KR20140096195A - Mn-doped zns nanoparticle conjugate, process for preparing the same and use of the same for multiphoton imaging - Google Patents

Abstract

The present invention relates to a zinc sulfide nanoparticle conjugate doped with manganese, a process for its preparation and its use as an optical imaging agent. More particularly, the present invention relates to a ZnS nanoparticle doped with manganese, an oleylamine layer bonded to the nanoparticle, a phospholipid layer coated with the oleylamine layer, and a functional biomaterial bonded to the phospholipid layer ZnS nanoparticle conjugates, methods for their preparation, and their use as multiphoton optical imaging agents.

Description

[0001] The present invention relates to a ZnS nanoparticle conjugate doped with manganese, a method for producing the same, and a use thereof as a multiphoton optical imaging agent. [0002] Mn-doped ZnS nanoparticle conjugate, Process for Preparing the Same and Use of the Same for Multiphoton Imaging [

The present invention relates to a zinc sulfide nanoparticle conjugate doped with manganese, a process for its preparation and its use as a multiphoton optical imaging agent. More particularly, the present invention relates to a ZnS nanoparticle doped with manganese, an oleylamine layer bonded to the nanoparticle, a phospholipid layer coated with the oleylamine layer, and a functional biomaterial bonded to the phospholipid layer ZnS nanoparticle conjugates, methods for their preparation, and their use as multiphoton optical imaging agents.

Optical imaging plays an important role in both preclinical research and clinical diagnostics. Complementing macroscopic imaging modes such as MRI, CT and PET, the main advantage of optical imaging is the ability to microscopy imaging at subcellular resolution.

Of the various optical imaging techniques, multiphoton imaging using a non-linear optical process with near-infrared pulsed laser excitation not only provides a high spatial resolution but also enables imaging of deep tissue in vivo.

Semiconductor nanoparticles are being studied as biomedical imaging probes due to their remarkable light stability, brightness and size-dependent photoluminescence. Moreover, due to the large two-photon excitation cross-section of semiconductor nanoparticles, they have been successfully applied to in vivo multiphoton imaging of vascular structures, tumor microenvironment, and cellular trafficking processes .

However, these semiconductor nanoparticles contain toxic elements such as Cd and Te, and these toxic elements are not metabolized in the human body. In this connection, since Zn and S are the main components of the human body, ZnS nanoparticles are attracting much attention.

Due to recent advances in doping techniques for nanoparticles, ZnS nanoparticles can be processed to emit visible light by doping phosphorescent centers.

However, their constituent-biocompatibility interferes with non-invasive in vivo imaging due to their ultraviolet absorption. This problem has been pointed out by the use of a three-photon excitation in which three photons are simultaneously absorbed by each nanoparticle through a highly efficient fifth-order nonlinear optical process of nanoparticles, a virtual state (S. Maiti, JB Shear, RM Williams JWM Chon, M. Gu, C. Bullen, and M. Wang Zipfel, WW Webb, Science 275, 530 (1997); GS He, PP Markowicz, T.-C. Lin, PN Prasad, Nature 415 , P. Mulvaney, Appl . Phys . Lett ., 84, 4472 (2004); J. He, W. Ji, J. Mi, YG Zheng, JY Ying, Appl . Phys . Lett ., 88, 181114 (2006); . He, GD Scholes, YL Ang , W. Ji, CWJ Beh, WS Chin, Appl Phys Lett 92, 131114 (2008);.. GS He, Q. Zheng, K.-T. Yong, F. Erogbogbo, MT Swihart, PN Prasad, Nano Lett . 8, 2688 (2008); GC Xing, W. Ji, Y. Zheng, JY Ying, Opt . Express 16, 5710 (2008)).

In particular, this three-photon excitation process can be applied to UV-luminophores using near-infrared pulsed lasers, which can prevent photodamage by ultraviolet light, And can compete with Rayleigh scattering. Furthermore, it is expected that the three-photon excitation process will yield a higher subdiffraction resolution than the two-photon excitation process.

Although three-photon fluorescence detection for endogenous biomolecules has been reported a decade ago (S. Maiti, JB Shear, RM Williams, WR Zipfel, WW Webb, Science 275, 530 (1997)), an inherently low quantum mechanical due to the efficiency of three-photon biomedical imaging it has not yet been realized (WR Zipfel, RM Williams, R. Christie, AY Nikitin, BT Hyman, WW Webb, Proc. Nat. Acad. Sci. 100, 7075 (2003)).

The present inventors have completed a high resolution in vitro and in vivo target imaging method using a three photon excitation process of ZnS nanoparticles utilizing visible light emission from a Mn ^{2 +} dopant.

A primary object of the present invention is to provide a nanoparticle-doped ZnS nanoparticle, an oleylamine layer bonded to the nanoparticle, a phospholipid layer coated with the oleylamine layer, and a functional biomolecule bonded to the phospholipid layer And a ZnS nanoparticle conjugate.

A second object of the present invention is to provide a method for preparing a nanoparticle comprising: (i) forming an oleylamine layer bound to manganese-doped ZnS nanoparticles; (ii) forming a phospholipid layer covering the oleylamine layer; And (iii) bonding the functional biomolecule to the phospholipid layer. The present invention also provides a method for producing a ZnS nanoparticle conjugate.

A third object of the present invention is to provide a ZnS nanoparticle doped with manganese, an oleylamine layer bonded to the nanoparticles, a phospholipid layer coated with the oleylamine layer, and a functional biomolecule bonded to the phospholipid layer And a ZnS nanoparticle conjugate comprising the ZnS nanoparticle conjugate.

A primary object of the present invention is to provide a nanoparticle-doped ZnS nanoparticle, an oleylamine layer bonded to the nanoparticle, a phospholipid layer coated with the oleylamine layer, and a functional biomolecule bonded to the phospholipid layer ≪ RTI ID = 0.0 > ZnS < / RTI > nanoparticle conjugate.

The size of the ZnS nanoparticles doped with manganese contained in the ZnS nanoparticle conjugate of the present invention is preferably 1 nm to 30 nm. The molar ratio of zinc to manganese (Zn: Mn) in the manganese-doped ZnS nanoparticles is preferably 1: 0.001 to 1: 1.

