Use of a dye for a method of treating a vitreous haze-related disease

Abstract

The present invention relates to a dye for use in a method of treating a vitreous opacity related disease in a subject. The method preferably comprises administering the dye to the vitreous of the affected eye of the subject; and irradiating at least part of the vitreous opacity, thereby causing destruction of the vitreous opacity in the subject. The present invention further relates to the use of a dye for photo-destroying a vitreous opacity in an eye of a subject, and a method of photo-destroying a vitreous opacity in an eye of a subject, the method comprising: administering the dye to the vitreous of the affected eye of the subject; and irradiating at least part of the vitreous opacity, thereby causing destruction of the vitreous opacity in the subject.

Description

TECHNICAL FIELD

[0001] The present invention broadly pertains to the medical field, more precisely to the ophthalmic field. In particular, the present invention relates to the use of a dye in a method of treating a vitreous haze-related disease in a subject. BACKGROUND

[0002] The vitreous or vitreous body in the eye is a transparent gelatinous mass composed mainly of water (99%) and formed by a network of collagen and glycosaminoglycans such as hyaluronic acid (HA). For certain diseases such as myopia or diabetes, and with age, the vitreous liquefies, which in some cases can lead to the formation of a posterior vitreous detachment and vitreous haze. Those vitreous hazy that scatter light onto the retina are the cause of a degenerative fly-eye vision consisting of the perception of various sizes and shapes of floaters. Although floaters (or muscae volitantes in Latin) are not considered an ophthalmic emergency, some patients who present symptomatic floaters often complain of a decrease in visual acuity and a serious negative impact on their quality of life.

[0003] There is currently no large number of therapeutic solutions to treat eye floaters. The most common strategy is based on restoring the patient's confidence on how to live and cope with the symptoms. Due to the lack of therapeutic and medical options, patients who present symptomatic floaters that are severe often resort to unconventional therapies. Currently, there are two main therapeutic options for patients with vitreous hazy. Based on the patient's eligibility, a pars plana vitrectomy (PPV) is performed (i.e. vitreous replacement with physiological saline or gas). The second strategy relies on the use of a neodymium: yttrium aluminum garnet laser (Nd:YAG) with limited efficacy. Only about 30% of patients treated have an improvement after using this therapy. Even if these two strategies are safe in most cases, they can be associated with side effects such as cataracts or endophthalmitis.

[0004] In 2002, a retrospective study showed that 38% of patients who received YAG laser treatment found a moderate improvement in their symptoms, while 93.3% of the eyes treated with PPV had complete relief of symptoms. However, surgical interventions such as PPV can be associated with complications such as cataracts, retinal tears or endophthalmitis.

[0005] In previous work, cationic gold nanoparticles and hyaluronic acid-coated gold nanoparticles (HA-AuNPs) were investigated for their efficacy to disrupt artificial and human vitreous opacities. Cationic gold nanoparticles were immobilized at the injection site and were unable to reach the vitreous opacity. Small HA-AuNPs (10 nm) were able to bind on the opacity and generate vapor nanobubbles (VNBs) upon nanosecond laser irradiation, leading to its disruption (Sauvage et al., 2019, ACS Nano., 13, 8401-8416). However, due to concerns about the toxicity of gold nanoparticles, especially since they are not biodegradable and fragment after laser irradiation, there remains a need in the art for further and / or improved treatment options to ablate vitreous opacities and concomitant treatment of vitreous opacity-related diseases. SUMMARY

[0006] The present inventors have discovered compounds for use in a method of treating a vitreous opacity-related disease, thereby addressing one or more of the above-mentioned problems in the art.

[0007] Accordingly, a first aspect of the present application relates to a dye for use in a method of treating a vitreous opacity-related disease in a subject.

[0008] Preferably, the present application provides a dye for use in a method of treating a vitreous opacity-related disease in a subject, wherein the method comprises:

[0009] - administering said dye to the vitreous of the affected eye of said subject; and

[0010] - irradiating at least part of said vitreous opacity, thereby causing vitreous opacity disruption in said subject.

[0011] As shown in the experimental section, the present inventors have discovered that a dye, such as trypan blue or indocyanine green, is able to diffuse within the vitreous and accumulate at the vitreous opacity after administration, and can generate vapor nanobubbles (VNBs) at the vitreous opacity when irradiated. As shown in the examples, the VNBs thus obtained can provide sufficient mechanical force to disrupt the vitreous opacity. Moreover, VNBs are only observable at the vitreous opacity and not in its surroundings, illustrating a targeting effect. Thus, the use of a dye advantageously avoids damage to the vitreous and to the ocular tissues surrounding the vitreous opacity.

[0012] As a further advantage, the dyes for use in the present method achieve the disruption of vitreous opacities when used at concentrations lower than those currently used in the clinic. Moreover, at these concentrations, no significant toxicity to retinal cells was observed, both in vitro and in vivo. Furthermore, compared to the current (YAG) laser treatment, the present treatment uses a greatly reduced number of pulses and laser energy (intensity), resulting in a significant disruption of the patient's collagen type I fibers and opacities, thus limiting the side effects on other tissues, such as the retina. Moreover, the dye is biodegradable and approved for clinical use, in particular for ophthalmic use.

[0013] Another aspect relates to a method of photo-disrupting a vitreous opacity in an eye of a subject, the method comprising:

[0014] - administering a dye to the vitreous of the eye of the subject; and

[0015] - irradiating at least part of the vitreous opacity, thereby causing a disruption of the vitreous opacity in the subject.

[0016] Another aspect relates to a dye for use in photo-disrupting (photo-excising) a vitreous opacity in an eye of a subject.

[0017] The above and other aspects and preferred embodiments of the present application are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated into this description. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 : A: graph showing the size measurements (left) and zeta potential (right) of indocyanine green (ICG)-loaded nanoparticles; B: MTT assay of Muller cells (MIO-M1) treated for 24 h with free and ICG-loaded nanoparticles (0.1-1 mg / ml); C: cell titer glo assay of Muller cells treated for 24 h with different concentrations of trypan blue (0.001-1 mg / ml). PAH: poly(allylamine) hydrochloride, HSA: human serum albumin, LIP-ICG: ICG-encapsulated liposomes.

[0019] Figure 2: A: dark field microscopy images of collagen type I fibers mixed with free ICG, different types of ICG nanoparticles (0.5 mg / ml) and TB (0.01 mg / ml) before and after irradiation with a nanosecond laser (4.5 J / cm 2 ; λ = 561 nm). Dashed rectangle indicates the position of the targeting optical fiber. Dashed circle indicates the position of the laser beam. B: average number of laser pulses required to disrupt collagen type I fibers using a nanosecond laser (4.5 J / cm 2 ). For this experiment, fibers with an average diameter in the range 500-1000 μm were selected. C: one laser pulse (4.5 J / cm2 Dark-field image of type I collagen fibers during the period. Bright spots corresponding to VNBs can be observed on the fiber, but not externally. Scale bar = 100 μm. PAH: poly(allylamine) hydrochloride, HSA: human serum albumin, LIP-ICG: liposomes encapsulating ICG.

[0020] Figure 3: A: at 4.5 J / cm 2 Dark-field images of free TB, free ICG, and nano-encapsulated ICG in water before and during laser pulses. B: Number of bubbles generated by TB, ICG, and nano-encapsulated ICG as a function of laser flux. PAH: Poly(allylamine) hydrochloride, HSA: Human serum albumin, LIP-ICG: Liposomes encapsulating ICG, TB: Trypan blue.

[0021] Figure 4 Vitreous opacities in humans treated with free ICG (0.5 mg / ml) were measured at 561 nm (4.5 J / cm²). 2 ) and 800nm ​​(1.1J / cm 2 Dark-field image at (). Rectangles represent target turbidity. Dashed circles represent the position of the laser beam.

[0022] Figure 5 Schematic diagram of mixing collagen fibers with free ICG (1.25 mg / ml aqueous solution) before intravitreal injection of ICG fibers into a rabbit eye (top image). Color fundus photographs, photoacoustic microscopy (PAM) images, and composite images of intravitreal injected ICG-labeled fibers show that these fibers can be imaged by PAM at 578 nm and 800 nm (bottom image).

[0023] Figure 6 : Schematic diagram of intravitreal injection of unlabeled collagen fibers and ICG (1.25 mg / ml) 5 days later (top). 3D photoacoustic microscopy (PAM) images and overlay images of the intravitreal injected fibers at 578 nm and 800 nm show the colocalization of ICG with the fibers (bottom).

[0024] Figure 7 A diagram illustrating intravitreal injection of collagen fibers into a rabbit eye, followed by intravitreal injection of ICG (day 0) and laser treatment (day 3) 5 days later.

[0025] Figure 8: Photographs show the diffusion of intravitreal injected ICG from the injection site and its gradual removal from the vitreous humor. ICG concentrations: A: 1.25 mg / ml; B: 0.625 mg / ml; C: 0.25 mg / ml. After 7 days, if ICG concentrations of 1.25 mg / ml or 0.625 mg / ml are used, ICG is still only detectable at the level of injected collagen fibers.

[0026] Figure 9 Injected collagen fibers without ICG injection (indicated by white arrows) were treated with pulsed laser (5 scans; <7ns; 800nm; 1.9J / cm). 2 2D optical coherence tomography (OCT) images before and after processing. The target area (4.5 × 4.5 mm) was scanned with a laser (white dashed rectangle). 2 (Contains injectable fibers).

[0027] Figure 10: Injection of collagen fibers (indicated by white arrows), followed by ICG injection at concentrations of A: 1.25 mg / mL and B: 0.625 mg / mL, using a pulsed laser (5 scans; <7 ns; 800 nm; 1.9 J / cm). 2 Two-dimensional optical coherence tomography (OCT) images of collagen fiber damage (5 scans) before and after processing are shown. The target area (4.5 × 4.5 mm) was scanned with a laser (white dashed rectangle). 2 (Contains injectable fibers). Detailed Implementation

[0028] As used herein, unless the context clearly indicates otherwise, the singular forms “a” and “the” include both singular and plural indicators.

[0029] As used herein, the terms “comprising,” “comprises,” and “compose of” are synonymous with “including,” “includes,” or “containing,” and are inclusive or open-ended and do not exclude additional, non-enumerated members, elements, or method steps. These terms also cover “compose of” and “substantially composed of.”

[0030] The range of values ​​listed by endpoints includes all numbers and fractions that fall within the corresponding range, as well as the listed endpoints.

[0031] When referring to measurable values ​​(e.g., parameters, quantities, durations of time, etc.), as used herein, the term "about" is intended to cover changes in the specified value and changes relative to the specified value, particularly changes of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less and relative to the specified value, provided such changes are suitable for implementation in the disclosed invention. It should be understood that the value referred to by the modifier "about" is itself specifically and preferably disclosed.

[0032] Given that the term "one or more," such as one or more members in a group of members, is self-evident, by further example, the term specifically covers references to any one of the members, or any two or more of the members, for example, any ≥3, ≥4, ≥5, ≥6, or ≥7 of the members, and up to all of the members.

[0033] All documents referenced in this specification are incorporated herein by reference in their entirety.

[0034] Unless otherwise stated, all terms used in this disclosure, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance may include terminology definitions to better understand the teachings of this invention.

[0035] Through extensive experimental testing, the inventors have discovered that dyes are excellent compounds for targeted disruption of vitreous opacities. Dyes, such as trypan blue and indocyanine green, have been found to be not fixed at the injection site but possess sufficient mobility within the vitreous to reach the vitreous opacity fibers. This discovery is unexpected given the presence of collagen in the vitreous and the known ability of dyes to stain the inner limiting membrane of the eye, which is composed of an interwoven network of collagen.

[0036] Therefore, a first aspect of the present invention relates to a dye used in a method for treating vitreous opacity-related diseases in a subject.

[0037] The relevant parties provided:

[0038] - A method for treating vitreous opacity-related diseases in subjects who require this treatment, including administering a therapeutically effective amount of dye to the subject.

[0039] - The use of dyes in the production of medicines for treating vitreous opacity-related diseases in subjects.

[0040] - Use of dyes in the treatment of vitreous opacity-related diseases in subjects.

[0041] As used in this article, the term "dye" or "coloring agent" refers to a compound that can bind to various substances in nature to induce color. Therefore, dyes can increase the visibility of substances.

[0042] As shown in the Examples section, the dye (e.g., indocyanine green) advantageously binds to the vitreous opacity, thereby causing the dye to accumulate in the vitreous opacity and act as a light absorber, thereby causing localized disruption of the vitreous opacity.

