JP4936508B2 - Vitreous staining agent and staining method of eye containing fluorescent nanoparticles - Google Patents

Description

  The present invention relates to a staining agent and method used to stain the vitreous body of human and / or non-human animal eyes.

  In recent years, it has been elucidated that many ophthalmic diseases are associated with physiological and / or pathological changes in the vitreous. For example, liquefaction of the vitreous due to aging can induce retinal tears and retinal detachment, and it is thought that the macular hole and the upper macular membrane are caused by the posterior vitreous cortex pocket. There is also macular disease caused by the vitreous towing the macula. Some of these diseases become severe and eventually lead to blindness.

  For this reason, it is very important to observe the liquefaction and turbidity of the vitreous body and the presence or absence of lesions. However, it is difficult to observe the vitreous because the vitreous is composed of a transparent gel substance.

  Slit lamp microscope, Scanning Laser Ophthalmoscope (SLO), Ultrasound diagnostic device, Optical Coherence Tomography (OCT), Spectroscopy, Dynamic light scattering measurement Although it is possible to observe with a device (Dynamic Light Scattering; DLS) (Non-patent Document 1), it is difficult to say that the vitreous body can be observed sufficiently and easily no matter which one is taken.

  Currently, there are triamcinolone acetonide, indocyanine green, sodium fluorescein, rose bengal, lissamine green, sulferodamine, etc., which are used in the ophthalmology field, but are used for clinically staining the vitreous. The only substance that can be obtained is triamcinolone acetonide.

  Even though triamcinolone acetonide is used for vitreous observation, it is mostly used during vitreous surgery, and it is used for treatment other than surgery as a therapeutic drug using its pharmacological action as a corticosteroid. Body injection is the main. Therefore, it cannot be said that it is used for vitreous body observation.

  Moreover, since triamcinolone acetonide is white, it is difficult to contrast with physiological / pathological changes such as Weiss Ring in the vitreous body, and it is difficult to observe the object. In addition, since triamcinolone acetonide is in the form of powder, the particles are scattered in the vitreous body even after being injected into the vitreous body, and the continuity between the particles is poor, and therefore the observation image becomes rough. is there. Furthermore, in recent years, side effects such as increased intraocular pressure after the injection of triamcinolone acetonide, the occurrence / aggravation of cataract, and endophthalmitis have been reported.

  Fluorescent nanoparticles are considered to have various applications as phosphors having high brightness and hardly causing photobleaching, and have been used as intracellular markers.

J. Sebag, Journal of Biomedical Optics January / February 2004 Vol. 9 No. 1 p38-46 Hoshino, A. et al., Biochem. Biophys. Res. Comm. 314 (2004) pp.46-53 Hanaki, K. et al., Biochem. Biophys. Res. Comm. 302 (2003) pp.496-501 Hoshino, A. et al., Nano Letters 2004, Vol. 4, No. 11, pp. 2163-2169

  An object of the present invention is to provide means and a method capable of easily and safely observing the vitreous body of an eye in daily diagnosis and / or surgery.

The present invention
[1] Vitreous staining agent for eyes containing fluorescent nanoparticles;
[2] The staining agent according to [1], wherein the fluorescent nanoparticles have a core monolayer structure or a core-shell multilayer structure;
[3] The staining agent according to [1] or [2], wherein the fluorescent nanoparticles include a semiconductor element;
[4] A method for staining the vitreous body of the eye, comprising injecting the staining agent according to any one of [1] to [3] into the vitreous body of the eye,
I will provide a.

  According to the present invention, it is possible to easily and safely observe a vitreous that has not been provided with an appropriate observation method in the past, and it is possible to easily detect the presence or absence and degree of abnormality of the vitreous. Specifically, the staining agent of the present invention is less likely to cause unnecessary diffusion, has low persistence, has a long fluorescence duration, and provides a transparent and high-contrast staining, so the vitreous changes are very easy to see, Have advantages such as. Therefore, the staining agent and staining method of the present invention are useful for ophthalmic diagnosis and treatment for humans and animals.

  Since the staining agent of the present invention uses fluorescent nanoparticles that are fluorescent substances having a fluorescent color such as red, having high brightness, and hardly causing photobleaching, lesions caused by physiological / pathological changes in the vitreous body It is easy to check the object. In addition, when observing the vitreous body or the liquefaction cavity, since the particles are small, it is easy to obtain a fine observation image. Furthermore, since it can be made translucent by adjusting the concentration, there is an advantage that the relative position with respect to surrounding tissues such as the retina can be easily confirmed. Due to these advantages, extremely safe surgery is possible when the staining agent of the present invention is used during vitrectomy.

