KR20130043718A - Ph-reactive optical imaging probe and method for producing the same - Google Patents

Abstract

The present invention relates to a pH-sensitive optical imaging probe of self-assembled particles in which fluorescent dyes are encapsulated, and a near-infrared fluorescent dye is enclosed in self-assembled particles formed of a pH-sensitive polymer and an anionic polymer. An increase in selective fluorescence signal for a specific cell can be detected by using a phenomenon in which the fluorescence signal of the dye disappeared in the H-sensitive optical image probe particle of the present invention is recovered by a pH change.

Description

PH-reactive Optical Imaging Probe and Method for Producing the Same

The present invention relates to a pH-sensitive optical imaging probe containing a near-infrared fluorescent substance, and also to a method of manufacturing the pH-sensitive optical imaging probe.

As a carrier or tracer used to selectively detect and image specific organs or cells inside the human body, magnetic resonance imaging (MRI) contrast agents or radioisotope technology (PET) is used. However, there are still problems such as high cost, rejection due to radiation exposure, and treatment of contaminants during the inspection, which requires the development of other simpler and more environmentally friendly molecular imaging methods.

Meanwhile, even in the development of optical imaging technology using fluorescent dyes, constraints due to low skin penetration and human toxicity remain. In addition, most of the particles serving as the tracer are mostly redistributed to specific organs or cells in the human body, and thus an increased signal is detected. That is, there is a problem that the sensitivity or efficiency of the signal is not high.

It is an object of the present invention to provide a pH-sensitive near infrared optical imaging probe that is simple, harmless to humans and excellent in signal sensitivity.

Another object of the present invention is to provide a method for preparing the pH-sensitive near infrared optical imaging probe.

In the present invention, nanoparticle probes are prepared using biocompatible polymers and harmless near-infrared fluorescent dyes for the human body, and the principle that the fluorescence signal is detected only in specific cells according to the pH change is developed so that the specificity and sensitivity of the optical imaging probes can be determined. I want to increase.

In one embodiment of the present invention, a hydrophobic pH-sensitive polymer (A), a hydrophilic anionic polymer (B), and an amphiphilic near infrared fluorescent dye (C), the hydrophilic anionic polymer (B) in the aqueous solution is hydrophobic pH Self-assembled particles formed to surround the sensitive polymer (A), pH of the fluorescent dye (C) is physically contacted between the polymer (A) and the polymer (B) is encapsulated in the particles and quenched (quenching) Provide a sensitive optical imaging probe.

In the pH-sensitive optical imaging probe of the present invention, self-assembled particles may be deassembled in weakly acidic conditions, and a fluorescent dye (C) may be emitted to fluorescence. That is, the amphipathic near-infrared fluorescent dye (C) is characterized in that it uses a material that fluoresces in the state of self-fluorescence (self-quenching) in the state encapsulated in the particle and released from the particles according to the pH change.

 The hydrophobic pH sensitive polymer (A) is polybetaamino ester (PBAE), poly acrylic acid (PAA), polymethacrylic acid (poly methacrylic acid (PMAA), polymaleic anhydride, PMA), N, N-dimethylaminoethyl methacrylate (DMAEMA), poly (N (ω) -methacryloyl glycyl-L-leucine, polyamide, poly (L-lysine isophthalamide) , Phenylalanine, a polymer material containing imidazole, and mixtures or copolymers thereof may be selected from, but is not limited thereto.

In addition, the hydrophilic anionic polymer (B) is a polyglutamic acid (Polyglutamic acid), polyacrylic acid (Polyacrylic acid), alginic acid (Alginate), carrageenan (Carrageenan), hyaluronic acid (Hyaluronic acid), polystyrene sulfonate (Poly (styrene sulfonate) )), Carboxymethylcellulose, Cellulose Sulfate, Dextran Sulfate, Heparin, Heparin Sulfate, Polymethylenecoguanidine (Poly (methylene-co-guanidine) ) And chondroitin sulfate (Condroitin sulfate) may be selected from, but is not limited thereto.

The amphipathic near-infrared fluorescent dye (C) may be a fluorescent dye that is self-quenched in the state encapsulated in the particle and emitted from the particle according to the pH change to fluoresce in the state. In one embodiment, cycine is cy3.5, cy5, cy5.5, cy7, ICG, Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, IRDye82 and Oxazine-based Cresy Violet, Nile Blue, Oxazine 750, Rhodamines Rhodamine 800, Texas Red, and mixtures or complexes thereof may be selected from, but is not limited thereto.