[0043] In embodiments of the uses and methods taught herein, the dye can diffuse within the vitreous body. In embodiments of the uses and methods taught herein, the dye can mix with the turbidity of the vitreous body. In embodiments of the uses and methods taught herein, the dye can accumulate at the turbidity of the vitreous body.

[0044] In embodiments, the dye is a light absorber or photosensitizer. In embodiments, the dye is capable of absorbing light, such as light in the visible or near-infrared range. Therefore, the laser radiation used in embodiments of the methods taught herein can be applied by using a laser that emits light in the visible spectrum, for example, at a wavelength of 561 nm. This advantageously makes the laser radiation used in the methods taught herein visible to clinicians, in contrast to prior art (Nd:YAG) laser therapy for treating floaters, which operates at 1064 nm outside the visible spectrum. The radiation used in embodiments of the methods taught herein can be applied by using a laser that emits light in the near-infrared spectrum, for example, at a wavelength of 800 nm. This advantageously reduces interference with surrounding tissues and thus reduces side effects.

[0045] Therefore, on the one hand, a dye is provided as a photosensitizer for a method of treating vitreous opacity-related diseases in subjects.

[0046] In embodiments of the uses and methods taught herein, the dye, when irradiated, is able to form vapor nanobubbles when treating a subject with vitreous opacity-related diseases.

[0047] The term "dye" encompasses one or more dyes, such as two or more, three or more, or four or more, such as five, six, seven, eight or more dyes.

[0048] The term "dye" encompasses its salts, such as its pharmaceutically acceptable salts.

[0049] Dyes can be used on living cells taken from an organism or introduced into the body, for example, by injection.

[0050] In implementations of the uses and methods taught herein, the dye may be a biocompatible dye.

[0051] In implementations of the uses and methods taught herein, the dye may be a live dye.

[0052] The term "live dye" generally refers to a dye that can bind to living cells or their components (such as vitreous opacities) without inducing immediate and obvious degenerative changes in the cells or their components.

[0053] Live dyes can be used on living cells taken from an organism or introduced into the body, for example, by injection.

[0054] In implementation, the dye (e.g., a live dye) can be a natural dye or a synthetic dye.

[0055] In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) may be a dye approved for ophthalmic use. In embodiments, the dye may be a live dye approved for ophthalmic use.

[0056] In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) may be a fluorescent dye. Such fluorescent dyes (e.g., ICG, fluorescein) enable further imaging / visualization of vitreous opacities.

[0057] In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) may be an amphiphilic dye. Such dyes advantageously diffuse through the vitreous humor and specifically bind to vitreous opacities (accumulated in) the subject's eye.

[0058] The term "amphiphilic" refers to the property of having both hydrophilic (water-loving, polar) and lipophilic (fat-loving) characteristics.

[0059] In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) may be selected from the group consisting of free azo dyes, arylmethane dyes, cyanide dyes, thiazide dyes, and xanthan dyes.

[0060] Examples of azo dyes include trypan blue (Vision Blue, CAS No.: 72-57-1) and Janus green B (Diazine Green S, Union Green B, CAS No.: 2869-83-2).

[0061] Examples of arylmethane dyes include gentian violet (crystal violet, methyl violet 10B, hexamethyl pararosaniline chloride, CAS No.: 548-62-9); bromophenol blue (CAS No.: 115-39-9); patent blue (Blueron, CAS No.: 3536-49-0); brilliant blue (acid blue, Coomassie brilliant blue, Brilliant Peel, CAS No.: 6104-59-2); pachygreen (pachygreen SF, pachygreen SF pale yellow, CAS No.: 5141-20-8); and fast green (fast green FCF, food green 3, FD&C green 3, green 1724, pure green FCF, CAS No.: 2353-45-9).

[0062] Examples of anthocyanin dyes include indocyanine green (Cardiogreen, Foxgreen, Cardio-Green, Fox Green, IC Green, CAS No.: 3599-32-4) and hypocyanine green. Hypocyanine green (IfCG) is a green dye with the same chemical formula and similar pharmacological properties as ICG. Compared to ICG, IfCG dyes have two pharmacological differences. First, IfCG does not contain sodium iodide, which must be added to ICG during dye synthesis. Second, the presence of sodium iodide in ICG solutions necessitates dilution with water to produce a hypotonic solution.

[0063] Examples of thiazine dyes include methylene blue (CAS No.: 61-73-4) and toluidine blue (CAS No.: 92-31-9).

[0064] Examples of saxanthin dyes include sodium fluorescein (CAS No.: 518-47-8); rose red (CAS No.: 4159-77-7); and rhodamine 6G (Rhodamine 590, Rh6G, CI Pigment Red 81, CI Pigment Red 169, Basic Rhodamine Yellow, CI45160, CAS No.: 989-38-8).

[0065] In embodiments of the uses or methods taught herein, the dye may be a live dye selected from the group consisting of: indocyanine green (ICG), trypan blue (TB), brilliant blue (BB), Jenners green B (JG), gentian violet (GV), bromophenol blue (BPB), patent blue (PB), light green (LG), fast green (FG), hypocyanine green (IfCG), methylene blue (MB), toluidine blue (ToB), sodium fluorescein (FS), rose red (RB), and rhodamine 6G (R6G). In embodiments, the dye may be indocyanine green, trypan blue, or brilliant blue. Such dyes are advantageously approved for use in ophthalmology.

[0066] Preferably, the dye is indocyanine green or trypan blue. Both ICG and TB achieve destruction of vitreous opacities when used at concentrations lower than those currently used clinically. Furthermore, destruction can be achieved using ICG or TB with significantly reduced pulse counts and laser energy (intensity) compared to existing laser treatments, thus limiting side effects on other tissues (e.g., the retina).

[0067] More preferably, the dye is indocyanine green. Indocyanine green advantageously has a wide range of absorbance. Therefore, ICG enables the laser wavelength to be tuned to, for example, near-infrared light, which helps to reduce interference with surrounding tissues and thus reduce side effects.

[0068] The term "living dye" encompasses one or more living dyes, such as two or more, three or more, or four or more, such as five, six, seven, eight or more living dyes.

[0069] The term "living dye" encompasses its salts, such as its pharmaceutically acceptable salts.

[0070] In embodiments of the uses or methods taught herein, the dye may be a free or unbound dye, an aggregate of dye (e.g., an H-aggregate or J-aggregate), or a crystal of dye; or the dye (including its aggregates or crystals) may be conjugated with an agent (e.g., a polymer, lipid, peptide, protein) and / or the dye (including its aggregates or crystals) may be contained in particles (e.g., nanoparticles or microparticles).

[0071] The dyes taught herein (e.g., living dyes) can be free dyes or can be combined with or chemically bonded to other elements or compounds. In embodiments of the uses or methods taught herein, the dye can be free or unbound. Free dyes advantageously achieve a localized effect at vitreous opacities. Free dyes achieve specific binding and effectively disrupt vitreous opacities, even at concentrations lower than those used clinically. Without being bound by theory, this phenomenon may be due to the binding (accumulation) of free dyes on vitreous opacities, which lowers the energy threshold for generating vapor nanobubbles.

[0072] The terms “free” or “unbound” mean that the dye is not combined or chemically bonded with other elements or compounds, such as the dye is not conjugated with another agent, or the dye is not coupled (e.g., grafted) or blocked (e.g., encapsulated) in the particles. Free or unbound dyes as taught herein include, but are not limited to, dyes in solution, and dried or lyophilized dyes, such as dye powders, such as lyophilized powders for injection.

[0073] In embodiments of the uses or methods taught herein, the dye can be an aggregate of dye (e.g., H-aggregates or J-aggregates) or crystals of dye. Such aggregates and crystals advantageously improve the deterioration of vitreous opacities because the aggregates and dyes remain in the vitreous for a longer period. Due to their larger size, the use of dye aggregates or crystals reduces or avoids dye entry into the retina, thereby limiting or avoiding retinal toxicity. Furthermore, dye aggregation shifts the absorption wavelength to higher wavelengths, such as further into the IR region (e.g., 800-900 nm), thereby reducing toxicity because tissue does not absorb or only absorbs very little in this region. Moreover, these higher wavelengths, such as in the IR region (e.g., 800-900 nm), correspond to the wavelengths of currently used lasers. A further advantage is the ease of synthesizing the aggregates, and that their structure contains only the dye (without any other agents, such as polymers or lipids).

[0074] Free or unbound dyes may be included in compositions or formulations, such as pharmaceutical formulations or kits of parts, as will be further described herein. Compositions may contain dyes in the following concentration ranges: from about 0.001 mg / ml to 5 mg / ml, for example, from 0.01 mg / ml to 1 mg / ml, or from 0.1 mg / ml to 0.5 mg / ml.

[0075] Although no toxicity of free ICG and free TB has been observed at clinically used concentrations, conjugating dyes to agents or encapsulating dyes in particles (e.g., nanoparticles or microparticles) may be advantageous to reduce or even prevent dye penetration into the retina. The internal limiting membrane covering the retina has pores that prevent compounds or particles (e.g., larger than 100 nm) from passing through (Peynshaert et al., 2017, Drug Delivery, 24:1, 1384-1394). Therefore, conjugating dyes to agents and / or encapsulating dyes in microparticles or nanoparticles reduces or prevents dye entry into the retina, thus limiting or avoiding retinal toxicity.

[0076] In embodiments of the uses or methods taught herein, dyes (including aggregates or crystals thereof) may be conjugated with agents (e.g., polymers, lipids, peptides, proteins) and / or dyes (including aggregates or crystals thereof) may be contained within particles (e.g., nanoparticles or microparticles). In embodiments of the uses or methods taught herein, dyes (including aggregates or crystals thereof) may be grafted onto particles and / or dyes (including aggregates or crystals thereof) may be encapsulated within particles.

[0077] In embodiments of the uses or methods taught herein, the dye (e.g., a living dye) may be conjugated with an agent. The nature of the agent is not limited, and the agent may be any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., a biological macromolecule).

[0078] In this embodiment, the agent can be a polymer, lipid, peptide, or protein. Because the size of the conjugate is larger than the size of the dye itself, applying the conjugate advantageously prevents the dye from migrating to other parts of the eye and thus reduces or even eliminates toxicity.

[0079] In embodiments, the polymer can be selected from the group consisting of: hyaluronic acid (HA), poly(ethylene glycol) (PEG), poly(DL-lactic-glycolic acid copolymer) (PLGA), poly(lactic acid) (PLA)), polycaprolactone, ethyl cellulose, cellulose acetylated phthalate, polylactic acid, cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginate, dextran, starch, polycarbonate, polyacrylate, polystyrene, poly(alkyl cyanoacrylate) (PACA), and polyoxazoline. Preferably, the polymer can be hyaluronic acid. For example, a dye (e.g., ICG) can be conjugated with the polymer (e.g., hyaluronic acid). Because the size of the ICG-HA conjugate is larger than the size of the dye itself, application of the conjugate advantageously avoids migration to other parts of the eye and thus reduces or even eliminates toxicity.

[0080] In this embodiment, the lipid can be anionic, neutral, or cationic lipid. In this embodiment, the lipid can be natural, synthetic, or bacterial lipid.

[0081] Suitable examples of anionic lipids include phosphatidylserine (PS) and phosphatidylglycerol (PG).

[0082] Suitable examples of neutral lipids include prostaglandins, arachidic acids, glycerides, glycosylated diacylglycerols, oxygenated fatty acids, very long-chain fatty acids (VLCFAs), palmitate of hydroxystearic acid (PAHSA), N-acylglycine (NAGly), and isopentenol.

[0083] Suitable examples of cationic lipids include multivalent cationic lipids; 1,2-di-O-octadecenyl-3-trimethylpropane ammonium (DOTMA); ethylphosphocholine (ethylPC); dimethylbis(octadecyl)ammonium (DDAB); pH-sensitive lipids; 1,2-dioleoyl-3-trimethylpropane ammonium (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol); N4-cholesterol-spermine (GL67); and 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA).

[0084] These lipids are commercially available from Avanti Polar Lipids (Alabama, USA). For example, a suitable multivalent cationic lipid is (N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylformamide)ethyl]-3,4-di[oleoxy]-benzamide). Examples of ethyl PCs include 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (chloride salt) (12:0 EPC Cl salt); 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (chloride salt) (14:0 EPC Cl salt); 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (chloride salt) (16:0 EPC Cl salt); 1,2-distearateloyl-sn-glycerol-3-ethylphosphocholine (chloride salt) (18:0 EPC Cl salt); 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (chloride salt) (18:1 EPC Cl salt); 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (chloride salt) (16:0-18:1 EPC Cl salt). Cl salt); and 1,2-dimyristoleoyl-sn-glycerol-3-ethylphosphocholine (Tf salt) (14:1EPC Tf salt).

