KR101731140B1 - Photoacoustic image agent comprising a melanin naoparticle - Google Patents

Abstract

The present invention relates to a photoacoustic imaging agent comprising melanin nanoparticles useful for diagnosis and / or treatment of diseases.

Description

The present invention relates to a photoacoustic imaging agent comprising a melanin nanoparticle,

The present invention relates to a photoacoustic imaging agent comprising melanin nanoparticles useful for diagnosis and / or treatment of diseases.

Photoacoustic imaging based on photoacoustic effects has received much attention as a biomedical imaging modality that provides biological information by visualizing intrinsic biological molecules such as melanin and hemoglobin. The high potential of photoacoustic imaging as biomedical imaging is based on the photoacoustic response of nanomaterials in terms of diagnosis and treatment, including real-time and targeted imaging with high sensitivity, multi-mode imaging and image-guided therapy It is being accelerated by the development of exogenous contrast agents.

In this regard, the development of suitable nanoprobes representing photoacoustic effects is an important technical issue in nanotechnology to improve the efficiency of photoacoustic imaging and optimize various biomedical applications. In addition, the development and artificial manipulation of nanoprobes that exhibit photoacoustic effects can provide another important insight into the fundamental understanding of photoacoustic imaging based on nanomaterials. In principle, the photoacoustic effect is due to the generation of sound waves through the absorption of short-pulsed radiation which causes local expansion of the target resulting in thermal expansion. Although the extinction coefficient and the conversion efficiency of converting the absorbed photons into heat are key factors in generating photoacoustic signals in photoacoustic imaging based on nanomaterials such as molecular level absorbers, Lt; / RTI > Since the photoacoustic effect associated with thermal expansion is generated from a locally heated surrounding medium through thermal conversion from the nanomaterial to the surrounding medium, the surface environment associated with the thermal transfer and dissipative dynamics creates a photoacoustic effect based on the nanomaterial It is a sensitive factor.

Thus, the development and manipulation of new forms of nanomaterials for a basic understanding of photoacoustic reactions based on nanomaterials can provide important clues to devising and optimizing contrast agents that cause photoacoustic imaging.

Melanin, on the other hand, is a bio-polymer present in various parts of life, including plants, animals, and protists. It is classified as black-brown eumelanin and yellow-red pheomelanin. The above eumelanin may be 3,4-dihydroxy-L-phenylalanine (L-DOPA) or 2- (3,4-dihydroxyphenyl) , 4-dihydroxyphenyl) ethylamine, dopamine), and the peomelanin is derived from L-DOPA or dopamine in the presence of a mercapto group (-SH) containing substance such as cysteine or glutathione will be. Herein, eumelanin is a predominant form of melanin in mammals, and is involved in the intramolecular addition of the amine group portion of o-quinones induced by the oxidation of catecholamine And is known as a biomolecule having a nonuniform structure including indole units formed by the polymer.

Since melanin exists in nature such as in squid ink and the like, it can be obtained by separating from the natural world, or can be obtained also by an artificial synthesis method including a reaction with an enzyme or a reaction with an oxidizing agent.

The present inventors have made intensive research to find a method for developing a nanomaterial for photoacoustic imaging and amplifying the signal, and as a result, it has been found that the melanin nanoparticle incorporating a compound having an amide group and a compound having a carboxylic acid on the surface is in an acidic condition And the photoacoustic signal of the nanoparticles can be rapidly increased during coagulation. Therefore, the present invention has been accomplished by confirming that the nanoparticles modified with the surface can be used as a contrast agent for detecting lesions showing acidity.
[Prior Art Literature]
Biomacromolecules. 14 (10), 3491-3497 (2013.08.29.)

The present invention is to provide a photoacoustic imaging agent comprising melanin nanoparticles which can be usefully used for diagnosis and / or treatment of diseases.

In order to solve the above problems, the present invention provides a photoacoustic imaging agent comprising melanin nanoparticles.