The phospholipid contained in the ZnS nanoparticle conjugate of the present invention may be selected from the group consisting of mPEG-PE (methoxypoly (ethylene glycol) -phosphatidylethanolamine), mPEG-PC (methoxypoly (ethylene glycol) -phosphatidylcholine), mPEG- (ethylene glycol) -phosphatidylglycerol), mPEG-PS (methoxypoly (ethylene glycol) -phosphatidylglycerol), mPEG-PS (methoxypoly ≪ / RTI >

In addition, the functional biomolecule contained in the ZnS nanoparticle conjugate of the present invention may be a peptide, a DNA, an RNA, or an antibody protein. Furthermore, the peptide may be a tumor-targeting peptide and the tumor-targeting peptide may be an arginine-glycine-aspartic acid (RGD), an internalizing RGD (iRGD), or LyP 1 (amino acid sequence: CGNKRTRGC (S-S Bonded)). The antibody protein may be a vascular endothelial growth factor (VEGF), an epidermal growth factor (EGF), a transferrin, or a Herceptin. In addition, the hydrodynamic diameter of the ZnS nanoparticle bonded body is preferably 3 nm to 1 μm.

A second object of the present invention is to provide a method for preparing a nanoparticle comprising: (i) forming an oleylamine layer bound to manganese-doped ZnS nanoparticles; (ii) forming a phospholipid layer covering the oleylamine layer; And (iii) bonding the functional biomolecule to the phospholipid layer. The present invention also provides a method for preparing a ZnS nanoparticle conjugate.

The size of the ZnS nanoparticles doped with manganese used in the ZnS nanoparticle bonded product manufacturing method of the present invention is preferably 1 nm to 30 nm. The molar ratio of zinc to manganese (Zn: Mn) in the manganese-doped ZnS nanoparticles is preferably 1: 0.001 to 1: 1.

The phospholipid used in step (ii) of the ZnS nanoparticle conjugate preparation method of the present invention may be used in combination with mPEG-PE, mPEG-PC, mPEG-PA, mPEG-PG, mPEG-PS, mPEG- Block copolymers.

In addition, the functional biomolecules used in step (iii) of the ZnS nanoparticle conjugate manufacturing method of the present invention may be a peptide, DNA, RNA, or antibody protein. Moreover, the peptide may be a tumor target peptide, and the tumor target peptide may be RGD, iRGD or LyP 1. The antibody protein may be vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transferrin or Herceptin. The hydrodynamic diameter of the thus prepared ZnS nanoparticle bonded body is preferably 3 nm to 1 μm.

A third object of the present invention is to provide a ZnS nanoparticle doped with manganese, an oleylamine layer bonded to the nanoparticles, a phospholipid layer coated with the oleylamine layer, and a functional biomolecule bonded to the phospholipid layer Lt; RTI ID = 0.0 > ZnS < / RTI > nanoparticle conjugate.

The size of the manganese-doped ZnS nanoparticles included in the multiphoton optical imaging agent of the present invention is preferably 1 nm to 30 nm. The molar ratio of zinc to manganese (Zn: Mn) in the manganese-doped ZnS nanoparticles is preferably 1: 0.001 to 1: 1. In addition, the hydrodynamic diameter of the ZnS nanoparticle bonded body is preferably 3 nm to 1 μm.

The phospholipid included in the multiphoton optical imaging agent of the present invention may be a block copolymer of mPEG-PE, mPEG-PC, mPEG-PA, mPEG-PG, mPEG-PS, mPEG-PI or their PEI .

In addition, the functional biomolecule contained in the multiphoton optical imaging agent of the present invention may be a peptide, a DNA, an RNA, or an antibody protein. Moreover, the peptide may be a tumor target peptide, and the tumor target peptide may be RGD, iRGD or LyP 1. The antibody protein may be vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transferrin or Herceptin. The hydrodynamic diameter of the multiphoton optical imaging agent is preferably 3 nm to 1 μm.

The most important advantages of ZnS nanoparticles on other semiconductor nanoparticles are potential in vivo clinical applicability due to biocompatibility. Despite these advantages, however, application of in-vivo imaging of ZnS nanoparticles is not yet available.

The in vivo imaging according to the present invention can be applied not only to the scattering of skin tissue which can interfere with the imaging of deep tissue which can not reach effectively from 50 [mu] m to 60 [mu] m beneath the skin with confocal microscopy, Photon excitation fluorescence and intrinsic emission including a two-photon self-fluorescence and second harmonic generation (SHG) with higher intrinsic efficiency than the three-photon excitation emission.

The ZnS nanoparticle conjugates of the present invention enable high resolution in vivo multiphoton imaging by first applying biocompatible semiconductor nanoparticles to the field of microscopic optical imaging.

