the above compound or a pharmaceutically acceptable salt thereof as an active ingredient is provided. The contrast agent composition may circulate through blood vessels in the body and emit near-infrared fluorescence by binding to tissues or cells containing calcium through alendronate, which can bind to calcium, specifically calcium ions.

The contrast agent composition according to the present invention can target bone tissue, calcific plaque, or osteogenic macrophages.

The contrast medium composition can be used for diagnosis of diseases related to vascular calcification selected from atherosclerotic calcification or medial calcification.

In the present invention, the term “calcific plaque” may refer to a deposit or limestone formed by the influx and deposition of calcium in a plaque, which is a deposit made of lipids such as cholesterol within blood vessels.

In the present invention, the term “osteogenic macrophage” may refer to a macrophage induced by RANKL (Receptor activator of nuclear factor kappa-Β ligand), and can specifically be formed by treating macrophages with RANKL. there is. More specifically, macrophages that have acquired osteoclast characteristics by treatment with RANKL promote the intake of calcium minerals from the surroundings. As a specific example, when macrophages in bone acquire osteoclast characteristics, they can promote the loss of bone rich in calcium minerals. However, when "ectopic" osteoclast characteristics are acquired in areas other than bone, as intracellular calcium particles increase, calcification of the surrounding area is promoted, and thus, it can be expressed as having osteogenic ability. Accordingly, It can be expressed as “osteogenic macrophage”.

As used herein, the term "vascular calcification" may refer to the formation, growth, or deposition of crystal deposits of hydroxyapatite (calcium phosphate), which is an extracellular matrix, within blood vessels. .

The vascular calcification-related disease may refer to a disease caused by death or calcification of vascular cells within blood vessels, and may include, but is limited to, atherosclerotic calcification or medial calcification. That is not the case.

The arteriosclerotic (atherosclerotic) calcification may refer to vascular calcification that occurs within atheromatous plaques along the intima layer of the artery. Additionally, the medial calcification, medial wall calcification, or Moenckeberg's sclerosis may refer to calcification characterized by the presence of calcium in the arterial media wall. Calcification progresses due to the deposition of limestone, which can be mediated by various diseases in various areas such as blood vessels, joints, and breasts, and can even progress to cancer.

The contrast agent composition may be formulated for oral or parenteral administration. The formulation for parenteral administration preferably includes a sterile aqueous solution or suspension containing the compound of the present invention or a pharmaceutically acceptable salt thereof, and various techniques for preparing sterile aqueous solutions or suspensions are known in the art. The sterile aqueous solution or suspension may also contain pharmaceutically acceptable buffers, stabilizers, antioxidants, and electrolytes such as sodium chloride. Formulations for parenteral administration can be directly injected or mixed with bulk parenteral formulations.

Formulations for oral administration can vary widely and are known in the art. The formulation for oral administration includes an aqueous solution or suspension containing a diagnostically effective amount of the contrast agent composition according to the present invention. The formulation for oral administration may optionally include a buffer, surfactant, adjuvant, thixotropic agent, etc. Additionally, formulations for oral administration may contain flavors and other ingredients to increase organoleptic properties.

The contrast agent composition may have various forms including, but not limited to, liquid, suspension, paste, powder, concentrate, granulated powder for mixing to an appropriate concentration, or solid form. For example, the contrast medium composition according to the present invention may be prepared in the form of an injection, a formulation for transdermal administration, a formulation for intubation, an oral formulation, or a formulation for rectal administration, preferably for injection, and more preferably for intravenous injection. .

The contrast agent composition according to the present invention is administered in an amount effective to achieve the desired contrast effect in imaging images. Such dosages can vary widely depending on the organ or tissue being subjected to the imaging procedure, the imaging device used, etc. For example, the administered concentration for use as a contrast agent composition may be 0.1 mM to 1 M.

The contrast agent composition according to the present invention is used in a conventional manner for diagnostic imaging analysis. For example, the contrast agent composition can be administered to the mammal systemically or locally to the organ or tissue being imaged in an amount sufficient to provide appropriate visualization, and then the mammal can be subjected to fluorescent imaging.

The contrast agent composition according to the present invention can be used by a near-infrared fluorescence imaging (NIR imaging) method known to those skilled in the art, but is not limited thereto, for example, by using a near-infrared optical imaging system (NIR optical imaging system). The near-infrared fluorescence spectrum can be measured using a fluorescence spectrophotometer (FluoroMate FS-2).

