KR102618470B1 - Drug delivery system for targeting to retina - Google Patents

Abstract

The present invention introduces chitosan, a biocompatible polymer based on temperature-sensitive polymers, distributes nucleotides on the surface of the nanogel using the charge interaction between the positive charge of chitosan and the negative charge of nucleotides, and makes the nucleotides specific to the P2Y receptor in the retina. This relates to a temperature-sensitive drug delivery system that allows nanogels to target and accumulate active ingredients in the retina due to their ability to bind to target substances.
Through this, the nanogel drug carrier developed in the present invention can target and deliver the target substance to the retina. Using a temperature-sensitive polymer, not only can delivery target substances such as proteins be loaded with high efficiency, but when the nanogel is dissolved at a high concentration, the viscosity can be increased, allowing sustained release of the drug while remaining within the eye for a long time. You can.

Description

{Drug delivery system for targeting to retina}

The present invention provides a chitosan-functionalized temperature-sensitive nanogel; and a retina-targeted nanocarrier containing one or more from the group consisting of purine-based nucleotides and variants containing the structure of the purine-based nucleotide and a method for producing the same.

The human eye is a very complex biological device. The structure of the eye is largely divided into two parts: the anterior segment and the posterior segment. The anterior segment consists of the sclera, iris, conjunctiva, and cornea, and the posterior segment consists of the vitreous humor, retina, choroid, and optic nerve. Each structure of the eye has various devices to prevent it from being exposed to excessive amounts of drugs. In the anterior segment, functions such as tear rotation, nasal tear drainage, esterases, and proteases dilute the concentration of drugs injected into the eye. The retinal layer located in the posterior segment plays a major role in preventing drugs from penetrating into the eye nerves.

The blood-retinal barrier (BRB), which surrounds the retinal layer, maintains retinal homeostasis and protects it from external stimuli. The retina is a light-sensitive component of the central nervous system, consumes large amounts of oxygen, and surrounds the innermost part of the eye. Therefore, the retina reacts sensitively to oxidative reactions, which can cause various retinal diseases such as age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy. Methods for delivering drugs to the posterior segment include injection through the circulatory system, manipulation of the blood-retinal barrier, and direct local administration (intravitreal injection). However, eye drops and injections through the circulatory system are very inefficient in delivering the drug to the end of the posterior segment and have little effect on the retina.

Therefore, the most commonly used method for injecting drugs into the posterior part of the eye is direct intravitreal injection. However, drugs injected through this route can be decomposed by proteolytic enzymes, and the highly viscous vitreous humor also prevents the drug from reaching the retina. Transport of drugs through the retina and RPE (retinal pigment epithelium) is particularly limited, and this applies especially strongly to protein drugs such as antibodies. Due to these reasons, multiple injections are required. Injections administered directly to the eyes create several small holes in the eyes, which can lead to various side effects. Side effects include hemorrhage, retinal detachment, optic nerve damage, increased intraocular pressure (IOP), and vision loss. Therefore, it is important to reduce the side effects and improve the results of this intravitreal injection method.

In recent years, much research has been done on various methods for delivering drugs to the posterior segment of the eye. One of the drug administration methods currently being studied is to synthesize therapeutic molecules into hydrogel and inject them, which is a sustained-acting drug injection method with minimal toxicity. For example, hydrogels that respond to temperatures equivalent to human body temperature, such as poloxamer and poly N-isopropyl acrylamide-co-acrylonitrile (PNIPAAm), are injected into the vitreous cavity.

These polymers have mucosal adhesion and optical transparency, so they can be effectively applied to administer drugs to the eye. However, this method does not provide a strategy for overcoming the numerous other barriers that must be overcome to reach the target retina. Research has been conducted on various nanoparticles to analyze how the particle's charge affects diffusion within the vitreous humor. As a result, it was found that anionic particles are more effective in penetrating through the vitreous humor barrier because the movement of cationic particles is restricted by electrostatic interactions with anionic glycosaminoglycans contained in the vitreous humor. . Additionally, it was shown that cationic particles could not penetrate into the retina, but anionic particles could penetrate the retinal barrier and be delivered to the RPE.

The retinal pigment epithelium (RPE) is an insulator of pigmented cells and is a part of the retina. RPE constitutes the outer blood-retinal barrier (BRB) formed by tight junctions. Tight junctions are complex structures that regulate cell polarity and intercellular diffusion of fluids and solutions, and play a role in preventing toxic molecules and plasma components from entering the retina. Purine receptors exist on the surface of the RPE layer.