[0085] Examples of pH-sensitive lipids include N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-amine (DOBAQ); 1,2-distearyl-3-dimethylammonium-propane (18:0DAP); 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0DAP); 1,2-dimyristoyl-3-dimethylammonium-propane (14:0DAP); and 1,2-dioleoyl-3-dimethylammonium-propane (18:1DAP or DODAP).

[0086] In embodiments of the uses and methods taught herein, the dye may be contained in particles (e.g., nanoparticles or microparticles).

[0087] In this embodiment, the particles can be nanospheres or microspheres. The particles can also be nanorods, microrods, nanostars, microstars, nanopyramids, micropyramids, nanoshells, or microshells. In this embodiment, the particles can be nanospheres. In this embodiment, the particle diameter can be in the range of 1 nm to 1000 nm, for example, 1 nm to 500 nm, for example, in the range of 50 nm to 500 nm, preferably in the range of 100 nm to 400 nm, for example, in the range of 150 nm to 300 nm. In this embodiment, the particle diameter can be in the range of 5 nm to 300 nm, for example, 10 nm to 250 nm, for example, 150 nm to 250 nm.

[0088] For example, particle sizes in the range of 100 nm to 300 nm, or in the range of 150 nm to 250 nm, are advantageous and can improve particle mobility in the glass.

[0089] In this embodiment, the nanoparticles, such as their core, may comprise polymeric materials, carbon, and / or titanium. The core may comprise melanin. The core may comprise polydihydroxyphenylalanine (DOPA).

[0090] Nanoparticles can be polymer nanoparticles, protein nanoparticles, or lipid nanoparticles (i.e., liposomes). The polymer can be poly(lactic-co-glycolic acid) (PLGA). For example, PLGA-based ICG nanoparticles (PLGA-ICGNP) can be prepared as described in Saxena et al., 2004, Int J Pharm, 278(2):293-301. The protein can be human serum albumin (HSA). For example, human serum albumin ICG nanoparticles (HSA-ICG NP) can be prepared as described in Sheng et al., 2014, ACS Nano, 8(12):12310-22. The lipid nanoparticles can be MC3-based lipid nanoparticles (Patel et al., 2019, J. Control. Release, 303, 91-100). ICG-encapsulated liposomes (Lip-ICG) can be prepared as described in Lajunen et al., 2018, J. Control. Release 284, 213-223.

[0091] Particles, such as particle size, can be characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), UV-Vis spectroscopy, and / or electrodynamic modeling using Mie theory. The obtained particle concentration can be estimated using experimental extinction intensity at the maximum wavelength and Mie theory calculations of the extinction cross-section of spherical particles. Encapsulation efficiency (i.e., dye loading efficiency) can be determined based on calibration curves of the free dye obtained via UV-Vis spectroscopy or fluorescence. The zeta potential can be measured via electrophoretic mobility.

[0092] In embodiments of the uses and methods taught herein, dyes may be grafted onto particles (e.g., nanoparticles or microparticles). In embodiments, dyes may be grafted onto nanoparticles. For example, dyes may be grafted onto particles via "click chemistry" on the particle surface (e.g., at the ends of polymer chains, such as poly(ethylene glycol) chains or hyaluronic acid chains). For example, dye grafting may occur at the ends of PEG chains grafted onto the particles.

[0093] In embodiments of the uses and methods taught herein, dyes may be encapsulated in particles (e.g., nanoparticles or microparticles). In embodiments, dyes may be encapsulated in nanoparticles. In embodiments, dyes may be encapsulated in particles by physical or chemical encapsulation. For example, physical encapsulation of ICG in liposomes can be achieved by adding ICG during lipid rehydration. For HAS-ICG particles, chemical encapsulation can be achieved by reacting ICG with the disulfide bonds of HAS.

[0094] The terms “vitreous body,” “vitreous fluid,” or “glassy substance” are used interchangeably herein and refer to the transparent, gel-like body that fills the space between the lens and retina of the eyeball in humans and other vertebrates. The vitreous body contains water (98-99% of its volume) and a network of collagen and glycosaminoglycans (such as hyaluronic acid (HA)).

[0095] In youth, the vitreous humor (HA) and collagen fibers form a supramolecular network that maintains transparency and gives the vitreous a colloidal state. With age, the reorganization of molecular components within the vitreous alters its structure, inducing colloidal liquefaction (senile vitreous liquefaction). This liquefaction may be accompanied by the collapse of the collagen network, which can induce the formation of other collagen-based structures as light-scattering opacities, leading to floaters or potentially posterior vitreous detachment (PVD), in which the vitreous membrane is released from the sensory retina. During this detachment, the constricted vitreous can mechanically stimulate the retina, causing the patient to see random flashes of light in their visual field, sometimes referred to as “flashes,” a symptom more formally known as photophobia. The eventual release of the vitreous around the optic disc sometimes results in large floaters, often in a ring shape (“Weiss’s ring”).

[0096] The terms “vitreous opacity,” “floaters,” “vitreous floaters,” or “floaters” are used interchangeably and refer to deposits in the vitreous fluid of the eye. The term “vitreous opacity” encompasses any type of floaters, such as floaters arising from vitreous liquefaction; floaters that may be caused by embryonic remnants; or floaters that may be acquired due to aging, trauma, iatrogenic, ocular, or systemic metabolic disorders.

[0097] Most floaters are caused by degenerative changes in the vitreous humor, where the vitreous network is disrupted, such as by collagen aggregates adhering to the vitreous framework within reticular masses, which impair normal vision. Floaters may appear as linear structures with nodules, or as reticular structures of linear structure, seemingly floating in front of the eyes, caused by shadows on the retina.

[0098] In one embodiment, the length of the vitreous opacity to be treated can be in the range of 0.5 mm to 5 mm, for example, in the range of 1 mm to 4 mm or 2 mm to 3 mm. In another embodiment, the vitreous opacity to be treated can be close to the retina or lens, for example, at a distance in the range of 0 mm to 5 mm, for example, at a distance in the range of 1 mm to 4 mm. For example, floaters may be present in the anterior macular bursa.

[0099] The term "vitreous opacity-related disease" refers to any disease or condition associated with vitreous opacities present in the subject's eye.

[0100] In this implementation, vitreous opacity-related diseases may include floaters or posterior vitreous detachment.

[0101] The terms "myodesopsia," "myodaeopsia," "myiodeopsia," or "myiodesopsia" refer to the perception of floating objects. The perception of floating objects can be characterized by shadow-like visual artifacts.

[0102] The term "posterior vitreous detachment" refers to an eye condition in which the vitreous membrane separates from the retina. PVD can be characterized by one or more symptoms selected from the following groups: flashes of light (photopsia), a sudden and dramatic increase in the number of floaters, and a ring of floaters or hairs just on the temporal side of central vision.

[0103] As used herein, the term “eye” has its common meaning in the field and refers to the organ of the visual system.

[0104] The terms “subject,” “individual,” or “patient” are used interchangeably herein and generally and preferably refer to humans, but may also encompass references to non-human animals, preferably warm-blooded animals, and even more preferably mammals, such as non-human primates, rodents, canines, felines, equines, sheep, pigs, etc. The term “non-human animal” includes all vertebrates, such as mammals, including non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals (e.g., chickens, amphibians, reptiles, etc.). In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a human subject. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and neonatal subjects, as well as fetuses, whether male or female. Examples of subjects include humans, dogs, cats, cattle, goats, and mice. The term subject is further intended to include transgenic species.

[0105] Suitable subjects may include, but are not limited to, subjects who visit a physician to screen for vitreous opacity-related diseases, subjects who visit a physician with symptoms and signs indicative of vitreous opacity-related diseases, subjects diagnosed with vitreous opacity-related diseases, and subjects who have received optional (unsuccessful) treatment for vitreous opacity-related diseases.

[0106] In implementations of the uses or methods taught herein, the method may include:

[0107] - The dye is applied to the vitreous humor of the affected eye of the subject; and

[0108] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0109] In one implementation, the method includes applying a dye (e.g., a live dye) to the vitreous humor of the affected eye of the subject.

[0110] In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) may be applied at a concentration of about 0.001 mg / ml to about 5.0 mg / ml. In embodiments, the dye (e.g., a live dye) may be applied at a concentration of about 0.01 mg / ml to about 1.0 mg / ml. In embodiments, the dye (e.g., a live dye) may be applied at a concentration of about 0.1 mg / ml to about 0.5 mg / ml. In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) may be applied at a concentration of about 0.001 mg / ml to about 0.5 mg / ml. Such concentrations are equal to or lower than dye concentrations commonly used clinically (e.g., concentrations commonly used clinically, particularly in ophthalmology, such as 0.6 mg / ml for TB and 1.25 mg / ml for ICG). This concentration advantageously allows treatment to be performed without toxicity to surrounding ocular tissues. This concentration is within the clinically acceptable and / or routinely used range.

[0111] In this embodiment, when ICG is used as the dye, the dye can be applied at a concentration of at least 0.01 mg / ml, for example, at least 0.1 mg / ml, preferably at least 0.5 mg / ml. For example, when ICG is used as the dye, the dye can be applied at a concentration of about 0.001 mg / ml to about 1.0 mg / ml, for example, at a concentration of about 0.001 mg / ml to about 0.5 mg / ml, or at a concentration of about 0.01 mg / ml to about 0.5 mg / ml, preferably at a concentration of about 0.1 mg / ml to about 0.5 mg / ml. This concentration is lower than currently used clinically and achieves treatment without causing any toxicity to the surrounding ocular tissues.

[0112] In embodiments of the uses and methods taught herein, when trypan blue is used as the dye, the dye can be applied at a concentration of at least 0.001 mg / ml, preferably at least 0.01 mg / ml. For example, when trypan blue is used as the dye, the dye can be applied at a concentration of about 0.001 mg / ml to about 0.5 mg / ml, for example, at a concentration of about 0.001 mg / ml to about 0.1 mg / ml, preferably at a concentration of about 0.001 mg / ml to about 0.01 mg / ml. This concentration is lower than the concentrations currently used clinically and achieves treatment without producing any toxicity to the surrounding ocular tissues.

[0113] In embodiments of the uses and methods taught herein, a dye (e.g., a live dye) can be administered to the vitreous body via intravitreal application. Intravitreal application advantageously achieves direct delivery of the dye (e.g., a live dye) to the vitreous body. In embodiments, the dye (e.g., a live dye) can be administered to the vitreous body via injection. In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) can be administered to the vitreous body via intravitreal injection. Intravitreal injection achieves direct delivery of the dye (e.g., a live dye) to the vitreous body through minimally invasive techniques, thereby reducing patient risk and pain and improving patient health.

[0114] As used herein, the term "intravitreal administration" refers to the process or procedure of directly implanting a drug (such as the dyes or compositions taught herein) into the vitreous cavity that is filled with the vitreous body.

[0115] In embodiments of the uses and methods taught herein, the dye (e.g., a live dye) is able to diffuse within the vitreous body after application, for example, via intravitreal injection. When a dye (e.g., a live dye) is used according to embodiments of the invention, the dye is able to diffuse within the vitreous body after application.

[0116] In embodiments of the uses and methods taught herein, a dye (e.g., a living dye) can bind to (accumulate in) vitreous opacities after application, for example, via intravitreal injection. When a dye (e.g., a living dye) is used according to embodiments of the invention, the dye can bind to (accumulate in) vitreous opacities after application.

[0117] The binding of dyes to vitreous opacities can occur through covalent bonding or non-covalent interactions.

[0118] In one embodiment, the method may include applying a dye to the vitreous humor of the affected eye of a subject, thereby inducing the binding of the dye to vitreous opacities (accumulation of the dye at the site of the vitreous opacity). In another embodiment, the method may include applying a dye to the vitreous humor of the affected eye of a subject, thereby inducing the diffusion of the dye within the vitreous humor and the binding of the dye to vitreous opacities (accumulation of the dye at the site of the vitreous opacity).

[0119] In embodiments of the uses and methods taught herein, the dye (e.g., a living dye) can form vapor nanobubbles in the vitreous turbidity upon irradiation. When a dye (e.g., a living dye) according to an embodiment of the invention is used, the dye can form vapor nanobubbles in the vitreous turbidity upon irradiation.