The contrast medium composition according to the present invention may additionally include a stabilizer. The stabilizer may include any compound that modifies the viscosity of a material, including any fluid, semi-fluid or solid. Stabilizers suitably used in the present invention may include, but are not limited to, cellulose, gelatin, natural hydrocolloids, bentonite, locust bean gum, or any compound that appropriately modifies the viscosity of the material to facilitate imaging processing. . Natural hydrocolloids suitable for use in the contrast medium composition according to the present invention include, but are not limited to, natural seaweed extracts such as carrageenan, alginate, agar, agarose, fucellan, and xanthan gum; natural seed gums such as guar gum, locust bean gum, tara gum, tamarind gum and psillium gum; natural plant exudates such as acacia, tragacanth, karaya and ghatti gum; natural fruit extracts such as low-methoxyl pectin and high-methoxyl pectin; Contains natural and refined clay. The contrast medium composition according to the present invention is not limited thereto, but may include about 0.001% by weight to about 70% by weight of the stabilizer, preferably about 0.05% by weight to about 25% by weight of the stabilizer, more preferably It may include about 0.1% to about 10% by weight of a stabilizer.

The contrast agent composition according to the present invention may further comprise an osmotic agent that promotes the transfer of fluid from the body into the anatomical part and at least substantially inhibits the reabsorption of fluid within the anatomical part by the body. Osmotic agents suitable for use in the present invention include, but are not limited to, sugar based compounds.

The sugar-based compounds include, but are not limited to, sucrose, glucose, fructose, mannitol, mannose, galactose, aldohexose, altrose, talose, sorbitol, and Silitol, lactose, nonionic seed polysaccharides, straight-chain mannans branched from each mannose by one galactose unit, beta-D-man, alpha-D-gal, D-glcA, D- Monosaccharides, disaccharides, including gal A, L-gul, beta-D-man, alpha-Dgal (4:1), D-glucuronic acid, D-galacturonic acid, and L-glucuronic acid. , and polysaccharides. The contrast medium composition according to the present invention may contain about 0.005% by weight to about 20% by weight of the osmotic agent, and preferably contains about 1% by weight to about 5% by weight of the osmotic agent.

When used in combination, stabilizers and osmotic agents have been found to work synergistically to form improved formulations for use in medical diagnostic procedures such as diagnostic imaging. It is believed that the osmotic agent facilitates the delivery of an appropriate amount of fluid into the anatomical portion of interest, and the stabilizing agent and osmotic agent ensure that a sufficient amount of fluid is retained within the anatomical portion of interest. Accordingly, the anatomical portion is sufficiently expanded or expanded for diagnostic imaging, such that when the anatomical portion is imaged, it is sufficiently delineated on the resulting diagnostic image.

The present invention also provides nanoparticles comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

[Formula I]

In formula I above

The definitions for X, a, b, R ₁ , R ₂ and R ₃ are defined the same as previously defined.

The nanoparticles may be formed by self-assembly of the compound represented by Formula I or a pharmaceutically acceptable salt thereof.

The size of the nanoparticles may be 50 to 500 nm.

The present invention also provides a contrast medium composition containing the nanoparticles as an active ingredient.

The compound represented by formula (I) according to the present invention may have selectivity and binding properties for calcium ions. Accordingly, it targets cells or tissues containing calcium ions due to its excellent affinity and emits near-infrared fluorescence for a long period of time in the body, thereby targeting bone tissue, calcific plaque, or osteogenic macrophages. It can be used as a contrast agent composition that can image the back.

Additionally, the contrast medium composition of the present invention can be used for diagnosis of diseases related to vascular calcification.

Additionally, the compound represented by Formula I according to the present invention can be administered into the body in the form of nanoparticles through self-assembly due to its amphipathic nature.