Purine receptor antagonists, especially ATP, have been reported to have a significant effect on the RPE layer. Since there is a P2Y2 receptor at the tip of the RPE cell membrane, ATP located outside the cell can bind to this receptor. The ATP-bound P2Y2 receptor reacts with the adjacent actin cytoskeleton and filamin A to induce rearrangement of the RPE layer. Taking advantage of this effect, research was conducted to induce the breakdown of tight junctions using nucleotides to allow drugs to pass through the RPE more easily. In particular, P1, P4-Di(adenosine-5') and AP4A were found to increase corneal epithelial barrier permeability. In addition, the P2Y receptor signaling pathway caused by the thermogenic binding substance was secured. In addition, the P2Y2 receptor antagonist can now be used as a treatment and adjuvant for edematous retinal disorders. Pyrogenic nucleotides are currently used for retinal detachment.

Since its development, chitosan-functionalized pluronic-based nanocarrier (NC) has been introduced to add various protein components. Nanocarriers have heat sensitivity and wide expansion ability, so they showed high protein encapsulation efficiency just by simple incubation with proteins while controlling temperature. In addition, in addition to its ability to target tumors and penetrate the skin, it was discovered that it could deliver drugs to the brain and increase the efficiency of oral administration through binding to specific peptides. However, nothing is yet known about a drug carrier that can effectively deliver drugs to the posterior retina using chitosan-functional pluronic-based nanocarriers.

Domestic Patent Publication No. 10-2015-0098539

The main purpose of the present invention is to penetrate the drug into the retina using a temperature-sensitive nanocarrier. Cationic nanoparticles have difficulty reaching the retina due to their interaction with hyaluronic acid in the vitreous humor. The present invention manufactures a temperature-sensitive nanogel by introducing chitosan, a biocompatible polymer, based on an amphipathic pluronic polymer, and mixes it with nucleotides that can specifically bind to the P2Y2 receptor present in the retina to form chitosan. The main purpose is to cause the positive charge of the nucleotide to bind to the negative charge of the nucleotide so that the nucleotides can be distributed on the surface of the chitosan-pluronic nanogel and targeted to the retina.

According to one aspect of the present invention, a chitosan functionalized temperature-sensitive nanogel; and purine-based nucleotides and variants containing the structure of the purine-based nucleotides.

In one embodiment, the purine-based nucleotide may be one or more selected from the group consisting of adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), and uridine diphosphate (UDP).

In one embodiment, the variant containing the structure of the purine-based nucleotide may be one or more selected from the group consisting of UDP-glucose and Ap4A (P1,P4-Di(adenosine-5')tetraphosphate). However, the variant is not limited thereto, and any variant containing a purine-based nucleotide structure may be selected.

In one embodiment, the chitosan-functionalized temperature-sensitive nanogel can be prepared by photo-crosslinking chitosan and a temperature-sensitive polymer having a photo-crosslinkable functional group.

According to a preferred embodiment of the present invention, the photocrosslinkable functional group is preferably acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, coumarin, thymine, or It is cinnamate, more preferably acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate or oligomethacrylate, and most preferably acrylate.

Initiators suitable for the method of the present invention are not particularly limited. Preferably, the initiator that can be used in the present invention is a radical photoinitiator that can induce a radical reaction by irradiation of ultraviolet or visible light. Examples of photoinitiators that can be used in the present invention include ethyl eosin, 2,2-dimethoxy-2-phenyl acetophenone, 2-methoxy-2-phenylacetophenone, 2-hydroxy-1-[4-( 2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959 or Darocur 2959), camphorquinone, acetophenone, acetophenone benzyl ketal, 1-hydroxycyclohexyl phenyl ketone , 2,2-dimethoxy-2-phenylacetophenone, xanthone, fluorenone, benzaldehyde, fluorene, anthraquinone, triphenylamine, carbazole, 3-methylacetophenone, 4-chlorobenzophenone, 4, 4'-dimethoxybenzophenone, 4,4'-diaminobenzophenone, benzoin propyl ether, benzoin ethyl ether, benzyl dimethyl ketal, 1-(4-isopropylphenyl)-2-hydroxy-2 -Methylpropan-1-one, 2-hydroxy-2-methyl-1-phenylpropan-1-one, thioxanthone, ethylthioxanthone, 2-isopropylthioxanthone, 2-chlorothiothioxanthone , 2-methyl-1-[4-(methylthio)phenyl]-2-morpholino-propan-1-one, 4,6-trimethylbenzoyl diphenylphosphine oxide and bis-(2,6-dime Contains (toxybenzoyl)-2,4,4-trimethylpentylphosphine oxide.