[0120] In one embodiment, the method may include irradiating a dye that binds to at least a portion of the vitreous opacity, thereby inducing the destruction of the vitreous opacity in the subject. In another embodiment, the method may include irradiating a dye that binds to at least a portion of the vitreous opacity, thereby forming vapor nanobubbles at the site of the vitreous opacity and inducing the destruction of the vitreous opacity in the subject.

[0121] The terms “inducing destruction,” “causing destruction,” or “destruction” are used interchangeably in this article.

[0122] In an implementation, the method may include:

[0123] - Applying the dye to the vitreous humor of the affected eye of the subject to induce the binding of the dye to vitreous opacities (accumulation of the dye at the site of vitreous opacities); and

[0124] - Irradiation with dye that binds to (accumulated in) at least part of the vitreous opacity, thereby inducing destruction of the vitreous opacity in the subject.

[0125] Dyes such as those taught in this article (e.g., live dyes) can be used as photosensitizers in methods for treating vitreous opacity-related diseases.

[0126] Treatment may include injecting a dye (e.g., a live dye) into the vitreous humor of the eye of a human or animal subject. Treatment may also include laser ablation therapy following the injection of a dye (e.g., a live dye) into the vitreous humor of the eye of a human or animal subject.

[0127] When using a dye (e.g., a live dye) according to an embodiment of the invention, the dye can specifically bind to vitreous opacities and can apply mechanical force to the vitreous opacities locally when irradiated by a laser during laser ablation treatment.

[0128] When a dye (e.g., a live dye) according to an embodiment of the invention is used, the dye can form vapor nanobubbles in the vitreous body when irradiated, thereby applying mechanical force to the turbidity of the vitreous body.

[0129] When a dye (e.g., a live dye according to an embodiment of the invention) is used as a photosensitizer in a method for treating vitreous opacities, the dye can accumulate around the vitreous opacity to concentrate energy deposition near and / or in the vitreous opacity by laser ablation treatment, such that the destruction of vapor nanobubbles releases mechanical force to remove and / or rupture the vitreous opacity.

[0130] In embodiments of the uses and methods taught herein, the method may include irradiating at least a portion of the vitreous opacity to induce destruction of the vitreous opacity in a subject. In embodiments, the method may include irradiating at least a portion of the vitreous opacity with radiation to remove and / or rupture and / or destroy the vitreous opacity in a subject.

[0131] As used in this article, “destruction of vitreous opacity” refers to the fragmentation of vitreous opacity.

[0132] In implementation, the use of a dye as taught herein (e.g., a bioactive dye) can cause the vitreous opacity to fragment into at least two fragments. For example, the use of a dye as taught herein can cause the vitreous opacity to fragment into two or more fragments, such as five or more, ten or more, twenty or more, fifty or more, or one hundred or more fragments. The size of the resulting fragments (i.e., the post-treatment fragments) can be up to 50% of the size of the treated vitreous opacity. For example, the size of the resulting fragments (i.e., the post-treatment fragments) can be up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, up to 1%, up to 0.1%, or up to 0.01% of the size of the pre-treatment vitreous opacity. The resulting fragments may be visible or may no longer be visible after treatment, for example, when observed by microscopic analysis.

[0133] When using dyes as taught herein (living dyes), due to the localized effect of the dye, it may be possible to irradiate only a portion of the vitreous opacity to break up the vitreous opacity.

[0134] In embodiments of the uses and methods taught herein, at least a portion of the vitreous opacity can be irradiated with electromagnetic radiation. In embodiments, at least a portion of the vitreous opacity can be irradiated with laser radiation. In embodiments, at least a portion of the vitreous opacity can be irradiated with pulsed laser radiation.

[0135] The terms “radiation” and “electromagnetic radiation” are used interchangeably in this document.

[0136] In this implementation, the electromagnetic radiation is infrared radiation (including near-infrared) or visible light.

[0137] Laser irradiation, such as pulsed laser irradiation, including picosecond, femtosecond, and / or nanosecond pulsed lasers, can be combined with dyes according to embodiments of the invention to effectively disrupt vitreous opacities, for example, through laser-induced vapor nanobubbles. While laser irradiation may be advantageous, it does not necessarily preclude achieving the same or similar effects through irradiation with another (intense) light source.

[0138] In implementations of the uses or methods taught herein,

[0139] - The laser pulse intensity can be at least 10 4 W / cm 2 ;

[0140] - The number of laser pulses can be at least 1 laser pulse;

[0141] - The duration of the laser pulse can be in the range of at least 10 fs; and / or

[0142] - Laser irradiation of the opaque areas of the vitreous reaches at least 0.1 mJ / cm². 2 The amount of bets.

[0143] In implementations of the uses or methods taught herein,

[0144] - The intensity of the laser pulse can be 10 4 Up to 10 15 W / cm 2 Or 10 7 Up to 10 15 W / cm 2 ;

[0145] - The number of laser pulses can range from 1 to 1000; and / or

[0146] The duration of the laser pulse can range from 10 fs to 10 ns.

[0147] In implementations of the methods or uses taught herein,

[0148] - The intensity of the laser pulse is 10 7 Up to 10 15 W / cm 2 Within a range; for example, within 10 10 Up to 10 15 W / cm 2 Within the range or within 10 12 Up to 10 14 W / cm 2 Within the range;

[0149] - The number of laser pulses is in the range of 1 to 1000 laser pulses per vitreous opacity; for example, in the range of 1 to 100 laser pulses or in the range of 1 to 10 laser pulses; and / or

[0150] - The duration of the laser pulse is in the range of 10 fs to 10 ns; for example, in the range of 10 fs to 1 ps or in the range of 1 ps to 10 ns.

[0151] The power density or intensity of each laser pulse can be 10 7 Up to 10 15 W / cm 2 Within a range, for example, in 10 12 Up to 10 15 W / cm 2 Within this range, or in other words, the injection volume is in the range of 10 μJ / cm² to 100 J / cm², for example, in the range of 10 mJ / cm². 2 Up to 10 J / cm 2 Within the range of 1J / cm 2 Up to 10 J / cm 2 Within the range.

[0152] For each vitreous opacity, the laser pulse can consist of 1 to 1000 laser pulses, such as 1 to 500 laser pulses, 1 to 100 laser pulses, 1 to 20 laser pulses, or 1 to 10 laser pulses. The number of laser pulses can depend on the dye and the size, composition, and shape of the vitreous opacity.

[0153] The duration of the laser pulse can be in the range of 10 fs to 100 ns, for example, in the range of 10 fs to 10 ns, or in the range of 10 fs to 1 ps or in the range of 1 ps to 10 ns.

[0154] The dyes taught in this article advantageously achieve effective and targeted disruption of vitreous opacities.

[0155] As used herein, the term "photodamage" refers to the process of using electromagnetic radiation (such as (visible) light or near-infrared radiation) to break down tissues (e.g., vitreous opacities). Electromagnetic radiation can be generated by lasers (e.g., pulsed lasers).

[0156] In this implementation, photo-induced damage can be laser-assisted photo-induced damage.

[0157] The use of dyes as taught herein can lead to vitreous opacity fragmentation or even destruction. Therefore, further aspects or embodiments relate to the use of dyes as taught herein in a photo-induced destruction method for vitreous opacity.

[0158] Therefore, further aspects or embodiments relate to the use of dyes, as taught herein, in methods for photo-induced vitreous opacities in the eyes of subjects.

[0159] On the other hand, a method is provided for treating vitreous opacities in the eyes of subjects whose eyes require photo-induced damage, including administering a therapeutically effective amount of dye to the subject.

[0160] The relevant parties provided:

[0161] - A dye used in a method for photo-induced damage to vitreous opacities in the eyes of subjects.

[0162] - The dye is used in the production of a drug for the treatment of vitreous opacities in the eyes of subjects with photoinduced damage.

[0163] - The use of dyes for photoinduced damage to vitreous opacities in the eyes of subjects.

[0164] In some implementations of the methods or uses taught herein, the method may include:

[0165] - Applying dye to the vitreous humor of the subject's eye; and

[0166] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0167] Therefore, one aspect involves a method for photoinduced damage to vitreous opacities in the eye of a subject, the method comprising:

[0168] - Applying dye to the vitreous humor of the subject's eye; and

[0169] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0170] In embodiments, treatment of vitreous opacity-related diseases may include laser irradiation of dyes as taught herein, particularly pulsed laser irradiation of dyes as taught herein. Therefore, in embodiments, treatments as taught herein include laser-assisted therapy. The terms “laser-assisted therapy,” “laser ablation therapy,” or “photoablation therapy” are used interchangeably herein.

[0171] Further aspects involve:

[0172] - Dyes used in methods for laser-assisted treatment of subjects with vitreous opacity-related diseases.

[0173] - Laser-assisted treatment is a method for treating vitreous opacities in subjects who require this treatment, including administering a therapeutically effective amount of dye to the subject.

[0174] - Use of dyes in the production of medicines for subjects with vitreous opacity-related diseases treated with laser-assisted therapy.

[0175] - Use of dyes in laser-assisted treatment of subjects with vitreous opacity-related diseases.

[0176] As taught in this article, dyes can be used to treat vitreous opacity-related diseases (e.g., laser-assisted therapy).

[0177] As used herein, phrases such as “subjects requiring treatment” include subjects who would benefit from treatment of a given condition, particularly vitreous opacity-related diseases. Such subjects may include, but are not limited to, those who have been diagnosed with the condition, those who are prone to developing the condition, and / or those who wish to prevent the condition.

[0178] The term "treat" or "treatment" encompasses therapeutic treatment of an established disease or condition, such as the treatment of an established vitreous opacity-related disease, as well as preventative or preventive measures aimed at preventing or reducing the chance of unintended suffering, such as preventing the occurrence, development, and progression of vitreous opacity-related diseases. Beneficial or desired clinical outcomes may include, but are not limited to, relief of one or more symptoms or one or more biomarkers, reduction of disease severity, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, and improvement of the disease state. The term may cover ex vivo or in vivo treatment.

[0179] The therapeutically effective amount of a dye as taught herein is administered to a subject with a vitreous opacity-related disease, which would benefit from such treatment, as taught herein. As used herein, the term “therapeutically effective amount” refers to the amount of an active compound or agent that a surgeon, researcher, veterinarian, physician, or other clinician is seeking to elicit a biological or medical response in a subject, which may in particular include the relief of symptoms of the disease or condition being treated.

[0180] The term “therapeutic effective dose” refers to an amount of agent (e.g., dye) as taught herein that produces a positive therapeutic response in a patient with a disease or condition being treated (e.g., vitreous opacity-related disease) when administered.

[0181] The appropriate therapeutic dose of an agent (such as a dye) as taught in this article can be determined by a qualified physician who takes into account the nature of the agent, the condition and severity of the disease, and the patient’s age, body size and condition.

[0182] In some embodiments, agents such as those taught herein (e.g., dyes) may be formulated into pharmaceutical preparations or compositions and administered as such. Such pharmaceutical preparations or compositions may be included in multi-component kits.

[0183] In this embodiment, the dye (e.g., a live dye) may be included in the pharmaceutical formulation.

[0184] Dyes or their pharmaceutically acceptable salts can be formulated into aqueous solutions.

[0185] Therefore, on the one hand, it relates to pharmaceutical preparations containing dyes as taught in this article.

[0186] On the other hand, the pharmaceutical preparations taught herein relate to methods for treating vitreous opacity-related diseases in subjects. Preferably, the subject is a human subject.

[0187] The terms “pharmaceutical composition,” “pharmaceutical formulation,” or “pharmaceutical preparation” are used interchangeably herein and refer to a mixture containing an active ingredient. The terms “composition” or “formulation” are also used interchangeably herein.

[0188] The terms "active ingredient" or "active component" are used interchangeably and broadly refer to a compound or substance that, when provided in an effective amount, achieves a desired outcome. The desired outcome can be therapeutic and / or preventative. Typically, the active ingredient achieves such an outcome by interacting with and / or modulating living cells or organisms.

[0189] The term "active" in the description of "active ingredient" or "active component" refers to "pharmacological activity" and / or "physical activity".

[0190] In addition to dyes, this pharmaceutical preparation may also contain one or more pharmaceutically acceptable excipients.

[0191] As used herein, the term “pharmaceutical acceptable” is consistent with the art and means compatible with other components of a pharmaceutical composition and harmless to its recipient.

[0192] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, solvents, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners, agents for achieving reservoir effects, coatings, antifungal agents, preservatives, antioxidants, tension control agents, absorption delay agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active substance, its use in therapeutic compositions may be considered.