Figure 1 shows FT-IR spectra of (a) N ₃ -PEG-NHS and (b) ALN-PEG-N ₃ .
Figure 2 shows ¹ H-NMR spectra of (a) N ₃ -PEG-NHS and (b) ALN-PEG-N ₃ .
Figure 3 shows (a) MALDI-TOF mass spectra of ALN-PEG-N ₃ (dotted line) and ALN-PEG-Cy5.5 (solid line) and (b) particle size distribution of ALN-PEG-Cy5.5 and nanoprobes. The SEM image is shown (scale bar: 200 nm).
Figure 4 shows the evaluation of the calcium binding affinity of the nanoprobe, (a) a photograph and fluorescence image comparing the degree of binding after reacting ALN-PEG-Cy5.5 with the inorganic minerals BCP, HAp, or MnO ₂ ; (b) This is the result of quantitative fluorescence signal analysis comparing the degree of binding of ALN-PEG-Cy5.5 to inorganic minerals.
Figure 5 shows the results of testing the cytotoxicity of the nanoprobe on (a) macrophages and (b) RANKL-induced osteogenic macrophages.
Figure 6 shows in vitro calcium ions detected in macrophages and RANKL-induced osteogenic macrophages using a nanoprobe.
Figure 7 shows the results of fluorescence intensity quantitatively comparing the degree of calcium binding of nanoprobes in macrophages and osteogenic macrophages.
Figure 8 shows (a) an in vivo fluorescence image to evaluate body distribution and excretion over time in Balb/c nude mice after intravenous injection of the nanoprobe, and (b) a liver extracted from the mouse 48 hours after nanoprobe administration. , shows ex vivo tissue distribution fluorescence images of the nanoprobe in the lung, spleen, kidney, and heart.
Figure 9 shows in vivo bone tissue fluorescence images obtained 48 hours after intravenous injection of ALN-PEG-Cy5.5 [ribs (R), spine (Ve), femur (F), sacrum (Sa), knee. (Kn)].
Figure 10 is a schematic diagram of (a) an in vivo atherosclerotic plaque molecular imaging protocol and (b) a multichannel fluorescence microscope in vivo molecular imaging system (Customized multichannel IVFM imaging system).
Figure 11 shows (a) the main experimental group (① mice taking warfarin; ALN-PEG-Cy5.5 injection), (b) the first control group (② mice not taking warfarin; injection of ALN-PEG-Cy5.5), (c) the two Multichannel fluorescence microscopy in vivo molecular imaging assessment (Multichannel IVFM imaging assessment) for the first control group (③ warfarin ingestion mouse; PEG-Cy5.5 injection) and (d) the third control group (④ warfarin ingestion mouse; saline injection) This is an image showing (scale bar: 1mm).
FIG. 12 shows (a) a merged image of the atherosclerotic plaque molecular imaging image of FIG. 11 (merger of FITC fluorescence filter and NIRF-Cy5.5 near-infrared filter; scale bar: 1 mm) and (b) Cy5.5 near-infrared for the merged image. This is a graph showing the results of quantitative analysis of signal strength.
Figure 13 shows (a) the main experimental group (① mice taking warfarin; ALN-PEG-Cy5.5 injection), (b) the first control group (② mice not taking warfarin; injection of ALN-PEG-Cy5.5), (c) the two (d) This image shows the results of von-Kossa staining (black dots) for the third control group (③ warfarin-ingested mouse; PEG-Cy5.5 injection) and (d) the third control group (④ warfarin-ingested mouse; saline solution injection) (scale bar) : 100μm).
Figure 14 shows (a) another image showing the von-Kossa staining results for the main experimental group (① warfarin-fed mice; ALN-PEG-Cy5.5 injection), (b) calcium deposition (von-Kossa) and cy5.5 near-infrared light. Graph of qualitative analysis results for signal correlation, (c) another image showing von-Kossa staining results, (a) a high-magnification image of dense calcification (d), and (d) another image showing von-Kossa staining results. This is a high-magnification image of spotty calcification in (a). (Scale bar: 100μm)
Figure 15 shows macrophages (marker: F4/80) and osteogenic macrophages (marker: RANK) for (a) Figure 13(a) (case 1) and (b) Figure 14(a) (case 2). This image shows the additional staining results.