The term "photoacoustic" used in the present invention means a phenomenon in which a substance absorbs light and locally rises in temperature and propagates through the substance as a pressure, so that the absorption of minute light can be measured with high sensitivity It is also used in spectroscopy because it is also possible to measure samples that are difficult to measure with ordinary optical methods. The term "photoacoustic imaging agent " used in the present invention means a material exhibiting such a photoacoustic effect, which means a material capable of measuring and imaging signals thereof, It is a hybrid biomedical imaging modality. The photoacoustic imaging irradiates a non-ionizing laser pulse, wherein the energy delivered by the laser is absorbed by the tissue or contrast agent and converted into heat to cause a transient thermoelastic expansion And further induces a wideband (e.g., MHz) ultrasound emission. At this time, the generated ultrasonic waves are detected by an ultrasonic transducer to form an image. The emitted ultrasonic emission, ie the intensity of the photoacoustic signal, is proportional to the local energy deposition. Since the photoacoustic imaging is a phenomenon generated by absorbing the irradiated light, optical absorption is an important factor for the photoacoustic imaging. In biological tissues, optical absorption is by an intrinsic molecule such as hemoglobin or melanin or by a contrast agent introduced from the outside.

The photoacoustic imaging agent according to the present invention is a substance to be administered for easy detection in medical imaging. Since it is administered into the body, it must be a very safe substance and should be able to be expressed immediately and rarely have allergic reaction in the subject. In the present invention, such a photoacoustic imaging agent uses melanin nanoparticles which are safe in the body and excellent in photoacoustic effect.

The term "melanin nanoparticles" as used herein means very small melanin particles having an average diameter on the order of nanometers. Preferably, the melanin nanoparticles may have a diameter of from 30 nm to 600 nm.

The melanin nanoparticles are highly reactive with functional groups such as an amine group or a thiol group, and thus the surface of the melanin nanoparticles can be easily modified with a compound having such a functional group.

The melanin nanoparticles can be separated and used in nature. For example, it can be extracted from squid ink and used. Or a melanin precursor of dopamine, DOPA or cysteine. For example, melamine can be produced by polymerizing dopamine while oxidizing dopamine formed by a chemical reaction between a dopamine · H ⁺ X ^- containing aqueous solution (where H ⁺ X ^- is an acid) and a base. When the molar ratio of dopamine · H ⁺ X ^- and base is adjusted during the addition of the base, melanin can be produced in a particle state having a uniform shape, and further, melanin particles having a particle size adjusted to a nanometer level can be produced.

The melanin nanoparticles used in the present invention can be used as a photoacoustic imaging agent without special modification due to its own photoacoustic effect. The melanin nanoparticle alone does not produce a signal specifically for a lesion of a disease such as a cancer or an inflammatory disease because the photoacoustic signal is weak, and the signal can be enhanced by agglutinating the melanin nanoparticles.

Surprisingly, it has been confirmed that the melanin nanoparticles according to the present invention are aggregated under specific conditions, thereby confirming that the photoacoustic signal is strengthened. The conditions under which the melanin nanoparticles according to the present invention coagulate may vary. For example, the melanin nanoparticles according to the present invention can be aggregated under acidic conditions, aggregated in the presence of metal ions, or aggregated in the presence of biomolecules. The conditions thereof will be described in detail.

First, the melanin nanoparticles according to the present invention can be aggregated under acidic conditions, which can be achieved by introducing functional groups capable of aggregating on the surface of melanin nanoparticles under acidic conditions.

Examples of functional groups capable of aggregating under acidic conditions include carboxyl groups and amine groups. Since carboxyl groups and amine groups are ionized under acidic conditions to attract each other, when a compound having such a carboxyl group and an amine group is introduced onto the surface of the melanin nanoparticles according to the present invention, ionization occurs at a specific acidic condition and the surface of each neighboring melanin nanoparticle Melanin nanoparticles can aggregate with each other due to carboxyl groups and amine groups and attraction.

The acidic condition is preferably pH 2 to 6. Due to this characteristic, the melanin nanoparticles according to the present invention can be usefully used for diagnosis of lesions caused by photoacoustic imaging. In general, the cellular environment becomes acidic locally in lesions in which various diseases such as inflammatory diseases and cancer diseases appear. Therefore, when the surface modified nanoparticles according to the present invention are injected into the body, the nanoparticles may circulate in the body and aggregate in acidic lesions to emit strong photoacoustic signals. In particular, the melanin nanoparticles have an advantage of being able to diagnose the lesion in vitro because the absorption wavelength is about 700 nm and absorbs a long wavelength near infrared which is relatively transparent, and emits a sound wave.