  By observing the vitreous using the staining agent of the present invention, vitrectomy can be performed simply, safely and accurately. In particular, in vitreous surgery, not only the vitreous body visibility is markedly improved by staining the transparent vitreous body, but also other tissues in the eye can be seen through, so the retina, retinal blood vessels, optic nerve head, hair It becomes easy to grasp the positional relationship between the cadaver and the posterior capsule of the lens and the vitreous being operated, and safe and accurate vitreous surgery can be performed. In addition, it is possible to easily confirm the minute vitreous body condition such as the insertion of the vitreous body into the wound opening and the traction / adhesion of the vitreous body, which makes it possible to perform an operation that is polite and has few complications. Furthermore, when there is a vitreous escape due to a cataract surgery or the like, the vitreous that is present in the anterior chamber or has escaped from the eye can be easily seen, so that it can be accurately determined and processed.

  Further, the staining agent of the present invention can be used not only for actual surgery but also for ophthalmic diagnosis and skill acquisition using a vitreous body such as an animal model.

  Further, the staining agent and staining method of the present invention are used in the fields of medicine, pharmacy, veterinary medicine, etc., the relationship between aging, various diseases, symptoms, physical conditions, etc. and the vitreous state of the eye, treatment or It can be used to study the relationship between preventive measures and effects, and is also useful as a research reagent and method.

Fluorescent Nanoparticles The fluorescent nanoparticles used in the present invention are nano-sized fine particles that can emit fluorescence due to the quantum size effect or the like, and include what are called quantum dots.

The fluorescent nanoparticles can be excited if the wavelength is shorter than the fluorescence wavelength, and have excellent light resistance so that the fluorescence can be stably emitted for about 30 minutes or more during continuous excitation. In addition, it has a feature of high brightness as compared with conventional organic fluorescent materials. The wavelength fluorescence wavelength depends on the particle size, and the smaller the wavelength, the shorter the wavelength. Therefore, the fluorescent nanoparticles used in the present invention are preferably composed of 10 to 10 ⁴ element atoms as described later, more preferably about 10 ² to 10 ^3, and the particle size is generally 1 to 4. The thickness is about 100 nm, preferably about 1 to 20 nm, more preferably about 2 to 10 nm.

  In general, the fluorescent nanoparticles are basically composed of a semiconductor element, but may contain non-metallic elements, metallic elements, or the like. In addition, as a structure, the fluorescent nanoparticles may have a core monolayer structure (that is, the whole particle is homogeneous), or a core-shell multilayer structure (that is, the particles have two or more different layers). It may be. Examples of the fluorescent nanoparticle having a core-shell multilayer structure include a quantum dot having a metal nanocrystal nucleus (core) and a transition metal coating (shell).

  For example, the core material may be a combination of one or more Group II metal elements (zinc, cadmium, mercury, copper, etc.) and one or more Group VI elements (selenium, sulfur, oxygen, etc.); A combination of group III elements (boron, aluminum, gallium, indium, etc.) and one or more group V elements (nitrogen, phosphorus, etc.); and one or more group IV elements (carbon, silicon, germanium, tin, lead) One type or a combination of two or more types selected from the group consisting of These materials may contain a trace additive composed of a transition metal element. Examples of the shell material include zinc sulfide, zinc oxide, indium, gallium, silicon, and phosphorus.

  The fluorescent nanoparticles having a core-shell multilayer structure may further have an additional layer (coating layer) outside the shell layer. For example, the function of such an additional layer includes imparting water solubility to the particle surface and preventing elution of metal, and the materials include various polymers (PEG, amino acid, oligos). Peptides and derivatives thereof), glass coatings (silane coatings, etc.), etc., and the thickness of each layer is generally about 10 nm or less. Moreover, although there are many coating layers, since fluorescence becomes bright, it is convenient, but generally it is about 1-4 layers as a coating layer.

  Since various kinds of such fluorescent nanoparticles are commercially available, commercially available products may be selected as appropriate. Examples of commercially available products include “QDot” (trade name: Quantum Dot Corporation), “EviDots” (trade name: Evident Technologies). Any known method may be used as the production method. Examples of such methods include vapor phase methods such as CVD, laser, and sputtering; liquid phase methods such as spraying, alkoxide, and reverse micelle methods.