1 is a transmission electron microscope (TEM) photograph (average size 5.5 nm, standard deviation 15%) of (A) ZnS nanoparticles doped with manganese of the present invention, (B) normalized 1 photon PLE (Red), (C) a multiphoton microscope spectral image of the ZnS nanoparticle film doped with manganese, (D) a log-log plot of the power-dependence obtained from FIG. 1C E) Yablonski diagram for one photon and three photon excitation luminescence of ZnS nanoparticles doped with manganese.
FIG. 2A is a graph comparing the photon emission spectra of ZnS nanoparticles doped with manganese and ZnS nanoparticles not doped according to the present invention, and FIG. 2B is a graph comparing the photon emission spectra of ZnS nanoparticles doped with manganese and time- resolved PL (collapse time is 950 μs).
3A is a graph showing power dependence of the 3PL (red circle) of ZnS nanoparticles doped with manganese of the present invention obtained from multiphoton fluorescence correlation spectroscopy (FCS) and the effective luminance of 2PL (black circle) of rhodamine 6G and, Figure 3B shows a wavelength-dependent three-photon action cross section (red diamond) and 1S _e -1S _h metastasis derived from the first photon PLE spectrum.
Figure 4 shows the power dependence of ZnS nanoparticle-doped manganese (17.85 [mu] M) and rhodamine 6G aqueous solution (10 [mu] M) of the present invention and Figure 4B shows the power- Shows the autocorrelation function for three photon emission of ZnS nanoparticles.
Figure 5 shows the results of dynamic light scattering analysis for manganese doped ZnS nanoparticles with unbonded (red), LyP 1 junction (blue), and c (RGDyK) junctions (black).
6 is a three photon emission multiphoton microscope image of MDA-MB 435 cancer cells incubated with manganese-doped ZnS nanoparticles conjugated (left), LyP 1 junction (middle) and c (RGDyK) junction (right).
FIG. 7 is (A) a multiphoton microscopic image (red) and (B) a corresponding spectral image of MDA-MB 435 cancer cells targeted with a conjugate of ZnS: Mn nanoparticles and Lyp- (FIG. 7B, circle 1, orange) and 2-photon self fluorescence (circle 2, green in FIG. 7B) of the target nanoparticles, and (D) the spectra obtained at 20 minutes, 120 minutes, 220 minutes, and 560 minutes (White arrows indicate targeted nanoparticles targeted to the cell surface), (E) cells targeted to the nanoparticles after fixation, and (F) FIG. 7E is an enlarged view of the box portion of FIG. 7E. (G) FIG. 7F shows a line profile (black) for three-photon signal intensity, a profile at an effective pixel size of 55 nm A smoothened profile (blue) averaged over every five points of the < RTI ID = 0.0 > A Gaussian profile (Gaussian profiling) (red).
Figure 8 is a graph of the change in fluorescence intensity of MDA-MB 435 cells targeted to manganese-doped ZnS nanoparticle-LyP 1 conjugates excited at wavelengths of 800 nm (left), 920 nm (middle) and 950 nm This shows the excitation wavelength dependency of the image.
9 is a three-photon image of a tumor targeted to a ZnS nanoparticle-RGD conjugate doped with manganese, (A) a spectral image of the tumor vasculature below the dermal base, (B) a SHG of the collagen fiber 9A), tissue autofluorescence (white second circle, green in FIG. 9A) of the nanoparticles with the nanoparticles in the deconvolution image of the tumor targeted with the nanoparticles (C) endothelial lining of the nanoparticles and (D) extravasation of the nanoparticles, (E) an in vitro image of the dissected tumor tissue, (F) contacting the nanoparticles with a balb / c nude mouse And the bio-distribution of the ZnS nanoparticles doped with manganese is shown.
10 is a spectral image according to the depth of the tumor vascular structure imaged through the skin. Tumor vascular structures filled with manganese-doped ZnS nanoparticles are seen at depths of 70 μm to 100 μm.
11 is a graph comparing the optical stability of ZnS nanoparticles doped with a general fluorescent stage and manganese of the present invention in a multiphoton excitation power of about 10 mW.
12 is a comparison of a spectral image (left) obtained from the same site of the tumor and an equimolar deconvolution image (right).
FIG. 13 shows (A) 293T, (B) HUVEC, (C) CdSe / CdZnS nanoparticles (green) and CdSe nanoparticles (blue) incubated with manganese doped ZnS nanoparticles ), RAW 264.7, (D) CHANG, (E) MCF-7, (F) MDA-MB 435, (G) A549 and (H) DU145 cell lines.
FIG. 14 is a histopathological examination result of liver, spleen, kidney, and lung of rats stained with hematoxylin and eosin after administering the ZnS nanoparticle conjugate of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples or drawings. It is to be understood, however, that the following description of the embodiments or drawings is intended to illustrate specific embodiments of the invention and is not intended to be exhaustive or to limit the scope of the invention to the precise forms disclosed.

Example  One. Colloidal ZnS : Mn  Synthesis of nanoparticles

ZnS: Mn nanoparticles were prepared by reacting ZnCl ₂ and MnCl ₂ with sulfur in a dibenzylamine coordinating-solvent. Dissolving 0.4 g of ZnCl ₂ and 0.015 g of MnCl ₂ in 54 mL of dibenzylamine and heated to 120 ℃ for 2 hours under vacuum. To the mixed metal-dibenzylamine complex solution was added 0.6 g of sulfur powder at 50 占 폚. The mixture was heated to 260 < 0 > C and aged at this temperature for 15 minutes. During the synthesis of ZnS: Mn nanoparticles, another zinc-dibenzylamine complex stock solution was prepared by heating a solution of 0.8 g of ZnCl ₂ in 10 mL of dibenzylamine to 120 ° C for 1 hour under vacuum. . After the ZnS: Mn nanoparticles were synthesized by aging at 260 DEG C for 15 minutes, the solution was cooled to 150 DEG C and 5 mL of the zinc-dibenzylamine complex storage solution was added. The reaction mixture thus obtained was heated to 260 DEG C and aged at this temperature for 15 minutes. Finally, the solution was cooled to 160 < 0 > C and hot ethanol or propanol was added to recover the nanoparticle powder (Figure 1A). The synthesized nanoparticles were post-treated with oleylamine to give high colloidal stability to each nanoparticle. The purified nanoparticles were dispersed in 40 mL of chloroform.

In the photoluminescence excitation (PLE) spectrum of the ZnS: Mn nanoparticles, 1 photon absorption is predominant by the ZnS nanoparticles, and 1S _e -1S _h exciton transition at 315 nm ultraviolet wavelength (Fig. 1B). Unpolarized ZnS nanoparticles emit blue fluorescence whereas efficient manganese doping improves photoluminescence Stokes-shift to 580 nm (internal transition from ⁴ T ₁ to ⁶ A ₁ ) (Fig. 1B, Fig. 2) with a high quantum efficiency of about 35% from Mn < ^{2 + &} gt ;. In terms of distinguishing the emission of the nanoparticles from the intrinsic background magnetic fluorescence, primarily in the blue-green wavelength region, as well as minimizing self-absorption, the enhanced Stokes shift has important advantages in the field of biomedical imaging .

Example  2. ZnS : Mn  Characterization of nanoparticles

The quantum efficiency of the ZnS: Mn nanoparticles synthesized in Example 1 was measured from an absolute photoluminescence measurement system. The zinc concentration obtained from the transmission electron microscope (TEM, JEOL EM-2010) photographic analysis and ICP-AES (inductively-coupled plasma atomic emission spectroscopy) was calibrated to determine the concentration of the nanoparticles. The nanoparticles were spin-coated onto a quartz substrate and placed in an integrating sphere. The nanoparticle thin film was excited using a 325 nm He: Cd continuous wavelength laser and emission was detected by a monochromator equipped with a photomultiplier tube. The decay kinetics of the nanoparticles were measured from a time-correlated single photon counting spectrometer (FLS920, Edinburgh) by excitation of the nanoparticle solution using a micro-flash lamp, and a microchannel plate-photomultiplier tube ). ≪ / RTI >

Photon emission characteristics of the ZnS: Mn nanoparticles were investigated after excitation with a titanium: sapphire near-infrared pulse laser. Figure 1C is a multiphoton micrograph spectra image excited at 920 nm for the nanoparticles solution cast on a glass substrate and the corresponding spectra show a clear Mn ^{2 +} ( T _d ) emission at 580 nm Degree). The slope obtained from the power-dependent PL intensity as a function of the incident power is about 2.9, which shows the characteristics of the 3 photon excitation light emission (3PL) (Fig. 1D). The multiphoton excitation wavelength (920 nm) is about three times the 1S _h -1 S _e transition (about 315 nm) of ZnS nanoparticles. Thus, the 3PL is due to 580 nm phosphorescence due to exciton energy transfer to the doped Mn < 2 + > ions after spontaneous 3 photon resonance absorption of 920 nm photons in ZnS nanoparticles carrying 1S _h -1S _e exciton transitions (Fig. .