Example

Below, preferred examples and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are provided only for easier understanding of the present invention, and the content of the present invention is not limited by the examples and experimental examples.

ingredient

Azide-PEG5000-NHS-ester (N ₃ -PEG-NHS) and dibenzocyclooctyne-Cy5.5 (DBCO-Cy5.5) were purchased from JENKEM Technology (Plano, TX, USA) and Lumiprobe Co., respectively. (Hallandale Beach, FL, USA). Alendronate sodium (ALN) and dimethyl sulfoxide (DMSO) were obtained from Samjin Pharmaceutical (Seoul) and Deoksan Science (Seoul). Biphasic calcium phosphate (BCP) was purchased from OssGen Corporation (Gyeongbuk). Hydroxyapatite (HAp) and deuterated chloroform (CDCl ₃ ) were purchased from Sigma (St. Louis, MO, USA). Manganese dioxide nanopowder (MnO ₂ , 98%, 50nm) was purchased from US Research Nanomaterials, Inc. (Houston, TX, USA). Phosphate buffered saline (PBS) was purchased from Lonza (Walkersville, MD, USA). RPMI 1640, fetal bovine serum (FBS), streptomycin, penicillin, and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Welgene Inc. Obtained from (Seoul, Korea). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Receptor activator of nuclear factor kappa-B ligand (RANKL); Fluorescent mounting medium Fluo-3; and 4',6-diamidino-2-phenylindole (DAPI) were purchased from R&D systems (Minneapolis, MN, USA), Dana Korea (Seoul, Korea), and GBI Labs (Bothell, WA, USA), respectively.

Example 1. Synthesis and characterization of calcium-binding nanoprobes

Calcium-binding nanoprobes were synthesized through step 2 of Scheme 1 below.

[Scheme 1]

(a) Alendronate-PEG5000-azide (ALN-PEG-N ₃ ) synthesis of

N ₃ -PEG-NHS (0.1 g, 0.02 mmol) and ALN (54.2 mg, 0.2 mmol) were dissolved in 10 mL of PBS (pH 7.4) and reacted for 24 hours. The resulting solution was purified using a dialysis membrane for 3 days and freeze-dried.

N ₃ -PEG-NHS and the obtained ALN-PEG-N ₃ were analyzed by Fourier transform infrared spectroscopy, and the results are shown in Figure 1, respectively. For FT-IR analysis, samples were prepared in the form of potassium bromide (KBr) pellets. FT-IR spectra were acquired from 4,000 to 400 cm ^-1 with a resolution of 4 cm ^-1 .

The FT-IR spectrum shows that the amide band (C=O stretching of N ₃ -PEG-NHS) disappears at 1745 cm ^-1 and the hydroxy peak (-OH stretching of ALN) at 3461 cm ^-1 increases in the product. gave.

¹ H-NMR spectra were acquired with ¹ H-nuclear magnetic resonance ( ¹ H-NMR) spectroscopy using CDCl ₃ . The NMR spectra of (a) N ₃ -PEG-NHS and (b) ALN-PEG-N ₃ are shown in Figure 2, respectively.

In ^{the 1H} -NMR spectrum, the methylene proton peak (2.72 ppm) present in NHS of N ₃ -PEG-NHS disappeared, and the methylene proton peak present in ALN was detected at 1.58 ppm. These data mean that ALN-PEG-N ₃ was successfully synthesized by combining N ₃ -PEG-NHS and ALN.

(b) Synthesis and characterization of ALN-PEG-DBCO-Cy5.5 (ALN-PEG-Cy5.5)

ALN-PEG-N ₃ (80 mg, 0.0152 mmol) dissolved in 8 mL of PBS (pH 7.4) and DBCO-Cy5.5 (31.9 mg, 0.031 mmol) dissolved in 2 mL of DMSO were reacted. After 24 h of reaction, the reaction was dialyzed using a dialysis membrane (MWCO; 3,500 Da) against distilled water containing 30% EtOH for 1 day and against distilled water for an additional 2 days. Then, the dialyzed solution was centrifuged (4,000 rpm, 20 minutes) to remove the precipitated material (unreacted DBCO-Cy5.5), and then the separated supernatant was freeze-dried to produce ALN-PEG-Cy5.5. got it

The synthesis of ALN-PEG-Cy5.5 was confirmed by measuring the molecular weight using MALDI-TOF mass spectrometry, and the results are shown in Figure 3(a). Specifically, the synthesis of ALN-PEG-DBCO-Cy5.5 (hereinafter referred to as ALN-PEG-Cy5.5) was performed by MALDI-TOF mass spectrometry, yielding ALN-PEG-N ₃ (m/z = 5,252) and ALN This was confirmed by the measured molecular weight of -PEG-Cy5.5 (m/z = 6,147) (Figure 3(a)).