  When producing fluorescent nanoparticles for use in the staining agent of the present invention, the following method can be given as an example. First, the raw material as described above is dispersed in a coordinating solvent containing tri-n-octylphosphine oxide (TOPO) or the like, and the fine particles of the raw material are micellized with TOPO (TOPO micelle). Next, the TOPO micelles are heated under an argon gas sealing condition to grow the fine particles of the raw material in the TOPO micelles, thereby producing fluorescent nanoparticles having TOPO fixed on the surface. The fluorescent nanoparticles in this state are soluble in an organic solvent such as toluene or tetrahydrofuran (THF).

  Thereafter, the prepared TOPO-fixed particles are dissolved in THF, heated to 85 ° C., and then dissolved in a polar solvent such as ethanol or chloroform (for example, amino acids, oligopeptides, aryl and derivatives thereof; In addition, examples of the derivatives include those subjected to amidation, deamination, decarboxylation, and / or biotinylation, and the like, and the mixture is refluxed for about 12 hours. Next, an aqueous NaOH solution is added, heated to evaporate the THF, and unreacted substances are removed using filtration and a column, etc., and the target product is purified and concentrated. Fluorescent nanoparticles are produced.

Staining agent The essential component of the staining agent of the present invention is only fluorescent nanoparticles as described above. The staining agent preferably contains a carrier as an additional component. Examples of the carrier include aqueous liquids such as water, physiological saline, various buffer solutions (PBS, Tris buffer solution, etc.), cell culture media (D-MEM, HBSS, etc.), or infusion solutions (lactic acid Ringer solution, etc.). . When used in the present invention, the fluorescent nanoparticles as described above are easy to handle when contained in a carrier at a concentration of about 0.001 to 100 μM, preferably about 1 to 100 nM.
In general use, about 100 to 250 μL of the dye in the form of such a solution is used at one time. This amount can be appropriately adjusted depending on the animal species to be applied or the size of the eyeball. Further, the larger the amount of the fluorescent nanoparticles, the stronger the fluorescence. However, since it is difficult to see if the amount is too much or too little, the amount of the staining agent is appropriately adjusted.

When using the staining method , the above-described staining agent is applied to the injection needle (for example, 27 gauge) from the sclera (transciliary flat portion) or cornea (transcornea) corresponding to the position of the ciliary flat portion of the eye. ) Or the like, or injected into the eye by other methods (eye drops, intravenous vein, etc.). After injection, the vitreous body stained with fluorescent nanoparticles diffused in the eye is observed with a slit lamp microscope or a surgical microscope. When observing with a slit lamp microscope, a front lens or the like may be used. Subsequently, the part where the abnormal tissue or lesion is observed can be excised and aspirated.

Manufacture of dyeing | staining agent The fluorescent nanoparticle (aqueous colloid quantum dot) was manufactured as follows. First, under an argon stream, 7.5 g of tri-n-octylphosphine oxide (TOPO) (Kanto Chemical), 2.9 g of stearic acid (Kanto Chemical), 620 mg of n-tetradecylphosphonic acid (AVOCADO), and cadmium oxide ( Wako) 250 mg was added and heated to 370 ° C. After naturally cooling this to 270 ° C., a solution in which 200 mg of selenium (STREM CHEMICAL) is dissolved in 2.5 mL of tributylphosphine (Kanto Chemical) is added in advance and dried under reduced pressure to obtain CPO particles coated with TOPO. It was.

  Next, 15 g of TOPO was added to the obtained CdSe fine particles and heated, and subsequently, a solution of 1.1 g of zinc diethyldithiocarbamate (Tokyo Kasei) dissolved in 10 mL of trioctylphosphine (Aldrich) was added at 270 ° C., and TOPO was added to the surface. Fluorescent nanoparticles (hereinafter referred to as “TOPO-fixed quantum dots”) having CdSe nanocrystals as the core and ZnS as the shell were obtained. In addition, the quantum dot of this state is soluble in organic solvents, such as toluene and tetrahydrofuran (THF).