From a practical point of view, a large three-photon cross-section and the corresponding 3PL should be evaluated in comparison to two photon emission (2PL) under multiphoton imaging conditions. In this respect, the 3PL of ZnS: Mn nanoparticles capped with phospholipid compared to Rhodamine 6G (Fig. 3A, Fig. 4) at the subdiffraction-limited spot Photon correlation spectroscopy (FCS) was performed. The 3 photon section (σ ₃ ) of the ZnS: Mn nanoparticles was determined from the cubic power-dependence of the 3PL intensity with respect to the incidence power (Equation 1):

(1)

( ₃ ) and action cross-section (? ₃ ?) Are 1.27 (± 0.50) × 10 ^-79 cm ⁶ s ² photon ^-2 and 2.54 0.10) x 10 ^-80 cm ⁶ s ² photon- ² . The three-photon cross-section of the nanoparticles is four orders of magnitude larger than the three-photon cross-section of a common ultraviolet fluorescent dye, indicating that the nanoparticles have a great potential to replace the ultraviolet fluorophore in the three-photon imaging field . ZnS: by comparing the 3PL luminance of Mn nanoparticles (f ₃₎ and 2PL luminance of Rhodamine 6G (f ₂₎ is provided with a practical means for the large three-photon cross-section, which, depending on input power, as shown in the following equation (2) Change:

(2)

In this connection, it is a non-saturated black area (nonsaturating power regime) 3-photon action cross section in the (σ parameter corresponding reflecting the three-photon emission luminance in photon imaging conditions, and the two-photon action cross section (σ ₂ η _{2) 3} η ₃₎ and the product of the input power (I _0). Equation 2 shows the importance of the excitation power as well as the operating section for three-photon imaging. In conventional three-photon fluorescence imaging of endogenous biomolecules, high incident power is used because of the low three-photon cross-section of the biomolecule. However, long-term imaging of living cells results in phototoxicity induced by high power excitation. In this experiment, due to the large three photon action cross section of the ZnS: Mn nanoparticles, the 3PL brightness of a single ZnS: Mn nanoparticle is about 1.0 (± 0.4) at a low nonsaturating excitation power of about 1.35 mW, Close to the effective luminance corresponding to a typical two-photon action cross section of the GM unit (Goepper-Mayer unit).

From the quantum mechanical point of view, the three-photon excitation has inherently lower efficiency compared to the two-photon excitation, but spectroscopic measurements show that the large three-photon cross-section of the ZnS: Mn nanoparticles results in low incidence Power can be used, which is compatible with two photon emission. In addition, at higher excitation power the three photon emission is increasingly saturated, which is due to the long phosphorescence decay time of the doped Mn ^{2 +} (T _d ) ions (FIG. 2). Furthermore, 900 nm to 3 photons at 1000 nm excitation spectrum is in agreement with the 1S _e -1S _h transitioning of the ZnS nanoparticles in the corresponding one photon PLE spectrum, which each in which the three-photon cross-section and the working section 950 nm 1.58 (± 0.63) × 10 ^-79 cm ⁶ s ² photon ^-2 and 3.16 (± 1.26) × 10 ^-80 cm ⁶ s ² photon ^-2 (FIG. 3B). The spectral measurement results clearly show that the three-photon excitation process of the nanoparticles can be applied in the field of biomedical imaging.

Example  3. ZnS : Mn  Nanoparticles Phospholipid  coating

The oleylamine-capped hydrophobic ZnS: Mn nanoparticles were transferred to water by incorporating ZnS: Mn nanoparticles in the mPEG-PE phospholipid. 50 mg of mPEG-PE powder was added to 2 mL of ZnS: Mn nanoparticle solution in chloroform. The chloroform was then evaporated using a rotary evaporator at 60 ° C and the flask was further vacuum dried at 100 ° C. After chloroform was completely removed, 20 mL of triple-deionized water was added to obtain a clear suspension containing mPEG-PE phospholipid micelles containing ZnS: Mn nanoparticles. The nanoparticles dispersed in water were centrifuged at 30,000 rpm for 2 hours. After the centrifugation was repeated 6 times, the supernatant containing the vacant micelles was discarded from the excess phospholipid. The remaining nanoparticles were filtered using a 0.25 μm cellulose acetate filter. For conjugation of the functional biomolecule to the ZnS: Mn nanoparticles, 5 mg of amino-PEG-PE was added along with 45 mg of mPEG-PE and all other procedures were the same. The emission quantum efficiency of the phospholipid-capped ZnS: Mn nanoparticles was 20% to 25%.

Example  4. ZnS : Mn  FCS of nanoparticles ( 여광체 correlation spectroscopy )

Multiphoton FCS measurements were performed to determine the three-photon luminescence brightness of the ZnS: Mn nanoparticles in water and the two photon luminescence intensities of rhodamine 6G in water. The nanoparticles in the aqueous solution and rhodamine 6G were excited with a titanium: sapphire pulse laser operating at a repetition rate of 76 MHz and an excitation wavelength of 920 nm and a pulse width of about 140 fs. During the spectral measurement, the laser beam was focused on a sub-micrometer diffraction limited spot and the luminescence formed in the spot at a wavelength in the range of 500 nm to 700 nm was focused on an avalanche photodiode, . ≪ / RTI >

Example  5. ZnS : Mn  Nanoparticles Of peptide  join( conjugation )