Particle size, polydispersity index and Z-average size were analyzed using a particle size analyzer after dispersing ALN-PEG-Cy5.5 at 0.25 mg/mL in distilled water. To observe the particle morphology of the nanoprobe, 1 mg of ALN-PEG-Cy5.5 was dispersed in distilled water and the dispersed solution was dropped on a glass cover slip. The sample was then dried and coated with platinum. The particle morphology of ALN-PEG-Cy5.5 was analyzed using a field emission scanning electron microscope operating at 15.0 kV, and the results are shown in Figure 3(b).

ALN-PEG-Cy5.5 dispersed in distilled water has a Z-average size of 275.2 nm, a hydrodynamic size of 275.8 ± 61.8 nm, and a polydispersity index (PDI) of 0.178. The SEM image showed that ALN-PEG-Cy5.5 was spherical (inset image in Figure 3(b)). These results demonstrated that the amphiphilic ALN-PEG-Cy5.5 molecules form nanostructures by self-assembly in an aqueous solution environment, because the intermediate hydrophobic DBCO-Cy5.5 molecules without sulfate groups have limited solubility in aqueous solution. .

Example 2. in vitro Calcium binding affinity test of nanoprobes in

ALN-PEG-Cy5.5 nanoprobes (1.5 mg) dispersed in 600 μL of PBS (pH 7.4) were added and 2 with 34.5 μmoles of BCP (14.7 mg), HAp (17.3 mg), and _MnO (3 mg). It was reacted for some time. The mixture was then centrifuged at 5,500 rpm for 30 min and washed three times with PBS to remove unreacted nanoprobes. To compare the binding affinity of the nanoprobes, the collected BCP, HAp, and MnO ₂ were dispersed in 200 μL of PBS (pH 7.4), and then photographs of the dispersed samples were acquired. Additionally, the fluorescence intensity of the dispersed samples was quantified and comparatively analyzed using a small animal imaging device. The affinity test results are shown in Figure 4.

Based on the fluorescence images and quantitative analysis results, stronger NIRF signals were observed in BCP and HAp than in MnO ₂ (Figure 4A (a) and (b)), and the PCP (hydroxyl methylene bisphosphonate) structure of the nanoprobe and the calcium phosphate showed a strong interaction. These data suggest that the nanoprobe can bind more strongly and selectively to minerals containing calcium phosphate.

Example 3. Evaluation of cytotoxicity of nanoprobes against macrophages and RANKL-induced osteogenic macrophages

The in vitro cytotoxicity of the nanoprobes against macrophages and RANKL-induced macrophages (referred to as osteogenic macrophages) was evaluated using the CCK-8 assay kit. RAW 264.7 cells were cultured in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin at 37 °C. Subcultured RAW 264.7 cells (2×10 ⁴ cells/well) were transferred to a 96-well culture plate and allowed to attach for 1 day. Additionally, macrophages were treated with RANKL (50 ng/mL) for 1 day to establish osteogenic macrophages. Macrophages and osteogenic macrophages were treated with various concentrations of nanoprobes (2, 5, 10, 20, and 50 μM). After culturing for 24 hours, the cells were washed, the CCK-8 solution was added to the cells, and the cells were cultured for an additional hour and then measured at 450 nm using a flash multi-mode reader.

The toxicity evaluation results for (a) macrophages and (b) osteogenic macrophages are shown in Figure 5, respectively. As a result of the measurement, it was confirmed that at all concentrations of macrophage and osteogenic macrophage nanoprobes, the cell viability reached or exceeded 100%.

Example 4. Nanoprobe in vitro Calcium binding assay

To evaluate the in vitro calcium binding of the nanoprobe, RAW 264.7 cells (2×10 ⁵ cells/well) were cultured in 4-well chamber slides. Additionally, osteogenic macrophages were established by treatment with RANKL (50 ng/mL) for 1 day. Macrophages and osteogenic macrophages were treated with nanoprobes (10 μM) and incubated for 1 hour. Cells were then washed three times with DPBS and fixed with 3.7% formalin for 30 min. Fixed cells were stained with 4 μM Fluo-3 (ex: 506 nm, em: 526 nm), a calcium indicator, and additionally treated with DAPI to stain cell nuclei for 10 minutes. The cells were then imaged using a confocal laser scanning microscope. An image of a cell bound to the nanoprobe is shown in Figure 6. To quantitatively compare and analyze the degree of in vitro calcium binding in macrophages and osteogenic macrophages, Image J software was used. The quantitative average fluorescence intensity of a single cell was calculated by dividing the total fluorescence intensity of randomly selected cells by the number of randomly selected cells (n = 5). The results of quantitative comparison of the degree of calcium binding of the nanoprobes in macrophages and osteogenic macrophages are shown in Figure 7.