In one embodiment, the target material that can be detected in the cell is coupled to the surface of the pH-sensitive optical imaging probe may be utilized for selective cell detection.

In another embodiment of the present invention, there is provided a method of manufacturing a pH-sensitive optical imaging probe comprising the following steps.

(i) obtaining an aqueous solution in which a solution containing an anionic polymer and a solution containing a near infrared fluorescent dye are mixed; And (ii) adding a solution comprising a pH sensitive polymer to form self-assembled particles.

The optical imaging probe according to the present invention is a pH-sensitive polymer assembly in which a near-infrared fluorescent dye is encapsulated, and thus the optical imaging probe can detect a selective fluorescent signal for a specific cell. In addition, the method according to the present invention can quickly and easily prepare a pH-sensitive self-assembled particles in which fluorescent dyes are encapsulated to observe the recovery of the fluorescence signal that has been extinguished by the change of the intracellular pH, and can detect the fluorescence signal in a specific cell. Particles that can be utilized as optical imaging probes can be prepared.

1 is a method for preparing pH-sensitive gamma-PGA / PBAE self-assembled nanoparticles containing an indocyanine green (ICG) fluorescent dye;
2 is a schematic of the fluorescence signal recovery process in intracellular migration of pH-sensitive gamma-PGA / PBAE self-assembled nanoparticles;
3 is a size distribution (left) and an electron transmission microscope (TEM) image of a pH-sensitive gamma-PGA / PBAE self-assembled nanoparticle containing ICG.
Figure 4 shows the absorbance (left) and fluorescence signal (right) spectrum of ICG (dotted line) at the same concentration as pH-sensitive gamma-PGA / PBAE self-assembled nanoparticles (solid line) containing ICG.
5 is a fluorescence signal spectrum obtained after redispersing the pH-sensitive gamma-PGA / PBAE self-assembled nanoparticles with ICG encapsulated in a pH 7.4 solution (dotted line) and a pH 5.2 solution (solid line), respectively.
FIG. 6 is a diagram obtained after treating an ICG-encapsulated pH-sensitive gamma-PGA / PBAE self-assembled nanoparticle to RAW264.7 cells, which are macrophages, followed by 2 hours of incubation in a 37 ° C. incubator. (Left) is a picture immediately after the treatment, and (right) is a picture 2 hours after the treatment (blue represents a stained nucleus, and red represents a fluorescent signal of ICG).

The present invention is a “on / off switchable” system in which a near-infrared fluorescent dye is encapsulated in a self-assembled particle using a biocompatible pH-sensitive polymer, and the fluorescent signal of the dye which has been extinguished in the particle is recovered in response to a pH change. Provides optical imaging techniques that can be used for cell detection.

Prior art related to fluorescent probes Japanese Patent Application Laid-Open No. 2010-053354 provides a probe in which the fluorescence intensity is changed while the conformation changes from a random coil type to a spiral in a pH-dependent manner as polyglutamic acid and a water-sensitive fluorescent substance. do. In the preceding document, the fluorescent properties of the fluorescent material itself change with the pH change due to the use of a water-sensitive fluorescent material.

In contrast, the fluorescent dye in the probe according to the present invention uses a fluorescent material exhibiting a self-quenching phenomenon at a high concentration, the fluorescent material itself does not appear differently depending on the pH, the fluorescent signal is encapsulated ( It utilizes the phenomenon that the fluorescence signal is extinguished or recovered by 'change of the concentration of phosphor' according to encapsulation and release.

pH-sensitive optical imaging probe

The present invention relates to a pH-sensitive optical imaging probe comprising a hydrophobic pH sensitive polymer (A), a hydrophilic anionic polymer (B), and an amphiphilic near infrared fluorescent dye (C).

The hydrophilic anionic polymer (B) surrounds the hydrophobic pH-sensitive polymer (A) in an aqueous solution to form self-assembled particles. In other words, the hydrophobic pH-sensitive polymer (A) in the aqueous solution is surrounded by the hydrophilic anionic polymer (B) dissolved in water to be protected from water. Therefore, the hydrophobic pH-sensitive polymer (A) inside the particles is hydrophilic to the outside. The form in which the anionic polymer (B) is wrapped is formed.