Examples of the compound having a carboxyl group or an amine group are compounds represented by the following formulas (1) and (2), respectively.

[Chemical Formula 1]

(2)

or

In this formula,

R is a C _{1 -6} alkylene

The above general formulas (1) and (2) all have amine groups and can be modified on the surface of the melanin nanoparticles through such amine groups (one amine group in the case of Formula (1)). Since the modified melanin nanoparticles have an amine group and a carboxyl group at the terminal ends of the above formulas 1 and 2, they are ionized under specific acidic conditions to form carboxy groups and amine groups and attraction groups on the surface of neighboring melanin nanoparticles, .

Preferably X is ethylene (-CH ₂ CH ₂ -), but is not limited thereto. The X can be selected without limitation as long as melanin nanoparticles form agglomerates so that they can generate a photoacoustic signal amplified by overlapping the thermal field of the individual particles while maintaining a constant gap between the particles have.

In addition, the melanin nanoparticles of the present invention may further be modified with polyethylene glycol on the surface thereof. The polyethylene glycol used in the present invention allows the melanin nanoparticles to be well dispersed in an aqueous solution. The molecular weight of the polyethylene glycol is preferably 1 kDa to 40 kDa. Since the nanoparticles having improved dispersibility in aqueous solution can be present in the body for a long time, sufficient time can be secured for the melanin nanoparticles to reach specific tissues or cells even by systemic administration with an injection or an oral administration agent . The polyethylene glycol may be modified on the surface of the melanin nanoparticles through the terminal thiol group, and therefore, it is preferable that the polyglycol is a general formula mPEG-SH.

As another condition for the melanin nanoparticles according to the present invention to aggregate, they can be aggregated in the presence of metal ions.

An example of a functional group capable of aggregating melanin nanoparticles in the presence of a metal ion is a functional group capable of coordinating with a metal ion. When a compound having a functional group capable of coordinating with metal ion is introduced on the surface of the melanin nanoparticle according to the present invention, the functional group is coordinatively bonded to the metal ion in the presence of the metal ion, and the metal ion is coordinated with another functional group As a result, the melanin nanoparticles can aggregate. The metal ion is preferably a transition metal such as Na, Mg, Al, K, Ca, Mn, Fe, Cu, Zn, Cd or Pb and an alkaline earth metal. Examples of the compound having a functional group capable of coordinating with the metal ion include a compound represented by the following formula (3) or (4).

(3)

[Chemical Formula 4]

Wherein, X is -COOH, -NH _2, catechol, or phosphate.

The above formulas 3 and 4 have an amine group or a thiol group and can be modified on the surface of the melanin nanoparticle through the amine group or the thiol group. Since the modified melanin nanoparticles have a functional group capable of coordinating with the terminal metal ion of the above formulas 3 and 4, when the metal ion is present, a plurality of melanin nanoparticles coordinate-bond with the metal ion to form melanin nanoparticles It can coalesce.

As another condition for the melanin nanoparticles according to the present invention to aggregate, they can be aggregated in the presence of specific biomolecules.

The photoacoustic imaging agent comprising the melanin nanoparticles according to the present invention can agglomerate under conditions of lesions such as cancer or inflammatory spots, thereby significantly improving photoacoustic signals. Also, it is well dispersed in water, has no cytotoxicity, and has a long residence time in vivo, so that it can be useful for diagnosis and / or treatment of diseases as a photoacoustic imaging agent.