(2). In this study, we investigated the effect of the amino-PEG-PE-encapsulated nanocrystal on the amino acid sequence of the sulfated-amino-PEG-PE-encapsulated nanocrystal The bond of ZnS: Mn nanoparticle micelle and peptide was formed by maleimide bond formation. 10 nmol of ZnS: Mn nanoparticles functionalized with amino-PEG-PE were added to the solution in aqueous solution using 50 mM phosphate buffer (NaH ₂ PO ₄ / Na ₂ HPO ₄ buffer, pH = 8.5) The nanoparticle micelles were purified through elution. 5 mg of sulfo-SMCC was dissolved in 50 mM phosphate buffer and mixed with ZnS: Mn nanoparticle phospholipid micelles functionalized with amino-PEG-PE. The mixture was incubated at room temperature for 90 minutes. After the incubation, the ZnS: Mn nanoparticles functionalized with sulfo-SMCC by eluting with phosphate buffered saline (PBS, pH = 7.2) using a PD-10 desalting column (GE Healthcare) The micelle was purified. 5 mg of Lyp-1 or 7 mg of c (RGDyK) was dissolved in 0.5 mL of PBS and mixed with the ZnS: Mn nanoparticle micelles functionalized with the sulfo-SMCC in PBS. The thus obtained mixture was incubated at room temperature for 90 minutes and then peptides-bonded ZnS: Mn nanoparticle micelles were separated by gel filtration using Sephacryl S-300 (GE Healthcare) (FIG. 5 And FIG. 6). The elution fractions of the peptide conjugated ZnS: Mn nanoparticle micelles were collected and further concentrated using a centrifuge filter 10K (Millipore) for animal experiments.

Example  6. ZnS : Mn  Of nanoparticles In vitro ( in vitro ) 3 photons  Imaging three - photon imaging )

200 μL of 1 μM ZnS: Mn nanoparticle-LyP 1 conjugate was added to MDA-MB 435 human breast cancer cells grown on a coverglass-bottom dish and incubated for 2 minutes at 37 ° C. The cells were washed three times with PBS buffer solution to remove the defective ZnS: Mn nanoparticle conjugate, and then the cover glass-bottom dish was filled with the cell growth medium. The nanocrystals-targeted cells were immersed in the cell growth medium with a water-immersion lens and subjected to LSM-780 multi-photon microscopy by 950 nm excitation. Lt; / RTI > Spectral images were acquired using a 32 channel GaAsP detector that collected photon signals at selected wavelengths from 411 nm to 691 nm at intervals of 8.7 nm and incorporated them into images corresponding to the color intensity at a given wavelength . The laser operating voltage was kept below 1 mW to minimize phototoxicity. To obtain images of the fixed cells, the nanoparticle-targeted MDA-MB 435 cells were fixed with 4% paraformaldehyde for 5 minutes. After the cell fixation, the cells were washed three times with PBS buffer.

Fig. 7 is a multiphoton microscopic image of MDA-MB 435 cancer cells incubated with a conjugate of Lyp-1 with ZnS: Mn nanoparticles. Cellular surface-lining of 3PL was visualized from a ZnS: Mn nanoparticle-Lyp-1 conjugate specifically bound to the cell receptor p32 (Fig. 7A). In addition, the orange 3PL spectrum of the ZnS: Mn nanoparticles targeted from the spectral image clearly recognizes the cell surface-targeted nanoparticle, enabling more sensitive detection (Figures 7B and 7C , Fig. 8). The experimental results show the usefulness of 3PL of ZnS: Mn nanoparticles for targeted molecular imaging. It should be noted that imaging of living cells was performed at low excitation and non-saturation power (approximately 0.6 mW) due to the high three photon action cross section of the ZnS: Mn nanoparticles. The excitation power in the imaging condition in this embodiment is smaller than the excitation power of the three-photon magnetic fluorescence imaging reported in the prior art by one or two powers. In this regard, the inventors of the present invention have found that dynamic clustering of p32 receptors (ZnS: Mn NC-Lyp-1 conjugates-targeted p32 receptors) targeted with a ZnS: Mn nanoparticle-Lyp- The dynamic fluid behavior was imaged. Long-term temporal imaging capabilities clearly demonstrate no phototoxicity to the 3PL cell imaging conditions of the ZnS: Mn nanoparticles due to low excitation power (about 1 mW) (Fig. 7D).

Moreover, imaging in the non-saturated excitation region makes it possible to exploit the three photon emission of the nanoparticles for imaging of the sub-diffraction resolution. Diffraction - Limited (

) As a method of imaging, three-photon imaging can be performed with two photon excitations at the near-

) Higher spatial resolution than

), Which has not yet been demonstrated in the biological medium. In this example, 3PL was successfully imaged for membrane connections surrounding the cell surface between two adjacent cells at sub-diffraction resolution up to 272 +/- 25 nm (Figs. 7F and 7G). Measured FWHM of 272 ± 25 nm is the theoretical resolution limit of 3 photon excitation (for 950 nm

= 279 nm, NA ~ 1.0), and the sub-diffraction limit of 1 photon emission (at 950 nm

= 475 nm, NA ~ 1.0) as well as the sub-diffraction limit of 2 photon emission (at 950 nm

= 335 nm, NA ~ 1.0) The border is higher resolution. Conventional attempts for three-photon imaging of a single micro-crystal, since the diameter of the micro-crystals have exceeded the wavelength size, Oh did not show a diffraction resolution (SW Hell et al., J. Biomed. Opt. 1, 71 (1996)). In this example, 3PL images were cured with "pin-like" resolution by effective targeting to small size nanoparticles (5.5 nm). According to the experimental results, the three-photon imaging of the nanoparticles provides a better spatial resolution in target cell imaging, compared to two-photon excitation.

Example  7. ZnS : Mn  Of nanoparticles In vivo ( in vivo ) 3 photons  Imaging

MDA-MB 435 human breast cancer cells (2 × 10 ⁵ cells / 100 μL of PBS) were injected into the shoulder of 4-week-old specific pathogen-free male balb / c-nu mice To produce breast cancer metastatic tumor xenografts. When tumor size ranged from 0.5 mm to 0.8 mm, 100 μL of a 40 μM ZnS: Mn nanoparticle-c (RGDyK) conjugate was injected into the tail vein.

To achieve non-invasive in vivo imaging conditions, the tumor xenografts were anesthetized by administration of Zolethyl 50 and the tumor site was placed between the cover glass and the bottom of a cover glass-bottom confocal dish. A gap between the cover glass and the bottom was filled with bone-wax and the dish was filled with water. The xenograft was placed on a microscope table, the dish was filled with water, and the immersion lens for imaging was immersed in the dish. The tumor site was then imaged in a LSM-780 multiphoton microscopy observation spectral imaging mode at 920 nm.