The near-infrared fluorescent dye (C) is amphiphilic bar is enclosed in the particles in physical contact between the polymer (A) and the polymer (B). In the present invention, the fluorescent dye (C) is encapsulated in the particles to increase the concentration is self-fluorescence (self-quenching) and released from the particles in accordance with the pH change is used to fluoresce when the concentration becomes low. Since the fluorescent dye (C) is in a phobic-phobic interaction state by physical contact rather than chemical bonding between the polymer (A) and the polymer (B), when the fluorescent dye (C) is released outside the particles The original fluorescence properties are retained and can be separated from the particles.

In the pH-sensitive optical imaging probe particles of the present invention, self-assembled particles are de-assembling under weakly acidic conditions, and fluorescent dyes (C) may be emitted and fluoresce at low concentrations. Here, the weakly acidic conditions correspond to the pKa value of the hydrophobic pH-sensitive polymer (A). For example, in a weakly acidic atmosphere having a pH of 6.5 or less, PBAE is decomposed due to the decomposition of the chain, and thus is self-assembled with PBAE. The particles produced show a phenomenon that they can no longer retain their particle form and collapse in a slightly acidic atmosphere below pH6.5. In one example, the weakly acidic conditions may range from pH6.5 to pH5.2.

In the present invention, the polymer (A) and the polymer (B) are both degradable by enzymes present in the living body so as to be suitable for in vivo use, there is no toxicity to the living body, it is preferable that there is no antigenicity.

Accordingly, the hydrophobic pH sensitive polymer (A) includes polybetaamino ester (PBAE), poly acrylic acid (PAA), polymethacrylic acid (poly methacrylic acid (PMAA), polymale anhydride ( pH sensitive organic polymers and polyamides such as poly maleic anhydride (PMA), N, N-dimethylaminoethyl methacrylate (DMAEMA), and poly (N (ω) -methacryloyl glycyl-L-leucine) It may be, but is not limited to, pH sensitive organic polymers or mixtures or copolymers thereof, including hydrophobic amino acids such as poly (L-lysine isophthalamide), phenylalanine, and a polymer material containing imidazole.

In addition, the hydrophilic anionic polymer (B) is a polyglutamic acid (Polyglutamic acid), polyacrylic acid (Polyacrylic acid), alginic acid (Alginate), carrageenan (Carrageenan), hyaluronic acid (Hyaluronic acid), polystyrene sulfonate (Poly (styrene sulfonate) )), Carboxymethylcellulose, Cellulose Sulfate, Dextran Sulfate, Heparin, Heparin Sulfate, Polymethylenecoguanidine (Poly (methylene-co-guanidine) ) And Chondroitin Sulfate (Condroitin sulfate) may be selected from, but is not limited thereto.

Amphiphilic near-infrared fluorescent dye (C) in the present invention is particularly limited if the high concentration in the state encapsulated in the particle is self-quenching (self-quenching) and released from the particles according to the pH change to fluoresce in a low concentration state It doesn't work. For example, cyanine may include cy3.5, cy5, cy5.5, cy7, and ICG. Other near-infrared fluorescent dyes used in the present invention include Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, IRDye82 and Oxazine-based Cresy Violet, Nile Blue, Oxazine 750 and Rhodamines-based Rhodamine800, Texas Red and mixtures thereof Or it may be an organic material selected from the group consisting of a composite, but is not limited thereto.

In addition, in the present invention, the particle size of the pH-sensitive optical imaging probe is not particularly limited, and may be about 20 to 2000 nm in size nanoparticles. If the particle size is less than 20 or more than 2000 nm, there is a problem of low efficiency of introduction into cells.

In one embodiment of the present invention, a pH-sensitive near-infrared optical nanoparticle probe is manufactured using polybetaaminoester (PBAE) and indocyanine green (ICG), which is a near-infrared fluorescent dye having high bio stability as a bio-friendly pH-sensitive polymer. It was.

The PBAE is a polymer having effective transfection properties into cells and exhibiting solubility corresponding to pH. In addition, since it has biocompatible degradability, it is known as a material that is used as a useful material for nanocarriers based on polymers, and has been utilized in research of various nanocarriers.