  Thereafter, the prepared TOPO-fixed quantum dots were dissolved in THF, heated to 85 ° C., and N-[(S) -3-mercapto-2-methylpropionyl] -L-proline (100 mg) dissolved in ethanol there. Sigma) was added dropwise and refluxed for about 12 hours. After refluxing for 12 hours, an aqueous NaOH solution was added, and the mixture was heated at 90 ° C. for 2 hours to evaporate THF. The obtained unpurified quantum dots are purified and concentrated using ultrafiltration (“Microcon”, Millipore) and Sephadex column (“MicroSpin G-25 Columns”, Amersham Biosciences). A hydrophilic quantum dot having N-[(S) -3-mercapto-2-methylpropionyl] -L-proline immobilized on the surface of the dot was produced.

  The obtained hydrophilic quantum dot aqueous solution (800 nM) was diluted with physiological saline, and 20 nM hydrophilic quantum dot / physiological saline aqueous solution was used as a staining agent in the subsequent experiments.

Vitreous observation and vitrectomy using the staining agent of the present invention 0.1 to 0.15 mL of the staining agent produced above was passed through the ciliary flat portion using a 27-gauge needle to remove the porcine eye. Injection into the vitreous cavity. Aged pig eyes (8 years old, large Yorkshire breed, female) and young pig eyes (6 months, edible hybrid (Duroc, Landrace, Yorkshire, Berkshire hybrid)) were used. Thereafter, the vitreous body was observed with a slit lamp microscope and compared with the case where the staining agent of the present invention was not used. In addition, vitrectomy of the eye was performed.

Observation result Immediately after the staining agent was injected, the staining agent diffused into the vitreous cavity. The structure of the vitreous body is reflected by the fluorescence (red) emitted from the fluorescent nanoparticles in the stain, and the desired part of the vitreous body can be easily observed with a slit lamp microscope. Could be determined. The fluorescence lasted stably for more than 30 minutes.

  In young pigs, the vitreous body was solid, and the vitreous cavity did not show liquefaction pockets (posterior vitreous cortex pocket) or posterior vitreous detachment, and the vitreous cavity appeared to be uniformly transparent.

  On the other hand, in aging pigs, physiological changes due to aging as described above have progressed, and particularly when posterior vitreous detachment occurs in the optic nerve head, the vitreous body adhered to the contour of the nipple is peeled off. A nipple contour (ie, ring-shaped) white turbidity (Weiss Ring) was observed in the vitreous cavity near the optic nerve head. This micrograph is shown in FIG. In FIG. 1, the rod-shaped light (indicated by an arrow (A)) visible at the center is the light of the slit lamp. The structure inside the white circle (indicated by arrow (B)) is the Weiss Ring. According to the present invention, the Weiss Ring can be clearly identified. Although not discovered this time, in general, conversely to the above, if the posterior vitreous cortex continuing to the Weiss Ring and Weiss Ring is observed with a slit lamp microscope, posterior vitreous detachment has occurred. You may think.

  It is difficult to find the Weiss Ring that exists in the transparent vitreous body when observing with a slit lamp microscope. However, when the stain of the present invention is used, the vitreous body is stained, so that the white Contrast with Weiss Ring, making it easier to find. In addition, the posterior vitreous cortex that continues to the Weiss Ring is confirmed while scanning little by little from top to bottom, left and right from the Weiss Ring, but it is transparent and easily lost. However, when the vitreous body was also stained, it became easier to confirm the continuity from the Weiss Ring.

Vitrectomy vitrectomy, Keike-like body to produce a perfusion port and two side ports, and treatment by piercing the light guide and vitreous cutter from side port in the vitreous cavity.
When the staining agent of the present invention is used, the stained vitreous body is easily viewed by being illuminated by the light guide illumination, and the degree of staining of the vitreous body is made translucent, so that the surrounding retina It was easy to grasp the positional relationship with the ciliary body and the vitreous body could be safely removed.

FIG. 1 is an observation image (photograph) of a vitreous body of an eye of an aged pig stained with the staining agent of the present invention, using a slit lamp microscope. The rod-shaped light (indicated by an arrow (A)) seen in the center is the light of the slit lamp. The structure inside the white circle (indicated by arrow (B)) is the Weiss Ring.

Claims (4)

A vitreous staining agent for eyes containing fluorescent nanoparticles composed of hydrophilic quantum dots .   The staining agent according to claim 1, wherein the fluorescent nanoparticles have a core monolayer structure or a core-shell multilayer structure.   The dyeing | staining agent of Claim 1 or 2 in which a fluorescent nanoparticle contains a semiconductor element. The particle size of the fluorescent nanoparticles is a 1 to 100 nm, dyeing agent according to any one of claims 1 to 3.

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