In situ spectral deconvolution imaging, which acquires an image of the spectral imaging mode and immediately releases it into a signal corresponding to a reference spectrum, (Carl-Zeiss) on the basis of a pre-stored reference spectrum. The background autofluorescence was then discarded during the image acquisition period.

In vitro (ex vivo imaging, the mice were sacrificed after in vivo imaging and tumor tissues were dissected and fixed with 4% paraformaldehyde and OCT compound-embedded. The tumor tissue was cut to a thickness of 20 μm and imaged by a multiphoton microscope. Spectral unmixing was performed using ZEN software (Carl-Zeiss) with known spectral information for the three-photon emission of the nanoparticles, SHG and background autofluorescence, and the background magnetic fluorescence was removed.

In this example, 3PL was visualized on the tumor vasculature targeted to the ZnS: Mn nanoparticles, located below the base of the dermis to a depth of about 100 [mu] m (Fig. 9A, Fig. 10). Due to efficient angiogenesis targeting by the ZnS: Mn nanoparticle-RGD conjugate, high-efficiency 3PL was clearly identified from ZnS: Mn nanoparticles in the vasculature via spectral imaging. The experimental results demonstrate that the three photon emission of the nanoparticles can be used for in vivo target imaging.

In terms of in vivo imaging conditions, most of the ultraviolet fluorophore is difficult to resolve because even though the three photon cross-sections of the ultraviolet fluorophore are large enough to be visualized, the intrinsic background self fluorescence corresponds to the blue-green wavelength . In this example, a large Stokes shift of the emission from ultraviolet to orange by nanocrystal doping results in a significant emission cross-talk between the 3PL of the nanoparticles and the two photon-magnetic fluorescence of the tissue under in vivo imaging conditions (Fig. 9B). In this regard, the bright 3PL of the nanoparticles can be spectrally distinguished from the background magnetic fluorescence through spectral imaging. In addition to the bright 3PL and increased Stokes shifts, the ZnS: Mn nanoparticles exhibit better light stability than a typical two-photon fluorescence stage in vivo in multi-photon imaging conditions requiring large incident power for deep tissue imaging (Fig. 11). Due to the remarkable light stability of the nanoparticles, concurrent imaging is possible in which temporal imaging in vivo as well as secondary harmonics of collagen fibers are generated, providing microscopic insights into the local tumor environment. Thus, in this example, the 3PL of tumor-targeted nanoparticles and the SHG of collagen fibers were simultaneously imaged through "in-situ spectral deconvolution imaging ", and at the same time as acquiring the spectral image, 3PL of nanoparticles and SHG of collagen fibers were extracted (Figs. 9C and 9D, Fig. 12). Two specific regions of the local tumor microenvironment, which is a signature for tumor targeting of nanoparticles, were observed: vasculature outlining by molecular angiogenesis targeting and enhanced permeation and retention (EPR Extravasation by extravasation. Figure 9C shows the 3PL of ZnS: Mn nanoparticles, showing the outline of the vessel wall after the nanoparticles filled in the vasculature were partially removed. The vascular lining is a signature of efficient angiogenic targeting of nanoparticulated RGD peptides directed against the overexpressed [alpha] _{v [} beta] ₃ integrin on the endothelial medium of the tumor vasculature. Under non-invasive imaging conditions, high excitation power is required to visualize tumors located beneath highly scattering dermis, which can reduce spatial resolution due to saturation of 3PL (FIG. 4). However, in this example, the vascular endothelium was visualized at a resolution of 2 mu m, demonstrating that the 3PL has sufficient spatial resolution at micrometer scale in in vivo imaging conditions.

Example  8. ZnS : Mn  Of nanoparticles Light stability ( 광성성 ) exam

The above ZnS: Mn nanoparticles and general fluorescent terminals (Rhodamine 6G, RITC and FITC) were solution-cast onto a cover glass substrate. The thin film thus obtained was photo-excited at 920 nm by focusing on a Plan-Apochromat 20X objective lens in a multiphoton microscope. The excitation power and pixel duration time were set to 10 mW and 12.6 s, respectively.

To test the photo-bleaching behavior, a region of interest (ROI) with a frame size of 82 x 51 pixels was defined in a multiphoton microscope image of the thin film. In the above defined region, the nanoparticles and the fluorophore were repeatedly excited 20 times at the emission signal detection interval, which was controlled by ZEN software. Then, the optical stability of the nanoparticles and the fluorophore was defined as a relative PL intensity compared with the PL intensity outside the ROI in the selected ROI. As shown in FIG. 11, the nanoparticles not only show better optical stability than general fluorescent lamps, but also increase the signal intensity at high power excitation due to photochemical annealing.

Example  9. ZnS : Mn  Evaluation of cytotoxicity of nanoparticles

The cytotoxicity of the ZnS: Mn nanoparticles was evaluated in comparison with CdSe and CdSe / CdZnS nanoparticles. 5 nm CdSe and CdSe / CdZnS nanoparticles were synthesized and coated with mPEG-PE phospholipid micelle, so that the physiological conditions of these nanoparticles were the same as the physiological conditions of the ZnS: Mn nanoparticles. (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) (Promega) assay for cytotoxicity . All cells were maintained in DMEM (Lonza) or RPMI 1640 (Gibco BRL). The medium was supplemented with 10% fetal bovine serum (FBS, Gibco BRL), 100 μg / mL streptomycin and 100 IU penicillin. Cells were monolayered in a 100 mm dish and subcultured 3 times a week for 1 week at 37 ° C in an atmosphere containing 5% CO ₂ , and these conditions were maintained at a low culture count of 3-15. For evaluation of in vitro cytotoxicity, the cells of the logarithmic growth phase were detached and attached to a 96-well flat bottom microplate at a density of 1,000 cells / well to 25,000 cells / well (180 μL / well) And maintained at 37 DEG C for 24 hours to initiate exponential growth. After 24 hours of recovery, 20 μL of PBS buffer (8 wells per plate control wells per plate) and various concentrations of nanoparticles were added to the wells three times. For the control wells, the same volume of complete culture medium was included in each experiment. The cells were exposed to the nanoparticles continuously for 48 hours under a 5% CO ₂ atmosphere at 37 ° C and then the cell viability was measured by MTS assay (FIG. 13) and the concentration-effect curve was plotted IC ₅₀ (concentration of ZnS nanoparticles inhibiting 50% cell growth) was obtained for each cell type (Table 1).