The indocyanine green (ICG) is a fluorescent dye that exhibits a fluorescent signal in the near infrared region (700-1300 nm) and is a very biocompatible fluorescent dye that is approved by the US FDA. In particular, since the skin permeability has high absorption and fluorescence characteristics in the near-infrared region, it can exhibit a high signal detection rate when used in the human body, and has an amphiphilic structure with both hydrophilic and hydrophobic groups.

Although the application of ICG has been made and attempted in several medical fields, its use in various fields is limited by some limitations. Although ICG has high biostability, it exhibits very low quantum efficiency, which makes it difficult to detect a signal. To overcome this problem, when the dye is treated at a high concentration, self-fluorescence disappears severely. In addition, since there is no structural functional bond with other materials, it is difficult to prepare or modify a probe in the form of particles including ICG, and exhibit low degradation in an aqueous solution due to rapid degradability in a humid environment. Accordingly, there is a demand for a technology that exhibits high quantum efficiency and stably encapsulates ICG in particles. In the present invention, ICG is stably encapsulated in self-assembled particles made of a polymer by using the amphiphilic nature of ICG. The advantage is that the technology has been developed.

In this regard, FIG. 1 shows a method of manufacturing a pH-sensitive optical imaging probe according to the present invention, and FIG. 2 shows a schematic diagram of recovery of a fluorescence signal of particles in an intracellular migration pathway.

First, referring to FIG. 1, polybetaaminoester (PBAE) was used as a pH-sensitive polymer, and indocyanine green (ICG), a near-infrared fluorescent dye that was harmless in vivo, was used as a fluorescent dye. To protect hydrophobic PBAE in a hydrophilic environment and to form more stable particles, gamma-PGA, a hydrophilic anionic polymer, was used together. When PBAE is introduced into a hydrophilic environment in which gamma-PGA and ICG are dissolved, particles are formed by intermolecular self-assembly and ICG is encapsulated inside the particle. Hydrophilic PGA is located on the outer surface of the particle, hydrophobic PBAE is located on the inner surface, and fluorescent dyes are located at the boundary between the two polymers.

Encapsulated fluorescent dye (ICG) has a characteristic that the fluorescent signal is reduced by self-quenching in the presence of high concentration. When the fluorescence-dissipated particles are placed in a low pH environment, the decomposition of PBAE that forms the particles is accelerated, and the ICG encapsulated inside the particles is unstable and released from the particles. The released ICG will exit the extinction state and recover the original fluorescence signal.

Here, the threshold value at which ICG shows self-fluorescence disappearance was determined to be approximately 0.05 mg / ml according to the experimental results of the present inventors. At 0.05 mg / ml or less, the fluorescence signal gradually increases with concentration, but at 0.05 mg / ml or more, the fluorescence signal decreases due to autofluorescence. In the practice of the present invention, ICG is dissolved in an aqueous solution at a concentration of 0.01 mg / ml, and then PABE is added to form particles to form a high concentration. Accordingly, the high concentration of ICG in the particles is concentrated at a high concentration above the threshold value indicating the self-fluorescence quenching effect and does not show a fluorescence signal. Thus, in one preferred example, the concentration of the fluorescent dye (C) in the self-assembled particles may be 0.05 mg / ml or more.

According to the experiments of the present inventors, when the absorption signal of the ICG encapsulated in the particle appears to shift slightly longer than the free ICG, it can be considered that a hydrophobic bond exists between the polymer and the ICG. When emitted outside, looking at restoring the original absorption wavelength, there may be physical bonding by phobic-phobic interactions rather than chemical bonding.

In addition, in the mixture of PGA and ICG alone, the absorption wavelength is not shifted, and the absorption wavelength shift is observed only after PBAE-added particles are formed. Therefore, ICG mainly forms a hydrophobic bond with PBAE. Accordingly, it was found that ICG was properly present in the particles even under the hydrophilic atmosphere of PGA and well encapsulated without disturbing the self-assembled structure of PGA and PBAE.

Referring to the behavior when the pH-sensitive optical imaging probe according to the present invention is introduced into the cell with reference to FIG. It shows the application of antibody-mediated endocytosis. In this case, it is possible to develop particles that selectively detect signals for specific cells.