1 is a diagram showing a photo-acoustic signal for (a) a TEM image of melanin-like nanoparticles synthesized according to a specific embodiment of the present invention, (b) molar concentration of the nanoparticles and (c) absorbance. Methylene blue and gold nanorods were used as comparative examples.
2 is a graph showing the influence of the melanin nanoparticles according to the present invention on the photoacoustic signal caused by agglomeration. (a) is a schematic diagram showing the surface modification and agglomeration of melanin nanoparticles designed to agglomerate under acidic conditions. Citraconamide, introduced on the surface of nanoparticles, hydrolyzes under acidic conditions to change the positive surface charge on the particle and consequently induces aggregation of the nanoparticles. (b) is a diagram showing photoacoustic slice vertical images of a tube comprising surface modified melanin nanoparticles under acidic conditions compared to neutral conditions. (c) shows the photoacoustic signal trajectory over time from the individual inclusions under acidic and neutral conditions.
Figure 3 shows the hydrodynamic radii and optical properties of the surface modified melanin nanoparticles according to the invention under acidic and neutral conditions. (a) shows changes in hydrodynamic radius over time under acidic and neutral conditions. (b) shows the change of absorbance (λ = 700 nm) due to optical scattering over time under acidic and neutral conditions. (c) is a graph showing changes in photoacoustic signals, hydrodynamic radius and absorbance (λ = 700 nm) of the surface-modified melanin nanoparticles over time under acidic conditions. Over time, agglomeration progressed and the hydrodynamic radius of the particles increased, resulting in a nonlinear increase in the photoacoustic signal.
FIG. 4 is a diagram showing the amplification of a photoacoustic signal caused by agglomeration of melanin nanoparticles and the mechanism thereof. (a) shows a change in photoacoustic signal according to a particle size change, and (b) shows a correlation between a concentration and a photoacoustic signal. (c) schematically illustrate the mechanism for explaining the photoacoustic signal generation from nanosized photoacoustic imaging agents and signal amplification due to particle agglomeration.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Manufacturing example  1: Preparation of melanin nanoparticles

Melanin nanoparticles were prepared according to the method described in Korean Patent No. 10-1227322.

Specifically, 180 mg of Dopamine hydrochloride (3,4-dihydroxyphenethylamine) HCl was dissolved in 90 ml of deionized water to obtain an aqueous solution containing about 10.5 mmol / l of dopamine · HCl (pH = 6.8). 7, the dopamine · HCl-containing aqueous solution was mixed with 760 μl of 1N NaOH at a temperature of 50 ° C. (dopamine · HCl: NaOH = 1: 0.8 molar ratio) The polymerization reaction was carried out by stirring in air. Subsequently, the reaction product obtained after stirring was purified by centrifugation at room temperature for about 20 minutes at a rate of about 18,000 rpm, which was repeated three times. Thereafter, size selection was carried out by centrifugation at about 4,000 rpm for about 10 minutes to obtain 70 mg of melanin particles dispersed in water. The obtained melanin particles had a size of about 80 to 100 nm It was spherical with diameter.

Here, as the mixed solution spontaneously oxidizes, the pH of the aqueous solution containing dopamine · HCl changed from about 6.8 to about 9.25 immediately after the addition of NaOH, and gradually became neutral. After about 5 hours from the addition of NaOH, Respectively. At this time, the color of the mixed solution changed from transparent to pale yellow to dark brown. A transmission electron microscope (TEM) image of the obtained melanin particles is shown in Fig. 1 (a).

In order to confirm the photophysical properties from the melanin nanoparticles, photoacoustic reaction was plotted against molar concentration at λ = 700 nm by applying a laser flow of 5 mJ / cm 2 to widely utilize the photoacoustic signal enhancement ability of melanin nanoparticles Were compared with gold nanorods and methylene blue, exogenous photoacoustic imaging contrast agents. The methylene blue and gold nanorods were 15.1 and 8.1 times higher than the melanin nanoparticles, respectively, in the photoacoustic signal increase rate corresponding to the molar concentration. Considering that the efficiency of the photoacoustic effect is critically dependent on the conversion of the absorbed and absorbed photons to heat, the thermal conversion of the melanin nanoparticles can be measured by plotting the photoacoustic signal on the corresponding absorbance. As shown in FIG. 1C, the nanoparticulate melanin nanoparticles, which are organic absorbers, exhibited slightly higher signals than the plasmon nanoparticles, while methylene blue exhibited a 2.2-fold higher response than the melanin nanoparticles. From the recently proposed structural model of polydopamine, described as a supramolecular nanoaggregate of monomers, it has been confirmed that the structure of melanin nanoparticles is similar to that of natural melanin associated with the photophysical properties of natural melanin.

Thus, the photoacoustic enhancement capability of melanin nanoparticles can be derived from its structural properties, which lead to broad band absorption and strong non-radioactive emission characteristics, which represent the photo-physical properties of natural melanin causing photoprotection in biological systems.