Cell line Characteristic IC ₅₀ (ZnS: Mn) IC ₅₀ (CdSe / CdZnS) IC ₅₀ (CdSe) HUVEC Human Umbilical Vein Endothelial 3669 403 108 293T Human Embryonic Kidney 2476 1472 358 RAW264.7 Mouse Leukaemic Monocyte Macrophage 1826 467.72 197.36 A549 Human Adenocarcinomic Human Alveolar Basal Epithelial 1275 509 413 MCF-7 Human Breast Cancer 1362 1343 157 MDA-MB435 Human Breast Cancer 1371 316 85 DU145 Human Prostate Cancer Cells 3294 1351 194 Chang Human Liver Cross-Contaminated with HeLa 2263 377 366

Example  10. In vivo  Toxicity assessment

To determine the in vivo cytotoxicity of the ZnS: Mn nanoparticles, pharmacokinetics, histopathology and serum chemistry were investigated.

To investigate the pharmacokinetics of the nanoparticles, 100 μL of 40 μM ZnS: Mn nanoparticles were injected into the tail vein of balb / c-nu mice. At 1, 3, 7, 10 and 14 days after the administration, the mice were sacrificed and reticuloendothelial system organs of liver, spleen and kidney were collected. The organ was digested with nitric acid and zinc ion concentration was determined from inductively-coupled plasma-optical emission spectrometry (ICP-OES). Histological examination of the organs was performed from the incision portion of kidney, liver and lung tissue collected from SD rats 7 days after administration of 10 nmol of the nanoparticles and stained with hematoxylin and eosin (H & E). 1 mL of blood samples were collected from the eyes of the SD rats on days 1, 3 and 7 after administration of 10 nmol of ZnS: Mn nanoparticles to detect hepatic function markers such as alkaline phosphatase (ALP), alanine amino (BUN) and creatinine (CREA), which are renal function markers, as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) Biochemical tests (serum biochemistry) were performed. The results of the serum biochemical test are shown in Table 2.

Week after death
Date normal
range AST (U / L) ALT (U / L) ALP (U / L) BUN (mg / dL) CREA (mg / dL) 74-143 ⁽²⁾ 35-80 ⁽¹⁾ 314.9-473.1 ⁽³⁾ 10-21 ⁽¹⁾ 0.5-1.0 ⁽¹⁾ 0 PBS 87.0 ± 27.9 43.2 ± 9.6 356.8 ± 59.6 11.9 ± 3.3 0.50 0.06 ZnS 93.5 ± 23.4 41.0 ± 5.7 394.5 ± 59.0 9.6 ± 1.1 0.33 ± 0.05 One PBS 97.0 + - 32.0 36.8 ± 5.3 319.0 ± 109.5 17.5 ± 3.3 0.40 + 0.06 ZnS 145.5 ± 36.4 48.0 + - 12.1 354.0 ± 60.0 18.1 ± 1.1 0.63 + 0.11 3 PBS 89.8 ± 25.2 40.5 ± 4.3 335.3 ± 117.5 12.7 ± 1.4 0.45 ± 0.05 ZnS 89.6 ± 26.1 43.3 ± 4.9 299.7 ± 30.6 12.2 ± 0.7 0.70 + 0.07 7 PBS 103.8 ± 45.5 37.7 ± 6.8 382.5 ± 77.2 14.5 ± 1.4 0.48 + 0.04 ZnS 95.3 ± 50.1 40.0 ± 6.9 368.0 ± 89.0 13.9 ± 1.2 0.47 ± 0.05

(1) Reference values for experimental animals (Research animal resources, University of Minnesota, USA) (http: // www. Ahc.umn.edu/rar/refvalues.html)

(2) Clinical parameters for Crl: WI (Han) (Giknis and Clifford, 2008. Charles river)

(3) SLC, Inc. (Http://www.labanimal.co.kr), a biochemical data sheet obtained from Japan (Japan)

The extravasation of the nanoparticles was visualized from the leaking tumor vasculature (Fig. 9D). Along with the active tumor targeting of the ZnS: Mn nanoparticle-RGD conjugate, which allows the nanoparticles to accumulate in the tumor vasculature, the small hydrodynamic size (about 23 nm) of the nanoparticle conjugate facilitates the extravasation of nanoparticles . The extravasated nanoparticles were visualized in the cellular components of the tumor tissue, demonstrating that the nanoparticle 3PL can provide cell and cell resolution in in vivo imaging conditions. The localization of the nanoparticles was further confirmed from the in vitro images of incised tumor tissues, and the 3PL of the nanoparticles was mainly located in the cellular constituents near the blood vessel structure (FIG. 9E). These results show that 3PL of ZnS: Mn nanoparticles can be used as an in vivo target imaging contrast agent to understand local tumor microenvironment at the level of the cells.

In addition to in vivo imaging capabilities, the biocompatibility of the nanoparticles is critical for clinical applications. The biodistribution of phospholipid-capped ZnS: Mn nanoparticles after intravenous administration was investigated (Fig. 9F). The basal level of Zn produced is higher in reticuloendothelial (RES) organs, as zinc ions actively intervene in the metabolism of these organs. After administration, the nanoparticles were mainly accumulated in the liver and spleen and gradually removed from the RES organs. In contrast to other semiconductor nanoparticles, the nanoparticles did not accumulate noticeably in the kidneys, which resulted in renal clearance and hepatobiliary clearance after degradation of the nanoparticles in the liver and spleen. . In addition, a series of in vivo biocompatibility tests, including histopathology (FIG. 14) and serum chemistry (Table 1), suggest that the ZnS: Mn nanoparticles are highly biocompatible and clinically applicable.

Example  11. Fluorescence Correlation Spectrum ( 여광체 correlation spectra  ( FCS )) From a multi-photon-excitation PL  Normalization of Century

Normalization of the FCS data of rhodamine 6G and ZnS: Mn nanoparticles on the number of particles within the focal volume resulted in a single (non-saturated) regression of 3PL ( F ₃ ) and 2PL ( F ₂ ) in the nonsaturating power regime The particle brightness was derived.