In Figure 6, the green fluorescence signal and NIRF signal increased in osteogenic macrophages compared to macrophages, and the NIRF signal of the nanoprobe shown in red matched well with the green fluorescence signal of Fluo-3 in macrophages. In Figure 7, the NIRF signal of osteogenic macrophages increased by 4.6 times compared to the NIRF signal of macrophages, which helps the nanoprobe of the present invention detect and visualize in vitro calcium levels by binding to in vitro calcium. It means it can be done.

Example 5. Nanoprobe in vivo Biodistribution and bone tissue imaging

To evaluate the temporal distribution and excretion of nanoprobes in the body, nanoprobes (2 nmoles/100 μL/mouse) were injected through the tail vein of Balb/c nude mice. Near-infrared fluorescence images were acquired using small animal imaging equipment at 0.5, 1, 2, 3, 6, 12, 24, and 48 hours after injection, and the results are shown in Figure 8.

Until the early hours after nanoprobe injection, a strong NIRF signal was observed, after which the intensity gradually decreased. However, the NIRF signal remained visible for up to 2 days, demonstrating prolonged in vivo circulation of the nanoprobe. 48 hours after injection, no fluorescent signal of the nanoprobe was detected in the lung, spleen, kidney, or heart, but NIRF signal was still detected in liver tissue due to its amphipathic nature.

Figure 9 shows the results of bone tissue fluorescence images obtained 48 hours after ALN-PEG-Cy5.5 injection. 48 hours after injection, the nanoprobe imaged the ribs, spine, femur, and sacrum. Additionally, the fluorescence signal of the nanoprobe was observed in cartilage tissue, including bone and knee joint, suggesting that the calcium-binding near-infrared fluorescent nanoprobe of the present invention successfully visualizes bone tissue in vivo .

Example 6. Nanoprobe in vivo Calcium-targeted imaging within atherosclerotic plaques

To evaluate the calcium-targeting properties of the nanoprobe in atherosclerotic plaques, calcium in atherosclerotic plaques was measured in 8-week-old ApoE(-/-) transgenic mice after a high-cholesterol diet for a total of 8 weeks and warfarin intake for a total of 4 weeks. promoted creation. Afterwards, signals from atherosclerotic plaques in both carotid arteries of mice were measured through in vivo molecular imaging of atherosclerotic plaques.

The in vivo atherosclerotic plaque molecular imaging protocol is shown in Figure 10(a). Specifically, an imaging experiment was conducted with one main experimental group (①) and a total of three control groups (②, ③, ④) as follows.

① In the main group, in vivo imaging was performed 48 hours after injection of 2.5 mg/kg of ALN-PEG-Cy5.5 nanoprobe into the tail vein.

② The first control group was the experimental animal control group, and in vivo imaging was performed 48 hours after injecting 2.5 mg/kg of Al-PEG-Cy5.5 nanoprobe into the tail vein in mice that did not consume warfarin.

③ The second control group was a nanoprobe material control group, and in vivo imaging was performed 48 hours after injection of 2.5 mg/kg of PEG-Cy5.5 nanomaterial without calcium targeting ligand into the tail vein.

④ The third control group was a nanoprobe material control group, and in vivo imaging was performed 48 hours after injection of saline solution into the tail vein.

In addition, molecular imaging was performed using a multi-channel fluorescence microscope in vivo molecular imaging system, and a schematic diagram of the imaging system is shown in Figure 10(b).

Specifically, in the imaging system, multiple laser wavelengths are generated in an excitation module and irradiated to the atherosclerotic plaque through an optical system to activate the nanoprobe. The laser emitted from the nanoprobe in the atherosclerotic plaque is transmitted to the emission module through the same optical system, and is separated into individual laser wavelengths and measured.