[0193] The pharmaceutical compositions as envisioned herein can be formulated for substantially any route of administration, such as, but not limited to, oral administration (e.g., oral ingestion), parenteral administration (e.g., subcutaneous, intravenous, or intramuscular injection or infusion), etc.

[0194] For example, for oral administration, the pharmaceutical composition may be formulated into pills, tablets, flakes, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions, or suspensions. In examples, but not limited to, the preparation of oral dosage forms may be suitably accomplished by uniformly and tightly blending appropriate amounts of the active compound in powder form together, optionally including one or more finely chopped solid carriers, and formulating the blend into pills, tablets, or capsules. Exemplary but non-limiting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (e.g., glucose, mannose, lactose, or sucrose), sugar alcohols (e.g., mannitol), dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low-melting-point waxes, and ion exchange resins. Compressed tablets containing the pharmaceutical composition may be prepared by uniformly and tightly mixing the active ingredient with a solid carrier as described above to provide a mixture with the necessary compressibility properties, and then compressing the mixture into the desired shape and size in a suitable machine. Molded tablets can be produced by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils, etc.

[0195] Preferably, the pharmaceutical formulation can be formulated for parenteral administration, such as intravitreal administration, for example, by injection. In embodiments, the pharmaceutical composition can be formulated as an aqueous solution. For example, for parenteral administration, the pharmaceutical composition can advantageously be formulated as a solution, suspension, or emulsion having suitable solvents, diluents, solubilizers, or emulsifiers. Suitable solvents are, but are not limited to, water, physiological saline solutions, or alcohols such as ethanol, propanol, glycerol, as well as sugar solutions (e.g., glucose, invert sugar, sucrose, or mannitol solutions), or optionally mixtures of the various solvents mentioned. Injectable solutions or suspensions can be formulated according to known techniques using suitable non-toxic, parenteral-acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or suitable dispersants or wetting agents and suspending agents such as sterile, mild, non-volatile oils, including synthetic monoglycerides or diglycerides, and fatty acids, including oleic acid. Dyes or pharmaceutically acceptable salts thereof can also be lyophilized. The obtained lyophilized product can be used, for example, in injections or infusions, or for the production of injections or infusions.

[0196] On the other hand, there are multi-component kits, as taught herein, relating to methods for treating vitreous opacity-related diseases in subjects. Preferably, the subject is a human subject.

[0197] As used throughout this specification, the terms "multi-component kit" and "kit" refer to a product containing components necessary for a specific purpose or method, and packaged for easy transport and storage. Suitable materials for packaging the components included in a kit include crystals, plastics (e.g., polyethylene, polypropylene, polycarbonate), bottles, flasks, vials, ampoules, paper, envelopes, or other types of containers, carriers, or supports. In kits containing multiple components, at least a subset of the components (e.g., two or more of a plurality of components) or all of the components may be physically separate, for example, contained in or on separate containers, carriers, or supports. The components included in a kit may be sufficient or insufficient for the specified purpose or method, such that external reagents or substances may or may not be necessary for performing these methods individually. Typically, kits are used in conjunction with standard laboratory equipment, such as liquid handling equipment, environmental (e.g., temperature) control equipment, analytical instruments, etc. In addition to dyes (optionally provided on an array or microarray) as taught herein, this kit may also include excipients, such as solvents useful in a specific purpose or method. Typically, kits may also include instructions for use, such as printed inserts or computer-readable media. As used herein, these terms are used interchangeably with the term "article," which broadly encompasses any man-made tangible structure.

[0198] This application also provides aspects and implementation methods set forth in the following statements:

[0199] Statement 1. The use of a dye in a method for treating vitreous opacity-related diseases in a subject.

[0200] Statement 2. A dye for use according to the purpose described in Statement 1, wherein the method comprises:

[0201] - The dye is applied to the vitreous humor of the affected eye of the subject; and

[0202] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0203] Statement 3. A dye for the purpose described in Statement 1 or 2, wherein the dye is a live dye; preferably wherein the dye is a live dye approved for ophthalmic use.

[0204] Statement 4. A dye for use according to any one of statements 1 to 3, wherein the dye is a living dye selected from the group consisting of: indocyanine green, trypan blue, brilliant blue, Jenners green B, gentian violet, bromophenol blue, patent blue, light green, fixed green, hypocyanine green, methylene blue, toluidine blue, sodium fluorescein, rose red, and rhodamine 6G; preferably wherein the dye is indocyanine green or trypan blue; more preferably wherein the dye is indocyanine green.

[0205] Statement 5. A dye for use according to any one of statements 1 to 4, wherein the dye is applied at a concentration of about 0.001 mg / ml to about 0.5 mg / ml.

[0206] Statement 6. A dye for use according to any one of statements 1 to 5, wherein the dye is a free dye or wherein the dye is conjugated with an agent and / or contained in particles, such as nanoparticles or microparticles; preferably wherein the dye is grafted onto the particles and / or wherein the dye is encapsulated in the particles.

[0207] Statement 7. A dye for use according to any one of statements 1 to 6, wherein the dye is applied to the vitreous body by intravitreal application; preferably wherein the dye is applied to the vitreous body by intravitreal injection.

[0208] Statement 8. A dye for use according to any one of statements 1 to 7, wherein the dye is capable of diffusing within the vitreous body after application.

[0209] Statement 9. A dye for use according to any one of statements 1 to 8, wherein the dye is capable of binding with the vitreous opacity after application.

[0210] Statement 10. A dye for use according to any one of statements 1 to 9, wherein the dye is capable of forming vapor nanobubbles at the turbidity of the vitreous body when irradiated.

[0211] Statement 11. A dye for use according to any one of statements 1 to 10, wherein at least a portion of the vitreous body is turbid by electromagnetic radiation; preferably wherein at least a portion of the vitreous body is turbid by laser radiation; more preferably wherein at least a portion of the vitreous body is turbid by pulsed laser radiation.

[0212] Statement 12. A dye for the purpose described in Statement 11, wherein:

[0213] - The laser pulse intensity can be at least 10 4 W / cm 2 ;

[0214] - The number of laser pulses can be at least 1 laser pulse;

[0215] - The duration of the laser pulse can be in the range of at least 10 fs; and / or

[0216] - Laser irradiation of the opaque areas of the vitreous reaches at least 0.1 mJ / cm². 2 The amount of bets.

[0217] Statement 13. A method for photoinduced vitreous opacity in the eye of a subject, the method comprising:

[0218] - Applying dye to the vitreous humor of the subject's eye; and

[0219] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0220] Statement 14. The method according to Statement 13, wherein:

[0221] -The dye is a living dye;

[0222] - The dye is a live dye approved for ophthalmic use;

[0223] The dye is a live dye selected from the group consisting of: indocyanine green, trypan blue, Jenners green B, gentian violet, bromophenol blue, patent blue, brilliant blue, light green, fast green, hypocyanine green, methylene blue, toluidine blue, sodium fluorescein, rose red, and rhodamine 6G; preferably, the dye is indocyanine green or trypan blue; more preferably, the dye is indocyanine green;

[0224] - The dye is applied at a concentration of about 0.001 mg / ml to about 0.5 mg / ml;

[0225] -This dye is a free dye;

[0226] -The dye is conjugated with the agent;

[0227] - The dye is contained in particles, such as nanoparticles or microparticles; preferably, the dye is grafted onto the particles and / or the dye is encapsulated in the particles;

[0228] - The dye is applied to the vitreous body via intravitreal administration; preferably, the dye is applied to the vitreous body via intravitreal injection;

[0229] - The dye is able to diffuse within the vitreous body after application;

[0230] - The dye can bind with the vitreous opacity after application;

[0231] - The dye is able to form vapor nanobubbles in the turbidity of the glass when irradiated;

[0232] - Irradiation of at least a portion of the vitreous body with electromagnetic radiation; preferably, irradiation of at least a portion of the vitreous body with laser radiation; more preferably, irradiation of at least a portion of the vitreous body with pulsed laser radiation;

[0233] - The laser pulse intensity can be at least 10 4 W / cm 2 ;

[0234] - The number of laser pulses can be at least 1 laser pulse;

[0235] - The duration of the laser pulse can be in the range of at least 10 fs; and / or

[0236] - Laser irradiation of the opaque areas of the vitreous reaches at least 0.1 mJ / cm². 2 The amount of bets.

[0237] Statement 15. Use of dyes for photoinduced vitreous opacities in the eyes of subjects.

[0238] Statement 16. The use of a live dye in a method for treating vitreous opacity-related diseases in a subject.

[0239] Statement 17. A living dye for use according to the purpose described in Statement 16, wherein the method comprises:

[0240] - The live dye was applied to the vitreous humor of the affected eye of the subject; and

[0241] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0242] Statement 18. A live dye for use as described in Statement 16 or 17, wherein the live dye is a live dye approved for ophthalmic use.

[0243] Statement 19. A living dye for use according to any one of Statements 16 to 18, wherein the living dye is selected from the group consisting of: indocyanine green, trypan blue, brilliant blue, Jenners green B, gentian violet, bromophenol blue, patent blue, light green, fast green, hypocyanine green, methylene blue, toluidine blue, sodium fluorescein, rose red, and rhodamine 6G; preferably wherein the living dye is indocyanine green or trypan blue; more preferably wherein the living dye is indocyanine green.

[0244] Statement 20. A live dye for use according to any one of statements 16 to 19, wherein the live dye is applied at a concentration of about 0.001 mg / ml to about 0.5 mg / ml.

[0245] Statement 21. A living dye for use according to any one of statements 16 to 20, wherein the living dye is a free dye or wherein the living dye is conjugated with an agent and / or contained in particles, such as nanoparticles or microparticles; preferably wherein the living dye is grafted onto the particles and / or wherein the living dye is encapsulated in the particles.

[0246] Statement 22. A living dye for any of the uses described in statements 16 to 21, wherein the living dye is applied to the vitreous body by intravitreal administration; preferably wherein the living dye is applied to the vitreous body by intravitreal injection.

[0247] Statement 23. A living dye for any of the uses described in statements 16 to 22, wherein the living dye is capable of diffusing in the vitreous body after application.

[0248] Statement 24. A living dye for use according to any one of statements 16 to 23, wherein the living dye is capable of binding with vitreous opacity after application.

[0249] Statement 25. A living dye for any of the uses described in statements 16 to 24, wherein the living dye is capable of forming vapor nanobubbles in a vitreous turbidity when irradiated.

[0250] Statement 26. A living dye for use according to any one of statements 16 to 25, wherein at least a portion of the vitreous body is turbid by electromagnetic radiation; preferably wherein at least a portion of the vitreous body is turbid by laser radiation; more preferably wherein at least a portion of the vitreous body is turbid by pulsed laser radiation.

[0251] Statement 27. A living dye for the purpose described in Statement 26, wherein:

[0252] - The laser pulse intensity can be at least 10 4 W / cm 2 ;

[0253] - The number of laser pulses can be at least 1 laser pulse;

[0254] - The duration of the laser pulse can be in the range of at least 10 fs; and / or

[0255] - Laser irradiation of the opaque areas of the vitreous reaches at least 0.1 mJ / cm². 2 The amount of bets.

[0256] Statement 28. A method for photoinduced damage to vitreous opacities in the eye of a subject, the method comprising:

[0257] - Apply a live dye to the vitreous humor of the subject's eye; and

[0258] - Irradiate at least part of the vitreous opacity, thereby causing damage to the vitreous opacity of the subject.

[0259] Statement 29. The method according to Statement 28, wherein:

[0260] - This live dye is an approved live dye for ophthalmic use;

[0261] - The living dye is selected from the group consisting of: indocyanine green, trypan blue, Jenners green B, gentian violet, bromophenol blue, patent blue, brilliant blue, light green, fast green, hypocyanine green, methylene blue, toluidine blue, sodium fluorescein, rose red, and rhodamine 6G; preferably, the living dye is indocyanine green or trypan blue; more preferably, the living dye is indocyanine green;

[0262] - The live dye is applied at a concentration of about 0.001 mg / ml to about 0.5 mg / ml;

[0263] -This living dye is a free dye;

[0264] -The living dye is conjugated with the agent;

[0265] - The live dye is contained in particles, such as nanoparticles or microparticles; preferably wherein the live dye is grafted onto the particles and / or wherein the live dye is encapsulated in the particles;

[0266] - The live dye is applied to the vitreous body via intravitreal administration; preferably, the live dye is applied to the vitreous body via intravitreal injection.