As described in the examples below, Irgacure 2959 was used in a specific example of the present invention, which is known to be an initiator with very low in vivo toxicity (Kristi S. Anseth, et al., Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro. J. Biomater. Sci. Polymer Edn., 2000. 11(5):P.439-457).

In one embodiment, the temperature sensitive polymer may be pluronic. The temperature sensitive polymer is most preferably a polymer represented by the following formula (1).

<Formula 1>

(PC1) - (PE) _x (PPO) _y (PE) _z - (PC2)

In Formula 1, PE represents ethylene oxide, PPO represents propylene oxide, PC1 and PC2 represent functional groups capable of photocrosslinking, and x, y, and z are each independently integers from 1 to 10,000.

In one embodiment, the nano-carrier may contain proteins, nucleic acid molecules, nanoparticles, or fluorescent substances therein as the transport target material.

Substances that can be transported by the sustained-release drug delivery vehicle of the present invention are not limited and include various substances that exhibit therapeutic efficacy. According to a preferred embodiment of the present invention, the material to be transported is a protein, nucleic acid molecule, nanoparticle, or fluorescent substance.

The protein transported by the drug delivery system of the present invention is not particularly limited and includes hormones, hormone analogs, enzymes, enzyme inhibitors, signal transduction proteins or parts thereof, antibodies or parts thereof, single chain antibodies, binding proteins or their binding domains, and antigens. , attachment proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulatory factors, blood coagulation factors, and vaccines, etc., but are not limited thereto. More specifically, the proteins carried by the drug delivery system of the present invention include insulin, IGF-1 (insulin-like growth factor 1), growth hormone, erythropoietin, G-CSFs (granulocyte-colony stimulating factors), GM-CSFs (granulocyte/macrophage-colony stimulating factors), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, EGFs (epidermal growth factors) ), calcitonin, VEGF (vascular endothelial cell growth factor), FGF (fibroblast growth factor), PDGF (platelet-derived growth factor), ACTH (adrenocorticotropic hormone), TGF-β (transforming growth factor beta), BMP ( bone morphogenetic protein), TNF (tumor necrosis factor), atobisban, buserelin, cetrorelix, deslorelin, desmopressin, dino Dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, growth hormone releasing hormone-II (GHRH-II), gonadorelin ), goserelin, histrelin, leuprorelin, lypressin, octreotide, oxytocin, pitressin, secretin ), sincalide, terlipressin, thymopentin, thymosine α1, triptorelin, bivalirudin, carbetocin , cyclosporine, exedine, lanreotide, LHRH (luteinizing hormone-releasing hormone), nafarelin, parathyroid hormone, pramlintide, T-20 (enfuvirtide), Thai Including, but not limited to, thymalfasin and ziconotide.

Nucleic acid molecules that can be transported by the sustained-release drug delivery system of the present invention include, but are not limited to, for example, DNA, DNA aptamers, RNA aptamers, antisense oligonucleotides, siRNA, shRNA, plasmids, and vectors. .

Nanoparticles that can be transported by the sustained-release drug delivery system of the present invention include, for example, gold nanoparticles, silver nanoparticles, iron nanoparticles, transition metal nanoparticles, and metal oxide nanoparticles (e.g., ferrite nanoparticles). However, it is not limited to this. For example, when the drug delivery system of the present invention carries ferrite nanoparticles, it can be used as a magnetic resonance (MR) contrast agent (imaging agent).

When transporting a fluorescent substance using the sustained-release drug delivery vehicle of the present invention, the fluorescent substance is preferably bound to the carrier. For example, a fluorescent substance can be used by binding it to a protein or metal nanoparticle (eg, magnetic nanoparticle). Examples of the fluorescent substances include fluorescein and its derivatives, rhodamine and its derivatives, lucifer yellow, B-phytoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4' -Isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyatophenyl)-4-methylcoumarin, succinimidyl-pyrenebutyrate, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivative, LC™-Red 640, LC™-Red 705, Cy5, Cy5.5, lissamine, isothiocyanate nate, erythrosine isothiocyanate, diethylenetriamine pentaacetate, 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidinyl- 6-naphthalenesulfonate, 3-phenyl-7-isocyanatocoumarin, 9-isothiocyanatoacridine, acridine orange, N-(p-(2-benzoxazoylyl)phenyl)melimide , benzoxadiazole, stilbene, and pyrene.