Example  1: Surface modification of melanin nanoparticles and aggregation of nanoparticles

In order to investigate the correlation between the aggregation of melanin nanoparticles and the photoacoustic response, the surface of the melanin nanoparticles was modified to incorporate citraconic amide, which is easily hydrolyzed on the surface of the particles, in an acidic environment 2a).

Since the surface of the melanin nanoparticles can be easily modified to molecules functionalized with amines or thiol groups via Schiff bases in Michael addition reactions, it is possible to use mPEG as an amine terminal group for potential biocompatibility and further reaction with citraconic anhydride A mixture of -SH and ethylenediamine was introduced.

Specifically, 30 mg of mPEG-SH (2 kDa) and 90 μmol of ethylenediamine were added to 10 mL of MelNP solution (1 mg / mL) and stirred vigorously for 1 hour. After several centrifugation and redispersion, mPEG- MelNP surface treated with SH and ethylenediamine could be obtained. Then, 5 mL of 30 μmol citraconic acid and phosphate buffer solution (pH 9) was added to the surface-treated 5 mL of MelNP solution (1 mg / mL), stirred vigorously for 12 hours, and centrifuged and redispersed several times Melanoprecipitated with citraconic acid was obtained.

The primary amine groups introduced on the melanin nanoparticles were reacted with citraconic anhydride to form amide bonds, where each step of surface modification could be monitored by specifying zeta potentials at each step. After all surface modification steps to melanin nanoparticles, the particles aggregated specifically in an acidic environment. On the other hand, unmodified melanin nanoparticles and PEGylated melanin nanoparticles showed nonspecific aggregation or stability, respectively, in both neutral and acid environments. FIG. 2 (a) shows the coagulation mechanism of the melanin nanoparticles according to the surface modification process and pH change.

The change of the photoacoustic signal from the surface of modified melanin nanoparticles in acidic condition was measured and compared with the result in neutral condition, the coagulation induction amplification of photoacoustic reaction was confirmed. As shown in FIG. 2, the photoacoustic cross-sectional image of the tube containing the surface modified melanin nanoparticles under acidic conditions exhibited increasing intensity over time, while the results under neutral conditions were stable. The time course of the photoacoustic signal from each inclusion showed signal intensity increasing up to 1 hour after exposure to acidic conditions, and the enhanced intensity was stable up to 90 minutes. Interestingly, the improved photoacoustic signal exhibited a 27-fold higher value compared to the results under neutral conditions (Figure 2c).

Example  2: due to aggregation of nanoparticles Photoacoustic  Nonlinear increase of signal

Significant amplification of photoacoustic signals from surface modified melanin nanoparticles was closely related to their physical agglomeration. As a result of the surface chemistry that enabled pH-induced aggregation of melanin nanoparticles, the hydrodynamic radius of melanin nanoparticles increased 10.8-fold higher than that of neutral conditions (Fig. 3a). In particular, the rate of increase in hydrodynamic radius in acidic conditions was saturated in about 60-90 min, consistent with the trend in amplification of photoacoustic signals.

Another factor involved in the change of the photoacoustic response is the absorption by the nanoparticulated absorber. In this regard, the increase in absorption (? = 700 nm) induced by the occurrence of optical scattering due to the increase in hydrodynamic radius may be an important factor associated with the cohesion-induced amplification of photoacoustic signals. However, the change in absorbance due to optical scattering is only 1.3 times higher compared to the results under neutral conditions and is negligible compared to changes in photoacoustic signals and hydrodynamic radius. Also, the optical scattering growth rate is stabilized within 30 minutes after exposure to acidic conditions, which is inconsistent with the results for photoacoustic signals and hydrodynamic radius. Thus, in terms of variation and trends, physical agglomeration can be a major factor in inducing a significant increase in photoacoustic signals over optical scattering.