[0267] - The living dye is able to diffuse within the vitreous body after application;

[0268] - The live dye can bind to the vitreous opacity after application;

[0269] - This living dye can form vapor nanobubbles in the turbidity of the vitreous body when irradiated;

[0270] - Irradiation of at least a portion of the vitreous body with electromagnetic radiation; preferably, irradiation of at least a portion of the vitreous body with laser radiation; more preferably, irradiation of at least a portion of the vitreous body with pulsed laser radiation;

[0271] - The laser pulse intensity can be at least 10 4 W / cm 2 ;

[0272] - The number of laser pulses can be at least 1 laser pulse;

[0273] - The duration of the laser pulse can be in the range of at least 10 fs; and / or

[0274] - Laser irradiation of the opaque areas of the vitreous reaches at least 0.1 mJ / cm². 2 The amount of bets.

[0275] Statement 30. Use of live dyes for photoinduced damage to vitreous opacities in the eyes of subjects.

[0276] The above aspects and implementation methods are further supported by the following non-limiting examples.

[0277] Example

[0278] Example 1: Study on the disruption of vitreous opacity by indocyanine green, trypan blue, and nanoparticles containing indocyanine green according to embodiments of the present invention.

[0279] This study investigated the ability of two FDA-approved photosensitizers, indocyanine green (ICG) and trypan blue (TB), to disrupt vitreous opacities. First, the efficacy of free ICG versus various ICG-loaded nanoparticles in disrupting type I collagen fiber artificial floaters was compared. Second, the efficacy of free ICG versus various ICG-loaded nanoparticles in treating vitreous opacities acquired after vitrectomy was compared. ICG nanoparticles limit ICG penetration into the retina by prolonging their residence time in the vitreous, thus limiting acute toxicity at the retinal level. Indeed, the internal limiting membrane covering the retina is porous, preventing nanoparticles larger than 100 nm from passing through. In this study, different types of ICG-loaded nanomedicines—polymer nanoparticles, albumin nanoparticles, and liposomes with various surface charges—were prepared, and their effects on floater disruption were compared with those of free ICG and free TB.

[0280] Materials and Methods

[0281] chemicals

[0282] The following chemicals were used: poly(allylamine) (PAH; 17000 g / mol) (Sigma-Aldrich, St. Louis, USA); human serum albumin (HSA) (Sigma-Aldrich, St. Louis, USA); indocyanine green (ICG) (Sigma-Aldrich, St. Louis, USA); trypan blue (TB) (Sigma-Aldrich, St. Louis, USA); rat tail collagen type I acid solution (Sigma-Aldrich, St. Louis, USA); ethanol (Chem-Lab NV, Zedelgem, Belgium) and DMSO (Sigma-Aldrich, St. Louis, USA).

[0283] Cell culture

[0284] MIO-M1 cells were cultured in DMEM Glutamax medium (Thermo Fisher Scientific, Waltham, USA). The medium contained 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, USA) and 100 IU / ml penicillin (…). -Invitrogen Life Technologies, Walthan, USA), 100 μg / ml streptomycin ( -Invitrogen Life Technologies, Walthan, USA) and 2 mM L-glutamine ( -Invitrogen Life Technologies, Walthan, USA). Before use, the culture medium was passed through a 0.2 μm PES membrane vacuum filter (VWR, Radnor, USA). Cells were seeded in polystyrene cell culture flasks (surface = 75 cm²). 2 Cells were placed in a culture flask (VWR, Radnor, USA) and incubated in an incubator (37°C, 5% CO2) (Thermo Fisher Scientific, Waltham, USA) until confluence (i.e., cell formation covering 80%-90% of the culture flask). Morphology was examined under a microscope (VWR, Radnor, USA). After confluence (more or less after 5 days) and morphology was examined, cells were aliquoted into new culture flasks. The culture medium in the confluence flask was removed, and the cells were washed with pre-warmed PBS (Thermo Fisher Scientific, Waltham, USA). Müller cells (adherent cells) were treated with 3 ml of trypsin (0.25%). - Invitrogen Life Technologies, Walthan, USA) treated and incubated for 5 minutes to isolate cells. Examine the cell isolation under a microscope. Add 7 ml of culture medium to the isolated cells and transfer the total contents (±10 ml) to a Falcon tube (15 ml) (Nerbe plus GmbH, Winsen, Germany). To remove (toxic) trypsin, centrifuge the Falcon tube (5 min, 0.2 rcf) until a clear precipitate is visible. Remove the supernatant and redisperse the precipitate in culture medium (approximately 4 ml). Place 9 ml of cell culture medium in a culture flask and add 1 ml of cell suspension. Mix the flask and place it in an incubator. Preheat all liquids (PBS, culture medium, trypsin) in a particle bath at 37°C.

[0285] Preparation of ICG-nanoparticles

[0286] PAH-ICG nanoparticles

[0287] A 2 mg / mL poly(allylamine) hydrochloride (PAH) solution and a 0.005 M Na₂HPO₄ (disodium hydrogen phosphate) solution were prepared (Merck, Leuven, Belgium). 200 μL of the PAH solution was mixed with 1200 μL of the Na₂HPO₄ solution. Then, 12 mL of deionized water was added and the mixture was vortexed for 10 seconds. Finally, 1200 μL of a 1 mg / mL ICG aqueous solution was added to the solution and the mixture was vortexed for 10 seconds. All solutions were pre-cooled at 4 °C. The suspension was aged at 4 °C for 2 hours. The total volume of the suspension was aliquoted into 14 Eppendorf tubes (1 mL per tube) and centrifuged (1 hour, 1000 G rcf) (Beckman-Coulter, California, USA). After centrifugation, the supernatant was removed and set aside. The precipitate in each Eppendorf tube was washed and redispersed with the same volume of PBS solution. Finally, centrifuge the solution again (under the same conditions), remove the supernatant, and set aside. Mix all the precipitates dispersed in different Eppendorf tubes together with 200 μL of PBS solution. Store the nanoparticles at 4°C in the dark. Prepare the same nanoparticles without ICG. Replace the ICG solution with deionized water.

[0288] HSA-ICG nanoparticles

[0289] ICG and HSA were dissolved in 50 mM GSH (Sigma-Aldrich, St. Louis, USA) solution at concentrations of 20 mg / ml and 80 mg / ml, respectively. 1 ml of ICG solution was mixed with 1 ml of HSA solution. Then, 2 ml of ethanol was added to precipitate HSA-ICGNP. The suspension was magnetically stirred (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) for 30 minutes at room temperature. The suspension was then transferred to an 8 ml dialysis cartridge with a cutoff of 10,000 Da (Thermo Fisher Scientific, Waltham, USA) using a syringe. After transferring the suspension, any remaining air was removed from the cartridge using another syringe. The dialysis cartridge, along with a Slide-A-Lyzer (to prevent the cartridge from sinking) (Thermo Fisher Scientific, Waltham, USA), was placed in a large beaker (approximately 1 L) filled with deionized water and placed on a magnetic stirrer (to create flow within the beaker) at 4°C for 24 hours. After dialysis, the suspension is removed from the box using a syringe and stored in a Falcon tube at 4°C in the dark.

[0290] Liposome-encapsulated ICG (Lip-ICG)

[0291] Positively charged liposomes loaded with ICG were prepared by mixing distearate phosphatidylcholine (DSPC) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) (1:1 molar ratio) using a thin-film rehydration method, as described in Lajunen et al., 2016, Mol. Pharm., 13, 2095-2107. The obtained liposomes were then sonicated for 1 min using a high-precision sonicator and purified by dialysis.

[0292] Characterization of nanoparticles

[0293] Encapsulation efficiency

[0294] ICG loading efficiency was determined based on free ICG calibration curves of two nanoparticles obtained by UV-Vis spectroscopy (NanoDrop 2000C, Thermo Fisher Scientific, Waltham, USA).

[0295] For PAH-ICG NP, multiple ICG concentrations (0.5 μg / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, and 7.5 μg / ml) were prepared from the free ICG stock solution in triplicate, and diluted in PBS (each concentration was prepared in triplicate). A blank assay was performed in PBS, and absorbance was measured at 780 nm. The supernatant was set aside to determine the loss of free ICG. The supernatant from the first washing step was diluted 1 / 10 in PBS, while the second supernatant did not require any dilution. The absorbance of both supernatants was measured at 780 nm. Based on the absorbance, the concentration of free ICG in the supernatant was determined, and the encapsulation efficiency of PAH-ICG NP(EE1) was calculated using the following equation:

[0296] EE1% = (Initial Mass ICG - Total Mass ICG Loss) / (Initial Mass ICG) × 100

[0297] Modifications were made to establish calibration curves and determine the encapsulation efficiency of HSA-ICG NPs. Measurements were performed in DMSO / H2O (9:1, V / V) (blank, calibration curve, and HSA-ICG NPs). Free ICG was diluted to concentrations ranging from 0.25 μg / ml to 5 μg / ml (0.25 μg / ml, 0.50 μg / ml, 1.0 μg / ml, 2.5 μg / ml, and 5.0 μg / ml), with each concentration prepared three times. Absorbance was measured at 780 nm and calibration curves were plotted. Nanoparticles were diluted 1 / 500 in DMSO / H2O (9:1, V / V), and absorbance was subsequently measured at 780 nm. DMSO was used to break down the particles, thereby releasing the ICG. The amount of free ICG was then measured by UV-Vis spectroscopy. The encapsulation efficiency (EE2) of HSA-ICG NPs was calculated using the following equation:

[0298] EE2% = (End of Quality ICG) / (Start of Quality ICG) × 100

[0299] Magnitude and ζ potential

[0300] PAH-ICG NPs and HSA-ICG NPs were diluted 1 / 100 in deionized water and their size and zeta potential were determined by DLS and electrophoretic mobility measurements, respectively, at 25°C using a Nanosizer (Malvern Instruments, Malvern, UK). For PAH-ICG NPs, these measurements were performed before the particles were stored at 4°C for 2 hours and after a centrifugation step to remove free ICG. The size and zeta potential of HSA-ICG NPs were measured after dialysis. The dispersions were prepared in a laminar flow hood to prevent dust from entering the sample. 1 ml of the dispersion was transferred to a folded capillary cell.

[0301] Toxicity study of human immortalized Müller cells (MIO-M1)

[0302] Free ICG

[0303] To test the toxicity of free and nano-encapsulated ICG, we wanted to seed 7,000 Müller cells in each well of a 96-well plate (VWR, Radnor, USA). Cells were counted using a Bürker counting chamber (BRAND, Wertheim, Germany) to obtain an appropriate number of cells. Cells were processed in the same manner as in Section 3.2. After redispersing the cell pellet (obtained after centrifugation) in 4 ml of medium, 50 μl was transferred to an Eppendorf tube for cell counting. 100 μl of trypan blue (an azo dye used to stain dead cells) (Sigma-Aldrich, St. Louis, USA) was added to the Eppendorf tube, and 10 μl of this mixture was transferred to both sides of the Bürker counting chamber. Unstained cells were counted in 6 large squares (3 per side of the chamber); each large square contained 0.1 μl of the mixture. The dilution factor (1 / 3) of trypan blue and the factor from 0.1 μL to 1 μL (factor 10,000) were taken into account to calculate the number of cells per milliliter. Based on the cell count, a dilution was prepared to obtain 7,000 cells in 200 μL cell suspension per well. Each well was loaded with 200 μL of diluted cell suspension and incubated for 24 hours.

[0304] After 24 hours of incubation, the cell culture medium was removed and the cells were treated with different concentrations of free ICG. A series of dilutions of free ICG were prepared in cell culture medium from a stock solution: 0.1 mg / ml, 0.3 mg / ml, 0.5 mg / ml, 0.75 mg / ml, and 1 mg / ml. The control contained cell culture medium and a specific amount of deionized water, i.e., the same amount of water added to obtain the 1 mg / ml ICG solution in cell culture medium (the most stringent concentration). Cell culture medium was removed from the 96-well plates and replaced with 200 μL of each solution (control and five different ICG concentrations). Each condition was performed five times. The plates were covered with aluminum foil and incubated at 37°C for 24 hours.