One of the biggest features of the present invention is that in the process of encapsulating the material to be transported in the nanocarrier, simply mixing the two materials results in natural encapsulation (spontaneous encapsulation). In other words, if an event is provided where the nanocarrier and the material to be transported can contact each other without any additional processing, the material to be transported is naturally encapsulated in the nanocarrier.

According to a preferred embodiment of the present invention, when trapping a drug in a nanocarrier, an organic dispersion phase is not used and the drug is entrapped in an aqueous dispersion phase.

According to a preferred embodiment of the present invention, the encapsulating step is carried out at a temperature of 0-20°C, more preferably 4-10°C, and most preferably 4-6°C. Natural entrapment in aqueous solution by the sustained-release drug delivery vehicle of the present invention has the advantage of greatly increasing the stability of the encapsulated drug, especially protein drugs. Although the drug is encapsulated in the drug delivery system of the present invention by natural entrapment, the encapsulation efficiency is very high at over 90%. In addition, since organic solvents are not used in the drug loading process and high-speed homogenization or ultrasonic treatment are not required, the method of the present invention can avoid denaturation or aggregation of the encapsulated drug.

According to another aspect of the invention,

(a) preparing a chitosan functionalized temperature-sensitive nanogel;

(b) cooling the temperature-sensitive nanogel obtained in step (a) to form a solution; and

(c) to the solutionized temperature-sensitive nanogel obtained in step (b),

Purine nucleotides and

A method for producing a retina-targeted nanocarrier is provided, comprising mixing one or more from the group consisting of variants containing the structure of the purine-based nucleotide.

In one embodiment, the purine-based nucleotide may be one or more selected from the group consisting of adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), and uridine diphosphate (UDP).

In one embodiment, the variant containing the purine-based nucleotide structure may be one or more selected from the group consisting of UDP-glucose and Ap4A (P1,P4-Di(adenosine-5')tetraphosphate).

In one embodiment, the chitosan-functionalized temperature-sensitive nanogel of step (a) is,

It can be prepared by adding a photoinitiator to a solution in which chitosan and a temperature-sensitive polymer component having a photo-crosslinkable functional group are dispersed and curing the solution.

In one embodiment, the temperature sensitive polymer may be pluronic.

In one embodiment, preparing a mixture by mixing the nanocarrier obtained in step (c) with a material to be transported; And it may additionally include the step of incubating the mixture to encapsulate the material to be transported inside the nanocarrier.

In one embodiment, the material to be transported may be a protein, nucleic acid molecule, nanoparticle, or fluorescent substance.

The present invention can target and deliver a target substance to the retina through a drug carrier distributed on the surface of a nucleotide capable of specifically binding to the P2Y2 receptor present in the retina. In addition, by using a temperature-sensitive polymer, not only can transport target substances such as proteins be encapsulated with high efficiency, but when the nanogel is dissolved at a high concentration, the viscosity can be increased, allowing sustained release of the drug while remaining within the eye for a long time. It can be delivered.

Figure 1 shows the preparation of nucleotide-binding nanocarriers (NC/NTP) through ionic interaction.
Figure 2 shows a retina-related disease targeting strategy using NC/NTP.
Figure 3 graphically shows the biocompatibility of nanocarriers and nanocarrier/nucleotide systems by WST testing.
Figure 4 shows a staining photograph of the rearrangement of cell layers after treatment. (a) AO/PI staining, (b) F-actin staining with phalloidin.
Figure 5 is a graph showing the distribution of in vitro nanocarriers and nanocarriers/nucleotides and the cumulative amount in the retina area in pig eyes. (n=3)
Figure 6 shows (A) retention amount of nanocarrier and nanocarrier/nucleotide in the body, (B) penetration amount of Cy5.5-NTP and Cy5.5-NC/NTP, (C) serum 90 minutes after administration of model drug FITC-BSA. The amount of penetration is shown graphically.
Figure 7 shows the penetration into the retina of cy5.5-NC and cy5.5 NC/NTP loaded with FITC-BSA 6 hours after nanocarrier administration. (Scale bar = 50 μm). ILN, inner nuclear layer; ONL, Outer nuclear layer; and RPE, retinal pigment epithelium. (n=3)
Figure 8 is a photograph of ZO-1 staining after treatment with nanocarrier or nanocarrier/nucleotide in the retina. (Scale bar: 20 μm)

Hereinafter, the present invention will be described in detail.