Considering that the amplification of the photoacoustic response from the melanin nanoparticles is promoted mainly by their physical agglomeration rather than the variation of the optical scattering at the excitation wavelength, the correlation between the photoacoustic response and the physical agglomeration is that the photoacoustic signal is converted to the physical By plotting with a function of the hydrodynamic radius associated with the agglomeration (FIG. 4A). The amplification of photoacoustic signals from melanin nanoparticles is nonlinearly proportional to the hydrodynamic radius that can be caused by an increase in local heating due to superposition of the thermal profile around the aggregated nanoparticles. The increase in photoacoustic response through the aggregation of melanin nanoparticles in the present invention is consistent with the result of amplification of the photoacoustic response through the aggregation of the recently reported silica and the proposed mechanism for this is shown in Figure 4c, It is reasonable to explain the amplification of the photoacoustic response induced by the aggregation of the absorber. Photoacoustic reactions are generally generated from an environment that is locally heated by nanoparticles that absorb the laser energy that is imparted by the fluid surrounding the nanoparticles in a pulse. The amplification of the photoacoustic response is approximately proportional to the slope of the temperature profile of the fluid around the nanoparticles within the time range of the laser pulse width and consequently the signal intensity of the photoacoustic response is proportional to the heat transfer from the nanoparticles to the surrounding solvent and the heat of the surrounding solvent It is sensitively affected by the surface environment that induces changes in divergence dynamics. However, there is no factor in the model system that contributes to surface environmental changes related to thermal dynamics except for physical agglomeration through melanin nanoparticles. Thus, amplification of the photoacoustic response through the aggregation of melanin nanoparticles is expected to result primarily from the superimposed thermal field of the fluid surrounding the melanin nanoparticles.

In addition, the nonlinear correlation of the concentration of melanin nanoparticles with the photoacoustic signal suggests that amplification of the photoacoustic signal is induced by the coupling of the thermal layer around the nanoparticles leading to the improved sound wave. As the concentration of nanoparticles increases, the chance of contact between each single nanoparticle in the Brownian motion can be increased, so that the potential for thermal coupling between each melanin nanoparticle under pulsed laser flow depends on the concentration of the melanin nanoparticles . As shown in Figure 4b, the uniformly dispersed melanin nanoparticles in water exhibited linear photoacoustic signal intensity at concentrations up to 1 mg / ml, but nonlinear light at concentrations exceeding 2 mg / ml Acoustic signal intensity.

As a result, the amplification of the photoacoustic signal from the melanin nanoparticles can be produced in a uniformly dispersed aqueous solution of melanin nanoparticles through an increase in concentration exceeding the critical concentration, which increases the probability of thermal coupling, Agglomeration leading to the amplification of the reaction could be confirmed by nonlinear correlation between the photoacoustic response and the concentration exceeding the critical concentration.

Claims (14)

As photoacoustic imaging agents comprising melanin nanoparticles,
Wherein the melanin nanoparticles are surface-treated to have functional groups capable of aggregating in the presence of functional groups or transition metals or alkaline earth metal ions that aggregate under acidic conditions so that photoacoustic signals can be amplified by aggregation of melanin nanoparticles. Acoustic imaging.
The method according to claim 1,
Wherein the functional groups that aggregate under the acidic conditions are represented by the following formulas (1) and (2):
[Chemical Formula 1]

(2)

or

In this formula,
X or R is _C1-6alkylene .
2. The photoacoustic imaging agent according to claim 1, wherein the functional group which coagulates in the presence of the transition metal or alkaline earth metal ion is represented by the following formula 3 or 4:
(3)

[Chemical Formula 4]

Wherein, X is -COOH, -NH _2, catechol or phosphate,
R is C _1-6 alkylene.
The photoacoustic imaging agent of claim 1, wherein the melanin nanoparticles have a diameter of from 30 nm to 600 nm.
The photoacoustic imaging agent according to claim 1, wherein the melanin nanoparticles are extracted from squid ink.
The photoacoustic imaging agent according to claim 1, wherein the melanin nanoparticles are synthesized from at least one melanin precursor selected from the group consisting of dopamine, DOPA, or cysteine.
delete The photoacoustic imaging agent according to claim 1, wherein the acidic condition is a pH of 2 to 6.
delete The photoacoustic imaging agent according to claim 1, wherein the melanin nanoparticles are further modified with polyethylene glycol on their surfaces.
delete The photoacoustic imaging agent according to claim 3, wherein the transition metal or alkaline earth metal is Mg, Al, Ca, Mn, Fe, Cu, Zn, Cd or Pb.
delete delete

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