The pH-sensitive optical imaging probe particles introduced into the cells migrate to the low pH endosome (pH5-6, late endosome), and ICG is released by the breakdown of PBAE, which is a structure in a low pH environment. The ICG remains in the state of fluorescence dissipation inside the probe particles, and when the pH is lowered, rapid decomposition of the self-assembled particles is achieved, and the fluorescence signal is recovered by the collapse of the structure.

In one embodiment, the pH-sensitive optical imaging probe of the present invention may have a cell-specific material bound to the particle surface. Accordingly, optical imaging nanoprobes can be manufactured that enable selective detection of specific cells.

Examples of the cell-specific substance include antibodies, fragmented antibodies, receptor binding molecules, and the like. The use of the probe of the present invention is not particularly limited, and enables, for example, pH-sensitive fluorescence sensing at sites of inflammation or tumor (pH5-6). The probe particles of the present invention are also useful as probes for understanding intracellular transport and proteolytic machinery. For example, it is a fluorescent probe capable of detecting the acidic environment in endosomes in cells. Such probes can be expected for application in the field of cell biology.

pH - Sensitivity  Optical imaging Of the probe  Manufacturing method

The present invention provides a method for preparing a self-assembled nanoparticles using a hydrophobic pH-sensitive polymer and a hydrophilic polymer quickly and easily and a pH-sensitive optical imaging probe in which a fluorescent dye is relatively stably encapsulated inside the particles.

In one embodiment, a method of manufacturing a pH-sensitive optical imaging probe comprising the following steps:

(i) obtaining an aqueous solution in which a solution containing an anionic polymer and a solution containing a near infrared fluorescent dye are mixed; And

(ii) adding a solution containing a pH sensitive polymer to form self-assembled particles.

In step (i), the reaction solvent is water, and in step (ii), the pH-sensitive polymer may be in a solution state dissolved in high concentration in methanol.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

Reagents and Materials

Polybetaaminoester (PBAE) is 1- (2-amino ethyl) pyrrolidine and was synthesized in this laboratory using Michael addition method using 1,4-butandiol diacrylate and 5-amino-1-pentanol. (Korea Polymer Testing Research Institute).

Gamma-PGA was provided by Bioleaders Co., Ltd. in a salt form with a molecular weight of 50,000 Da, and indocianin green was purchased from Donginang Pharmaceutical Co., Ltd.

PBS (phosphate buffered saline, pH7.4) solution was used for the redispersion of the particles, 1N HCl solution was added to the PBS solution in the preparation of pH5.2 solution.

[ Example  One]

pH - Sensitivity  Optical imaging Probe  Particle manufacturing

Gamma-PGA and ICG prepare stock solutions in aqueous solutions (DW) at concentrations of 4 mg / ml and 1 mg / ml, respectively. PBAE prepares stock solutions on methanol solutions at a concentration of 12 mg / ml.

Prepare 18.3 ml of DW, stir 1 ml and 0.5 ml of gamma-PGA and ICG stock solution, respectively, and add 0.5 ml of PBAE (methanol) solution. Final concentrations of each sample should be gamma-PGA, ICG and PBAE of 0.2, 0.01 and 0.3 mg / ml, respectively. The mixed solution is stirred at a speed of 500 rpm at room temperature for 1.5 hours, and the ICG is not exposed to light for a long time in all procedures.

3 is a particle size distribution (left) and an electron transmission microscope (TEM) image (right) through particle size analysis of the formed particles. The particles formed appeared to have a distribution of approximately 250 nm size.

[ Example  2]

pH - Sensitivity  Optical imaging Probe  Spectroscopic analysis of particles

For absorbance and fluorescence signal analysis of pH-sensitive nanoparticles, UV-Vis spectrophotometer and fluorescence spectrometer (Shinko, Korea) were used, and fluorescence signal was measured with excitation wavelength of 765 nm. Absorbance and fluorescence signal were measured immediately after particle preparation.

In order to determine the change in the fluorescence signal due to the pH of the particles, an equal amount was aliquoted from the prepared particle solution and freeze-dried, and then the same amount of pH7.4 and pH5.2 solutions were added to each particle powder and dispersed. Immediately after dispersion, the fluorescence signal was measured.