[0305] For MTT assay, a stock solution of 5 mg / ml MTT reagent (Sigma-Aldrich, St. Louis, USA) was prepared in PBS. For each well, 30 μL of MTT reagent (5 mg / ml) was mixed with 200 μL of cell culture medium. The solution in each well was carefully removed, and each well was washed twice with 100 μL of PBS, followed by the addition of 200 μL of MTT solution. Control wells were treated similarly. Cells were incubated at 37°C wrapped in aluminum foil for 3 hours. After incubation, the solution in the wells was removed again and replaced with 100 μL of LDMSO. 96-well plates were covered with aluminum foil and placed on a track-mounted shaker (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) for 30 minutes. MTT assays were performed using a Victor3 microplate reader (PerkinElmer, Waltham, USA). Absorbance was measured at 595 nm. The metabolic activity of cells treated with ICG solution was compared with that of the control. After receiving the results of the first MTT assay, it was noted that ICG adhered to the 96-well plate (even after two washes with PBS) and contributed to the absorbance measured at 595 nm using a Victor3 microplate reader. To address this, an additional control was implemented to account for ICG background. Additional wells were treated with the same dilutions of free ICG (0.1 mg / ml, 0.3 mg / ml, 0.5 mg / ml, 0.75 mg / ml, and 1 mg / ml), but after 24 hours of incubation, cell culture medium was added to the additional wells instead of the MTT reagent. Thus, it was possible to measure the absorbance of ICG adhered to the 96-well plate. The background absorbance of ICG was subtracted from the absorbance obtained from the wells incubated with the MTT reagent and then compared with the control. This experiment was performed in triplicate.

[0306] Nano-encapsulated ICG

[0307] Optimization experiments were performed on PAH-ICG NP, HSA-ICG NP, and LIP-ICG. Several modifications were made. For both types of nanoparticles, each condition was performed in triplicate. Another issue with HSA-ICG NP was the relatively low ICG concentration obtained after dialysis in HSA-ICG NP, resulting in significantly less cell culture medium in the dilution series. For this reason, each concentration of HSA-ICG NP had its own control: the amount of water added to the control cell culture medium was specific for each concentration (i.e., not just the most stringent conditions).

[0308] Poly(allylamine) hydrochloride

[0309] To test the toxicity of the PAH polymer itself (i.e., without ICG), the amount of polymer in which cells were exposed was calculated for each concentration of PAH-ICG NP. The same experiment was performed as described above, except that an additional control for determining the ICG background signal was excluded (because ICG was not used).

[0310] Photoablation of artificial and human vitreous opacities

[0311] Preparation of artificial floaters (Type I collagen fibers)

[0312] As described by Sauvage et al., 2019, ACS Nano, 13, 8401-8416, collagen fibers were prepared from rat tail type I collagen (GIBCO; concentration 3 mg / ml). 5 ml of PBS was pipetted into a 15 ml Falcon tube. 330 μL of PBS solution was replaced with 330 μL of type I collagen, and the solution was vortexed. Sodium hydroxide (0.1 M) (VWR, Radnor, USA) was added to the collagen solution to adjust the pH to 7.4. The solution was vortexed again and incubated at 37°C for one hour. The final collagen concentration was 0.2 mg / ml.

[0313] human vitreous opacity

[0314] Vitreous samples containing human floaters were obtained from the VMR Institute (Huntington Beach, CA, USA), where the patient underwent vitrectomy. The human vitreous turbidity was diluted 1:1 (v / v) with a 1 mg / ml free ICG stock solution to a final ICG concentration of 0.5 mg / ml. The sample was placed on a glass dish and covered with a coverslip.

[0315] Laser treatment of artificial and human vitreous opacities

[0316] First, dark-field microscopy imaging was performed to locate and align a nanosecond laser (Opolette HE 355LD, OPOTEK Inc., CA, USA) on the artificial collagen fibers in the sample. Then, the artificial or human vitreous opacity sample treated with TB, ICG, and ICG nanoparticles was irradiated with the laser (<7 ns). The laser wavelength was 561 nm and the energy was set to ±800 μJ. A beam expander (#GBE05-A, Thorlabs) was combined with a variable aperture (#D37SZ, Thorlabs) to adjust the laser beam diameter to 150 μm (so that 800 μJ corresponds to 4.5 J / cm²). 2The laser pulse energy was monitored using an energy meter (J-25MB-HE&LE, Energy Max-USB / RS sensor, Coherent) synchronized with the pulsed laser. The setup involved irradiating samples one by one. Videos of the samples were created during the irradiation process using NIS software. The same experiment was performed on human vitreous opacities.

[0317] Comparison of free ICG, PAH-ICG NP, and HSA-ICG NP in water and bovine vitreous.

[0318] Quantitative analysis of bubble number as a function of laser fluence

[0319] VNBs were generated using the same laser setup described in the previous section. VNBs scatter light efficiently, making them easily detectable by dark-field microscopy. The video shows VNBs generated from single laser pulses at two different laser energies (100 μJ and 800 μJ). The experiment was conducted in water and bovine vitreous. The number of bubbles was then plotted as a function of laser fluence using a graphpad prism.

[0320] In vivo imaging and photoablation of collagen fibers

[0321] Animal preparation

[0322] All animal experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) guidelines regarding the use of laboratory animals in ophthalmology and vision research. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan (Protocol PRO00008566, PI Paulus).

[0323] Twenty-four New Zealand white rabbits (3-6 months old; weighing 2.45-3.15 kg; both sexes) were used. The rabbits were randomly assigned to seven groups: control group A received laser treatment only (n=3); group B received intravitreal intubation (IVIT) with ICG (n=9); group C received IVIT with ICG-labeled collagen fibers (n=3); group D received IVIT with collagen fibers followed by IVIT with ICG (n=3); group E received IVIT with HA-AuNP-labeled floaters (n=3); and group F received IVIT with floaters followed by IVIT with HA-AuNP (n=3). During the in vivo experiments, the animals' condition, such as mucosal color, heart rate, body temperature, and respiratory rate, was monitored every 15 minutes. The animals were anesthetized by intramuscular injection of ketamine (40 mg / kg) and toluenethiazide (5 mg / kg). Fifteen minutes before the imaging procedure, one drop of tropicamide (1%) and phenylephrine hydrochloride (2.5%) was instilled into the rabbit eye to dilate the pupil. For topical anesthesia, one drop of topical tetracaine (0.5%) was used. To prevent corneal dehydration during the experiment, a lubricant solution (Systane, Alcon Inc., TX, USA) was added to the eye per minute using a syringe with a plastic needle. To maintain anesthesia, one dose of ketamine (13 mg / kg) was injected every 45 minutes. A circulating heating blanket was used to maintain the animal's body temperature.

[0324] Intravitreal injection

[0325] First, to observe the rabbit retina, a plastic contact lens was placed on the cornea. While selecting the target location in the vitreous humor under a microscope, intravitreal injections were then performed on anesthetized rabbits. A 27-gauge needle was used for the intravitreal injections. In one series of experiments, 40 μL of ICG fibers (which were collagen fibers treated with ICG) were injected; the collagen concentration in the dispersion was 0.02 mg / mL. In another series of experiments, 40 μL of collagen fibers (0.02 mg / mL) were injected intravitreally. Five days after fiber injection, 40 μL of ICG (0.25–1.25 mg / mL) was injected into the vitreous humor. The fiber position was monitored using OCT to ensure that ICG was injected into the vitreous humor near the fiber location. Three days after ICG injection, laser treatment was performed.

[0326] Color fundus photography, fluorescence imaging, PAM and OCT imaging

[0327] Rabbits were monitored one minute after intravitreal injection of collagen fibers and on day 4 after intravitreal injection of ICG. Rabbits receiving only intravitreal ICG were followed up for 14 days post-injection. Rabbit eyes were evaluated using color fundus photography, fluorescence imaging, optical coherence tomography (OCT), and photoacoustic microscopy (PAM), as described below. The rabbit's head and body were placed on two separate platforms to minimize respiratory and other motion artifacts. The same scan area for PAM and OCT imaging was monitored by a fundus camera integrated into the OCT system. To detect photoacoustic signals, an ultrasonic transducer was positioned in contact with the conjunctiva, allowing it to move freely in 3D without applying any physical pressure to the rabbit eye. The scan area (i.e., the area containing the injected fibers) was selected by the fundus camera and captured by PAM.

[0328] Color fundus photography was performed using a 50-degree color fundus photography system (Topcon 50EX, Topcon Corporation, Tokyo, Japan). An EOS 5D camera (resolution 5472×3648 pixels, pixel size 6.55μm) was used. 2 (Canon, Japan) The Topcon 50EX system captures images of the retina and fundus. Several locations in the eye are imaged, including the optic nerve, the superior retina above the optic disc, the inferior retina below the optic disc, the temporal medullary rays, and the nasal medullary rays. Fluorescence imaging is performed using appropriate excitation and emission filters with a Topcon 50EX system.

[0329] For photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging, a self-developed integrated PAM and OCT system was developed to track the position of fibers in the vitreous body. Briefly, for PAM, a tunable nanosecond pulsed laser generated by a solid-state Q-switched Nd:YAG laser (NT-242, Ekspla, Lithuania) was used as the light source. The wavelength of the light could be adjusted (405–2600 nm), the pulse repetition rate was 1 kHz, and the pulse duration was 3–5 ns. The output laser was diffused, filtered, and collimated through the iris to form a uniform beam of 2 mm. The laser was then passed through a galvanometer and telescope consisting of a scanning lens and an eyepiece, and focused onto the fundus of the retina, estimated to be 20 μm in diameter. To detect the photoacoustic (PA) signal, a custom-designed needle-shaped ultrasonic transducer (center frequency 27 MHz, bidirectional bandwidth -60%, Optosonic Inc., Arcadia, CA, USA) was used. The detected PA signal was amplified using a 1.4dB preamplifier (AU-1647, L3 Narda-MITEQ, NY). The analog data was then converted into a digital signal and digitized using a DAQ card (PX1500-4, Signatec Inc., Newport Beach, CA) at a sampling rate of 500MHz.

[0330] For PAM imaging, light at 578 nm (detecting retinal and choroidal vessels) and 800 nm (detecting ICG) with average energies of 80 nJ is shone into the eye. According to the American National Standards Institute (ANSI) definition, this is approximately half the maximum energy of a single laser pulse applicable to the retina (approximately 160 nJ at 570 and 800 nm). Using the line spread function (LSF) and the full width at half maximum (FWHM) of the A-line signal (one-dimensional point spread function), the lateral and axial resolutions are 4.1 μm and 37.0 μm, respectively. Both 2D and 3D PAM images can be acquired using an optical scanning galvanometer with an acquisition time of 65 s.

[0331] The OCT setup used in this study was constructed using a commercially available spectral-domain Ganymede-II-HR OCT device (Thorlabs, Newton, NJ), with the addition of dispersion-compensating glass and an eyepiece. Two superluminescent diodes (SLEDs) with center wavelengths of 846 nm and 932 nm were used to excite the sample. The incident beam was coaxially aligned with the PAM laser beam to achieve simultaneous acquisition of PAM and OCT at the same location, and co-registration of OCT and PAM images on the same orthogonal imaging plane. The OCT lateral and axial resolutions were 3.8 μm and 4.0 μm, respectively. At a scan rate of 36 kHz, a cross-sectional B-scan OCT image with a resolution of 512 × 1024 A lines was acquired in 0.103 seconds. A volume of 4.5 × 4.5 × 1.8 mm was acquired within 2 minutes. 3 3D stereoscopic OCT image (512×512×1024 pixels) (average rate 3 times).

[0332] In vivo rabbit eye laser treatment

[0333] Prior to laser treatment, the rabbit's eye was imaged using PAM and OCT (see previous section). For laser treatment, the anesthetized rabbit was placed on a custom-designed, stable platform. After acquiring images and locating the opacities (collagen fibers) via OCT, an 800 nm (ICG) laser pulse (<7 ns; 1.9 J / cm²) was used. 2 NT-242, Ekspla, Lithuania) Irradiation area for the eye (4.5×4.5mm) 2 Collagen fibers are destroyed; the scanning laser has a step size of 9 μm and a beam size of 20 μm. During treatment, real-time OCT is activated to monitor the location of collagen fibers. (4.5 × 4.5 mm) 2 The area was scanned several times (3-7 times) with a laser until the collagen fibers were completely destroyed. After laser treatment, PAM and fundus images were performed to evaluate potential damage to the retinal vessels. In addition, the rabbits' vital signs were monitored and recorded until the animals fully recovered from anesthesia.

[0334] In vivo safety evaluation

[0335] Immediately after laser treatment, the treated eye underwent an ophthalmic evaluation and was followed up for one month. The anterior segment structures, including the eyelids, iris, conjunctiva, cornea, anterior chamber, and lens, were comprehensively examined using a slit-lamp biomicroscope (SL120, Carl Zeiss, Germany). Additionally, the posterior segment structures (i.e., vitreous body, optic nerve, and retina) were evaluated using a fundus contact lens (Volk Optical Inc, Mentor, OH, USA).