The FCS intensity ratio of 3PL ( F ₃ ) and 2PL ( F ₂ ) is described as follows:

Where F ₃ and F ₂ are the FCS intensities of the ZnS: Mn nanoparticles and rhodamine 6G, respectively _, and V _{eff , ZnS} and V _{eff , R 6 G} are the 3 photon excited ZnS: Mn nanoparticles and 2 photon excited rhodamine 6G is the effective focal volume. In this case, the concentrations of the ZnS: Mn nanoparticles and the rhodamine 6G (ρ ₃ and ρ ₂ , respectively) were 17.85 μM and 10 μM, respectively.

Considering the illumination at the focus as a 3D-Gaussian shape and integrating over all three dimensions, the effective volume of the two-photon and three-photon excitation is approximated as:

In the above two equations,? Is 5 as the elongation of the volume along the optical axis, and a ₁ is 0.21 μm as the focal size. Then, the two-photon effective focus volume V _{eff , R 6 G} and the three-photon effective focus volume V _{eff , ZnS} are 0.73 μm ³ and 0.40 μm ^3, respectively. Further, the effective focal volume ratio of the three-photon to the two-

≪ / RTI > This expression means that the three-photon excitation has a higher spatial resolution compared to the two-photon excitation.

Considering both the effective volume and the solution concentration, the number of rhodamine 6G ( N ₂ ) and the number of ZnS: Mn nanoparticles ( N ₃ ) are 4325 and 4231, respectively.

Given the number of luminophore particles within the calculated focus volume, the FCS is normalized from the fluorescence correlation spectrum as follows:

Due to the low amplitude of g (0) from the autocorrelation function 3PL (Fig. 4), which can be influenced by various disturbing effects (Fig. 4) The correlation function 3PL was not considered. The single particle brightness ratio in the above equation was derived from a two-photon absorption cross-section of Rhodamine 6G and was 5.7 GM at 920 nm.

Claims (27)

ZnS nanoparticles doped with manganese, an oleylamine layer bonded to the nanoparticles, a phospholipid layer coated with the oleylamine layer, and a functional biomolecule bonded to the phospholipid layer. Junction. The ZnS nanoparticle conjugate according to claim 1, wherein the size of the ZnS nanoparticles doped with manganese is 1 nm to 30 nm. The ZnS nanoparticle conjugate according to claim 1, wherein the mole ratio of zinc to manganese (Zn: Mn) in the manganese-doped ZnS nanoparticles is 1: 0.001 to 1: 1. The method of claim 1, wherein the phospholipid is selected from the group consisting of mPEG-PE, mPEG-PC, mPEG-PA, mPEG-PG, mPEG-PS, mPEG-PI and block copolymers thereof with PEI ZnS nanoparticle conjugate. The conjugate of claim 1, wherein the functional biomolecule is selected from the group consisting of peptides, DNA, RNA, and antibody proteins. The ZnS nanoparticle conjugate according to claim 5, wherein the peptide is a tumor-targeting peptide. 7. The conjugate of claim 6, wherein the tumor targeting peptide is selected from the group consisting of RGD, iRGD and LyP 1. [Claim 6] The conjugate of claim 5, wherein the antibody protein is selected from the group consisting of vascular endothelial growth factor, epidermal growth factor, transferrin, and Herceptin. The ZnS nanoparticle conjugate according to claim 1, wherein the hydrodynamic diameter of the ZnS nanoparticle conjugate is 3 nm to 1 μm. (i) forming an oleylamine layer bound to manganese-doped ZnS nanoparticles;
(ii) forming a phospholipid layer covering the oleylamine layer; And
(iii) conjugating the functional biomolecule to the phospholipid layer.
ZnS nanoparticle conjugate. 11. The method of claim 10, wherein the size of the ZnS nanoparticles doped with manganese is 1 nm to 30 nm. 11. The method of claim 10, wherein the molar ratio of zinc to manganese (Zn: Mn) in the manganese-doped ZnS nanoparticles is 1: 0.001 to 1: 1. 11. The composition of claim 10, wherein the phospholipid is selected from the group consisting of mPEG-PE, mPEG-PC, mPEG-PA, mPEG-PG, mPEG-PS, mPEG- Wherein the ZnS nanoparticles are prepared by a method comprising the steps of: [Claim 11] The method according to claim 10, wherein the functional biomolecule is selected from the group consisting of peptides, DNA, RNA, and antibody proteins. 15. The method of claim 14, wherein the peptide is a tumor-targeting peptide. 16. The method of claim 15, wherein the tumor targeting peptide is selected from the group consisting of RGD, iRGD, and LyP 1. 15. The method according to claim 14, wherein the antibody protein is selected from the group consisting of vascular endothelial growth factor, epidermal growth factor, transferrin, and Herceptin. [Claim 11] The method according to claim 10, wherein the hydrodynamic diameter of the ZnS nanoparticle bonded body is 3 nm to 1 [mu] m. ZnS nanoparticles doped with manganese, an oleylamine layer bonded to the nanoparticles, a phospholipid layer coated with the oleylamine layer, and a functional biomolecule bonded to the phospholipid layer. A multiphoton optical imaging agent comprising a conjugate. 20. The multiphoton optical imaging agent according to claim 19, wherein the size of the ZnS nanoparticles doped with manganese is 1 nm to 30 nm. 20. The multiphoton optical imaging agent according to claim 19, wherein the molar ratio of zinc to manganese (Zn: Mn) in the manganese-doped ZnS nanoparticles is 1: 0.001 to 1: 1. 20. The method of claim 19, wherein the phospholipid is selected from the group consisting of mPEG-PE, mPEG-PC, mPEG-PA, mPEG-PG, mPEG-PS, mPEG-PI and block copolymers thereof with PEI A multiphoton optical imaging agent. 20. The multiphoton optical imaging agent according to claim 19, wherein the functional biomolecule is selected from the group consisting of peptides, DNA, RNA, and antibody proteins. 24. The multiphoton optical imaging agent of claim 23, wherein the peptide is a tumor-targeting peptide. 26. The multiphoton optical imaging agent of claim 24, wherein the tumor targeting peptide is selected from the group consisting of RGD, iRGD, and LyP 1. 24. The multiphoton optical imaging agent according to claim 23, wherein the antibody protein is selected from the group consisting of vascular endothelial growth factor, epidermal growth factor, transferrin, and Herceptin. The multiphoton optical imaging agent according to claim 19, wherein the hydrodynamic diameter of the multiphoton optical imaging agent is 3 nm to 1 μm.

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