Figure 11 is an image showing the results of multichannel fluorescence microscopy in vivo molecular imaging assessment (Multichannel IVFM imaging assessment).

Referring to Figure 11(a), the Cy5.5 near-infrared signal was measured to be very high in the atherosclerotic plaques of the main experimental group (① warfarin-ingested mice; ALN-PEG-Cy5.5 injection) that promoted calcium production, and ALN-PEG -It can be confirmed that the uptake rate of Cy5.5 nanoprobes into atherosclerotic plaques is high. In addition, referring to Figure 11(b), the intensity of the Cy5.5 near-infrared signal was measured to be low in the atherosclerotic plaques of the first control group (② mice not receiving warfarin; ALN-PEG-Cy5.5 injection) that did not promote calcium production. Therefore, it can be seen that if calcium production is not promoted, the uptake rate of ALN-PEG-Cy5.5 nanoprobes into atherosclerotic plaques decreases. In addition, referring to Figure 11(c), as an arteriosclerotic plaque that promoted calcium production, Cy5. 5 It can be seen that the near-infrared signal is measured at a low level, which suggests that the uptake rate of the nanoprobe into the atherosclerotic plaque is reduced when it does not contain the targeting ligand, alendronate. Additionally, referring to Figure 11(d), it can be seen that the Cy5.5 near-infrared signal was not detected in the third control group (④ warfarin-ingested mouse; physiological saline injection) that was not injected with Cy5.5, a near-infrared ray-emitting material.

Figure 12 shows (a) a merged image (merger of FITC fluorescence filter and NIRF-Cy5.5 near-infrared filter) among the atherosclerotic plaque molecular imaging images of Figure 11 and (b) quantitative analysis of Cy5.5 near-infrared signal intensity for the merged image. This is a graph showing the results.

Figure 12(a) presents the main imaging results of the main experimental group (①) and a total of three control groups (②, ③, ④). In addition, referring to Figure 12(b), as a result of comparing the target to background signal ratio (TBR) for each group, the atherosclerotic plaque Cy5.5 near-infrared signal intensity was higher in the control group (②, ③, ④). Compared to this, it was statistically significantly higher in the main experimental group (①) (p-value <0.05). The target was defined as an atherosclerotic plaque, and the background was defined as a normal blood vessel without an atherosclerotic plaque. As a statistical analysis method, Kruskal-Wallis test was performed, and post-hoc analysis was performed with Mann-Whitney U-test (**P-value <0.05 by Mann-Whitney U-test, ## P-value <0.05 by Kruskal- Wallis test).

Example 7. Histopathologic validation of Cy5.5 near-infrared signal in atherosclerotic plaque (1)

To histologically verify the Cy5.5 near-infrared signal in the atherosclerotic plaque of the nanoprobe, von-Kossa staining was performed on the main experimental and control groups and compared with the NIRF-Cy5.5 near-infrared filter image. , the results are shown in Figure 13.

Referring to Figure 13(a), the main experimental group (① warfarin-fed mice; ALN-PEG-Cy5.5 injection) showed a high degree of agreement between calcium deposition in the atherosclerotic plaque (black area stained by Von-Kossa) and Cy5.5 near-infrared signal. see. This suggests that targeted imaging of calcium in atherosclerotic plaques is possible through the ALN-PEG-Cy5.5 nanoprobe. In addition, referring to Figure 13(b), in the first control group (② mice not taking warfarin; ALN-PEG-Cy5.5 injection), calcium deposition in the atherosclerotic plaque was not clear, and the intensity of the Cy5.5 near-infrared signal in the atherosclerotic plaque was low. It can be seen that the results are lower than those of the main experimental group. In addition, referring to Figure 13(c), the second control group (③ warfarin-ingested mouse; PEG-Cy5.5 injection) showed calcium deposition in the atherosclerotic plaque (black Von-Kossa staining area), but Cy5. 5 It can be confirmed that the near-infrared signal was not detected. This suggests that the targeting of calcium in atherosclerotic plaques is limited when it does not contain the targeting ligand alendronate. In addition, referring to Figure 13(d), it can be seen that the third control group (④ warfarin-ingested mouse; saline injection), which was not injected with Cy5.5, a near-infrared ray-emitting material, did not detect Cy5.5 near-infrared signal in the atherosclerotic plaque. You can.