[0336] Statistical analysis

[0337] One-way ANOVA is used to calculate statistical significance. Data are considered statistically significant when p < 0.05.

[0338] result

[0339] Characterization and toxicity screening of ICG-loaded nanoparticles

[0340] After preparation, the sizes of poly(allylamine) hydrochloride (PAH)-ICG nanoparticles, human serum albumin (HAS)-ICG nanoparticles, and ICG-encapsulated liposomes (LIP-ICG) were 183, 250, and 153 nm, respectively. Therefore, the obtained sizes exceeded 100 nm (>100 nm) because particles with sizes >100 nm cannot penetrate the inner limiting membrane (ILM). Figure 1A (Left). The zeta potentials of PAH-ICG, HSA-ICG, and LIP-ICG were found to be -51, -11, and +37 mV, respectively. Figure 1A ,right).

[0341] To gain a preliminary understanding of the toxicity of free and nano-encapsulated ICG, MTT assays were performed on immortalized human Müller cells. This type of cell is located directly at the vitreoretinal interface, thus serving as a good model for retinal toxicity screening. The initial observation was that PAH-ICG exhibited toxicity and reduced metabolic activity. Figure 1C This toxicity can be attributed to PAH, as PAH nanoparticles have also been found to be toxic. Free ICG exhibited reasonably acceptable toxicity at concentrations up to 0.5 mg / ml, and at all studied concentrations, cellular metabolic activity was slightly decreased compared to free ICG (HSA-ICG treatment). Figure 1B ).

[0342] Comparison of the damage to type I collagen fibers caused by free and nano-encapsulated ICG after nanosecond laser irradiation

[0343] Type I collagen fibers are well-suited as an in vitro model for creating artificial floaters. Therefore, the ability of ICG-loaded NPs and free ICG to disrupt type I collagen fibers in water after nanosecond laser irradiation was investigated. Based on the results of the viability assay, the in vitro toxicity of free ICG at a concentration of 0.5 mg / ml was acceptable. Figure 1B The first observation is that, in the absence of free ICG or nanoparticles, the laser is insufficient to destroy type I collagen fibers (data not shown). However, when the fibers are mixed with free ICG, HSA-ICG, or PAH-ICG, they may be able to be destroyed using nanosecond lasers with pulse numbers less than 10 (<10) (4.5 J / cm). 2 The fibers were damaged after irradiation. However, although some bubbles were observed during the pulses, LIP-ICG was not found to effectively damage the fibers. The fiber structure was affected after five pulses; however, no effect was observed after 15 pulses. Figure 2A (last column).

[0344] To better understand the effectiveness of nanoparticles, the average number of pulses required to destroy a single fiber was measured. Figure 2B For both free and ICG-loaded nanoparticles, an average of 4-5 pulses appears sufficient to destroy a fiber. Figure 2B ).

[0345] It is also important to note that in these experiments, some aggregates can be visualized using HSA and PAH nanoparticles, while free ICG and LIP-ICG cannot.

[0346] Another interesting observation is that VNBs can be observed at the fiber level with free ICG, while for ICG-loaded nanoparticles, they can be observed at different locations within the laser beam.

[0347] ICG can trigger VNB on type I collagen fibers.

[0348] To examine whether free ICG could target and trigger VNB (i.e., on collagen fibers), dark-field microscopy images of free ICG and nano-encapsulated ICG were obtained in water and bovine vitreous. The first observation was that bright spots could be observed in some particles. Figure 3A The number of VNBs generated in the water was plotted as a function of laser intensity. Figure 3B Clearly, at this injection rate, free ICG and free TB did not generate VNB in ​​the water. Figure 3B Therefore, free ICG is interesting because it can disrupt type I collagen fibers without generating bubbles in the surrounding medium. However, PAH-ICG and HSA-ICG can be produced at energies of 4.5 J / cm². 2A single pulse induces VNB generation ( Figure 3B In the case of LIP-ICG, no VNB was observed in any of the tested light intensities, which is likely because a portion of the ICG is free within the aqueous core. Figure 3B ).

[0349] ICG can effectively destroy vitreous opacities acquired after vitrectomy.

[0350] Because the composition of ocular opacities varies and is a mixture of different types of collagen, the test aimed to determine if ICG could disrupt these opacities in a manner similar to that of type I collagen fibers. Therefore, vitreous humor containing opacities obtained from patients with ocular floaters was mixed with ICG (0.5 mg / ml) and analyzed at 561 nm (4.5 J / cm²). 2 ) and 800nm ​​(1.1J / cm 2 Nanosecond laser irradiation. (e.g.) Figure 4 As shown, vitreous opacities can obviously be disrupted at both wavelengths.

[0351] VNB produced by ICG damages collagen fibers in the body.

[0352] Next, we investigated the extent to which the combined use of laser pulses and ICG could destroy intravitreal collagen fibers (IVIT) injected into rabbit eyes. In the first series of experiments, ICG-labeled collagen fibers were prepared by mixing collagen fibers with free ICG (1.25 mg / mL aqueous solution). Then, the ICG fibers were injected into rabbit eyes. Figure 5 (See the image above). The first observation is that ICG-labeled fibers injected with IVIT can be easily imaged by PAM at 578 and 800 nm. At λ578 nm, both blood vessels and ICG fibers can be detected, while at λ800 nm, only ICG fibers are observed. Figure 5 (See the image below).

[0353] In a series of subsequent experiments, IVIT (unlabeled) collagen fibers were injected, followed by ICG (1.25 mg / mL) 5 days later. Figure 6 (See image above). Although collagen fibers can be observed via OCT, PAM imaging shows that ICG co-localizes with the fibers. These results confirm that ICG injected with IVIT can reach and bind to collagen fibers (…). Figure 6 (See the image below).

[0354] In further experiments, such as Figure 7 As shown, IVIT (unlabeled) collagen fibers were injected, followed by ICG injection 5 days later (day 0). Subsequently, the collagen fibers were irradiated with laser pulses 3 days after the ICG injection (day 3).

[0355] As shown in Figure 8, after intravitreal injection, ICG was visualized via fundus imaging at all ICG concentrations (0.25, 0.625, and 1.25 mg / ml). One day later, we observed that most of the injected ICG was no longer visible, likely due to vitreous clearance. At an ICG concentration of 0.25 mg / ml, ICG was no longer visible at the collagen fiber level 7 days after injection. Figure 8A Interestingly, when sufficiently high ICG concentrations were injected (i.e., 0.625 and 1.25 mg / ml), ICG was only visible at the collagen fiber level after 7 days. Figure 8B and 8C ).

[0356] Therefore, after intravitreal injection of ICG, the binding of ICG to collagen fibers was confirmed in vivo; the binding of ICG to collagen fibers could still be observed at concentrations as low as 0.625 mg / mL. Figure 8B This is even lower than the concentrations used for ILM peeling (>1 mg / mL). This is attractive because using lower ICG concentrations will reduce its phototoxicity. Equally important, at all concentrations of ICG injected intravitreally, most of the “free” ICG (i.e., unbound collagen) was no longer visible in the vitreous cavity 3 days after injection. Figure 8A -C).

[0357] Subsequently, three days after the ICG injection (day 3), the collagen fibers were irradiated with laser pulses. Figure 9 As shown, applying laser pulses without ICG does not damage the fibers. However, after IVIT injection of ICG (1.25 mg / mL), 5 laser pulses (i.e., 5 scans; 1.9 J / cm²) resulted in fiber damage. 2 ) is sufficient to completely destroy the injected collagen fiber clusters. Figure 10A Because retinal toxicity can be observed at high ICG concentrations, a lower concentration (0.625 mg / mL) was tested, and complete destruction of collagen fibers was again observed. Figure 10B Experiments showed that ICG is capable of destroying collagen fibers in vivo using laser settings (i.e., pulse number (scans), laser dose), similar to laser settings using HA-AuNP, and at ICG concentrations lower than those currently used clinically for ILM stripping (typically between 1 and 5 mg / mL). In all rabbits tested (n=3), 3.3 + / - 1.5 pulses appeared sufficient to destroy IVIT-injected fibers.

[0358] Security research

[0359] Visual examination confirmed that the laser-treated eyes (with and without ICG) showed normal cornea, eyelids, anterior chamber, conjunctiva, and clear lens. Color fundus imaging showed no significant changes or damage to the posterior segment structures; specifically, there was no hemorrhage, retinal detachment, vascular abnormalities, or pigmentary abnormalities in the retinal pigment epithelium (RPE). Furthermore, B-scan OCT images did not indicate any disruption of the retinal layer structure; normal retinal anatomy was observed after laser treatment. No retinal detachment or RPE hypertrophy was observed; the retinal and choroidal thicknesses in the rabbits after laser treatment differed slightly from pre-treatment values ​​(N=6, p>0.05).

[0360] in conclusion

[0361] Experiments showed that free ICG and free trypan blue could bind to and effectively destroy type I collagen fibers at concentrations of 0.5 mg / ml and 0.1 mg / ml, respectively. Both concentrations were lower than those used clinically (ICG: 1.25 mg / ml; TB: 0.6 mg / ml). Figure 2A Furthermore, no significant toxicity to immortalized human retinal cells (MIO-M1) was observed at these concentrations. Figure 1B At 4.5 J / cm 2 At this dosage, significant damage can be observed with fewer than 10 pulses. This is 1000 times lower than current YAG laser therapy and comparable to gold nanoparticle settings.

[0362] A significant advantage of ICG over TB and spherical gold nanoparticles is its broad absorbance range. Therefore, ICG allows for the tuning of the laser wavelength to near-infrared light. One advantage of using near-infrared light is its lower tissue interference and thus fewer side effects. The TB concentration used in this study was significantly lower than clinically used concentrations (0.01 mg / ml vs. 0.6 mg / ml). The ICG concentration used in this study was also lower than clinically used concentrations (0.5 mg / ml vs. 1.25 mg / ml).

[0363] Furthermore, when ICG is encapsulated in nanoparticles, bubbles can form in water after irradiation, while with free ICG, bubbles are only observed at the fiber level, indicating a targeting effect. Therefore, the use of free ICG can reduce or even avoid damage to the vitreous structure and surrounding ocular tissues. Figure 2C This phenomenon is likely due to the accumulation of live dyes on the fiber lowering the energy threshold for bubble generation.

[0364] Following the injection of ICG into the vitreous humor of rabbits previously injected with collagen fibers, it is noteworthy that a 4.5 × 4.5 mm scan within the vitreous humor was performed. 2 The collagen fiber regions can effectively destroy collagen fibers in the body (Figure 10), which is not the case in the absence of ICG. Figure 9Importantly, ICG enables the destruction of fibers in the vitreous humor with a significantly lower light dose than YAG lasers currently used in clinics. This reduction in light energy makes floater treatment safer and opens up possibilities for treating vitreous opacities close to the retina. Furthermore, the ability to “see” ICG-marked vitreous opacities via PAM imaging allows for more precise application of laser irradiation, further reducing the light dose required for YAG laser treatment. Equally important, the cornea, eyelids, anterior chamber, conjunctiva, and clear lens in rabbits returned to normal 30 days post-treatment. In conclusion, injecting ICG into the vitreous humor and applying laser pulses appears to be an effective and safe strategy for eliminating opacities.

[0365] In summary, this work demonstrates that ophthalmologically approved live dyes can be used for pulsed laser treatment of vitreous opacities that cause floaters.

Claims

1. Use of a dye in the manufacture of a composition for treating a vitreous haze-related disease in a subject, wherein the vitreous haze-related disease is floaters or posterior vitreous detachment, and wherein the dye is indocyanine green or trypan blue, and wherein the dye is a free dye or is contained in a protein nanoparticle.

2. The use of claim 1, wherein the composition is formulated for administration of the dye to the vitreous of an affected eye of the subject.

3. The use of claim 1, wherein the dye is formulated at a concentration of about 0.001 mg / ml to about 0.5 mg / ml.

4. The use of claim 1, wherein the composition is formulated for administration of the dye to the vitreous by intravitreal administration.

5. The use of claim 1, wherein the protein nanoparticle is an albumin nanoparticle.

6. The use of claim 1, wherein the dye is grafted on a nanoparticle and / or wherein the dye is encapsulated in a nanoparticle.

7. The use of claim 1, wherein the composition is formulated for administration of the dye to the vitreous by intravitreal injection.

Images