Fig. 4 shows the absorbance (left) and fluorescence spectrum (right) of the ICG used for particle formation, and shows the state change as the pure ICG (dotted line) is introduced into the particle (solid line) as the environment around the dye changes. As shown in the fluorescence spectrum, the ICG encapsulated in the particle can be seen that the signal is not detected because the fluorescence disappears completely.

Figure 5 shows the change in fluorescence signal when redispersed the formed particles in the pH7.4 (dotted line), pH5.2 (solid line) solution after freeze-drying, respectively, if the redispersed in the pH7.4 solution before lyophilization The state of fluorescence disappearance was maintained as in the state, and when redispersed in the pH5.2 solution, the increase in fluorescence signal due to particle collapse was detected as mentioned above. This signal difference is approximately 4.5 times, indicating that the particles can be used as an "ON / OFF switchable" system in response to pH.

[ Example  3]

pH - Sensitivity  Optical imaging Probe  Cell experiment of particles

As an "in vitro" experiment on the formed particles, the prepared particles were treated with RAW264.7 cells, which are macrophages at a rate of 25v / v%. Images were taken immediately after treatment, incubated for 2 hours in a 37 ° C. incubator and observed again. All images were observed without washing the extracellular particles. The filter used for observing the ICG fluorescence signal of intracellular particles was a filter having an excitation wavelength of 775/60 nm and a detection wavelength of 845/55 nm.

6 is a diagram showing the particles treated with macrophage RAW264.7 cells after the particles move into the cell by the phagocytosis of the cells. The figure on the left is immediately after the particle treatment, and the figure on the right is 2 hours after the particle treatment, and the particles treated in the cells are not washed. The particles treated in the cell are not detected by the fluorescent signal outside the cell, but the particles introduced into the cell can be seen that the fluorescent signal is recovered.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

Hydrophobic pH sensitive polymer (A), hydrophilic anionic polymer (B) and amphipathic near infrared fluorescent dye (C),
Hydrophilic anionic polymer (B) in the aqueous solution is a self-assembled particles formed to surround the hydrophobic pH-sensitive polymer (A),
The fluorescent dye (C) is physically contacted between the polymer (A) and the polymer (B) and is embedded in the particles, characterized in that the self-quenching (self-quenching), pH-sensitive optical imaging probe. The method of claim 1,
PH-sensitive optical imaging probes, characterized in that the self-assembled particles are deassembled in weakly acidic conditions, the fluorescent dye (C) is released and fluoresce. The method of claim 1,
The hydrophobic pH sensitive polymer (A) is
Polybetaamino ester (PBAE), poly acrylic acid (PAA), poly methacrylic acid (PMAA), poly maleic anhydride (PMA), N, N-dimethylamino Polymers containing ethyl methacrylate (DMAEMA), poly (N (ω) -methacryloyl glycyl-L-leucine, polyamide, poly (L-lysine isophthalamide), phenylalanine, imidazole PH-sensitive optical imaging probe, characterized in that selected from the group consisting of materials and mixtures or copolymers thereof. The method of claim 1,
The hydrophilic anionic polymer (B) is polyglutamic acid, polyacrylic acid, alginate, carrageenan, hyaluronic acid, polystyrene sulfonate, polystyrene sulfonate. Carboxymethylcellulose, Cellulose Sulfate, Dextran Sulfate, Heparin, Heparin Sulfate, Polymethylene-co-guanidine, and PH-sensitive optical imaging probes, characterized in that selected from the group consisting of chondroitin sulfate (Condroitin sulfate). 3. The method of claim 2,
The amphipathic near-infrared fluorescent dye (C) is cycine-based cy3.5, cy5, cy5.5, cy7, ICG, Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, IRDye82 and Oxazine-based Cresy Violet, Nile Blue, Oxazine 750, Rhodamines family of Rhodamine 800, Texas Red, and mixtures or complexes thereof, characterized in that the pH-sensitive optical imaging probe. The method of claim 1,
A pH-sensitive optical imaging probe, characterized in that the target material is detected on the surface is bound to the cell. A method of manufacturing a pH-sensitive optical imaging probe according to claim 1 comprising the following steps:
(i) obtaining an aqueous solution in which a solution containing an anionic polymer and a solution containing a near infrared fluorescent dye are mixed; And
(ii) adding a solution containing a pH sensitive polymer to form self-assembled particles.

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