Example 8. Histological verification of Cy5.5 near-infrared signal in atherosclerotic plaque (2)

To histologically verify the Cy5.5 near-infrared signal in the atherosclerotic plaque of the nanoprobe, von-Kossa staining was performed on the main experimental group and compared with the NIRF-Cy5.5 near-infrared filter image, and the results were compared. is shown in Figure 14.

Referring to Figures 14(a), (c), and (d), there is an overall high degree of agreement between calcium deposition (black Von-Kossa staining area) and Cy5.5 near-infrared signal in the atherosclerotic plaque of the main experimental group (①). You can check it. Morphologically, calcium deposition in atherosclerotic plaques is classified into dense calcification (area a indicated by a dotted line in Figure 14(a) and its enlarged figure in Figure 14(c)) and spotty calcification (Figure 14(a)). It can be classified into area b indicated by a dotted line and its enlarged view (Figure 14(d)). It can be confirmed that the ALN-PEG-Cy5.5 nanoprobe is capable of targeted imaging of both types of calcium deposition in atherosclerotic plaques. In addition, referring to Figure 14(b), calcium deposition (von-Kossa) in the atherosclerotic plaque and Cy5.5 near-infrared signal showed a statistical correlation at the severity level (r=0.529, p<0.001).

Meanwhile, the ALN-PEG-Cy5.5 nanoprobe signal was detected, but verification was performed through additional tissue staining in Example 8 for areas where visual von-Kossa calcium deposition was not evident.

Example 9. Histological verification of macrophage distribution and bone formation performance evaluation in atherosclerotic plaques

Figure 15 shows macrophages (marker: F4/80) and osteogenic macrophages (marker: RANK) for (a) Figure 13(a) (case 1) and (b) Figure 14(a) (case 2). This image shows the additional staining results.

Referring to Figure 15, it can be seen that there is a high overall consistency between calcium deposition in the atherosclerotic plaque (black Von-Kossa staining area) and Cy5.5 near-infrared signal (NIRF-Cy5.5). This suggests that targeted imaging of calcium in atherosclerotic plaques is possible through the ALN-PEG-Cy5.5 nanoprobe, but in some cases, there was a discrepancy between the Cy5.5 near-infrared signal and calcium deposition.

To confirm this discrepancy, additional staining was performed. As a result of additional staining, the Cy5.5 near-infrared signal in the area where calcium deposition was not evident reflected the distribution of macrophages (F4/80-positive area; staining area) and osteogenic capacity (RANK-positive area; staining area) in the atherosclerotic plaque. It was found that Through this, it was confirmed that when using the ALN-PEG-Cy5.5 nanoprobe, it is possible to target not only calcium deposition in the atherosclerotic plaque but also osteogenic macrophages with osteogenic ability.

Claims (12)

A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:
[Formula I]

In formula I above
X is ego;
a is 3;
b is an integer from 4 to 450; and
R ₁ , R ₂ and R ₃ are each independently -OH, -O ^- or -OR _c , and R _c is C ₁ to C ₃ alkyl. delete delete According to paragraph 1,
The compound represented by Formula I is a compound or a pharmaceutically acceptable salt thereof that self-assembles to form nanoparticles. According to clause 4,
The size of the nanoparticles is 50 to 500 nm. A compound or a pharmaceutically acceptable salt thereof. A contrast medium composition comprising the compound of any one of claims 1, 4, and 5 or a pharmaceutically acceptable salt thereof as an active ingredient. According to clause 6,
The contrast agent composition emits fluorescence of near-infrared (NIR). According to clause 6,
The contrast agent composition binds to calcium ions. According to clause 8,
The contrast medium composition targets bone tissue, calcific plaque, or osteogenic macrophages. According to clause 9,
The contrast medium composition is for diagnosing a disease selected from atherosclerotic calcification or medial calcification. Nanoparticles containing a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:
[Formula I]

In formula I above
X is ego;
a is 3;
b is an integer from 4 to 450; and
R ₁ , R ₂ and R ₃ are each independently -OH, -O ^- or -OR _c , and R _c is C ₁ to C ₃ alkyl. A contrast medium composition comprising the nanoparticles according to claim 11 as an active ingredient.