The present inventor neutralizes the charge of the cationic nanocarrier to prevent the nanocarrier from being disturbed by the vitreous humor, and to induce recombination of the tight junction of RPE to make protein transport more effective, ATP is converted to anionic nucleotide (negatively). charged nucleotide: NTP) and used as an agonist for the P2Y2 receptor, ATP and nanocarrier were mixed, and the gel-like mixture was injected into the vitreous humor (Figure 1). This nanocarrier/nucleotide system quickly reaches the retinal layer and selectively delivers the protein encapsulated in the nanocarrier across the RPE layer (Figure 2).

That is, the present invention provides a chitosan-functionalized temperature-sensitive nanogel and a chitosan-functionalized temperature-sensitive nanogel; and a retina-targeted nanocarrier comprising one or more from the group consisting of purine-based nucleotides and variants containing the structure of the purine-based nucleotide, wherein the purine-based nucleotides include ATP (adenosine triphosphate), UTP (uridine triphosphate), and ADP. (adenosine diphosphate) and UDP (uridine diphosphate), and variants containing the structure of the purine nucleotide include UDP-glucose and Ap4A (P1,P4-Di(adenosine-5')tetraphosphate) Provided is one or more retina-targeted nanocarriers selected from the group consisting of and a method for producing the same.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Example]

1. Preparation steps of chitosan-functionalized pluronic-based nanocarrier (NC) and nanocarrier/nucleotide complex (NC/NTP)

Chitosan-functionalized pluronic-based nanocarrier (NC) was prepared by UV photo-crosslinking of diluted diacrylated pluronic (DA-PF 127) and glycidyl methacrylated-chitosan (GMA-Chitosan) with photoinitiator Irgacure 2959 (photo -crosslinking). Diacrylated pluronic was prepared by reacting pluronic 127 (PF 127) with acrylyl chloride (98% based on NMR), and GMA-chitosan ( 15% based on NMR) was prepared.

Ultraviolet irradiation using Irgacure 2959 (0.05 wt%) was performed on 0.77 wt% of DA-PF 127 and 0.14 wt% of GMA-chitosan for 15 minutes. Through the ninhydrin reaction, the total amount of chitosan contained in GMA-chitosan was measured to be 15.38 wt%. The nanocarrier solution was mixed with ATP or GTP at 4°C to obtain a nanocarrier/nucleotide complex (NC/NTP) (0.5 mg of nucleotide per 1 g of nanocarrier in 100 μl Deionized Water).

2. Cytotoxicity of nanocarriers and nanocarriers/nucleotides

ARPE19 (human retinal pigment epithelial fluid) cells were cultured in a 96-well plate (number of cells per well: 5 × 10 ³ ) overnight in a cell incubator at 37°C. Afterwards, the culture medium (DMEM) from each well was removed and replaced with fresh medium containing various concentrations (0, 12.5, 25, 50 μg/mL) of nanocarriers and nanocarriers/nucleotides. After 24 hours, the culture supernatant medium was removed, and fresh medium containing 10% WST was added to each well. Afterwards, the plate was incubated for an additional 2 hours at 37°C. Finally, the absorbance at 570 nm was measured using a microplate reader. All experiments were conducted three times.

3. Effect of rearrangement of purinergic nucleotides on retinal cell layer

ARPE19 cells were cultured in a 12-well plate (number of cells per well: 10 × 10 ⁴ ) for 3 weeks and succeeded in creating a dense monolayer similar to the retinal pigment epithelial layer. Afterwards, ATP solution (pH 7.4, 100 μl, 10 mM) was injected into the well and left for 30 minutes. Next, 1 μl of 0.5 mg/ml acridine orange/propidium iodide (AO/PI) reagent was added to each well along with fresh medium and incubated at room temperature for 10 minutes. Afterwards, the results were analyzed using fluorescence microscopy (AO: ex 460 nm, em 650 nm, PI: ex 525 nm, em 595). After the experiment, cell rearrangement of ARPE was analyzed through phalloidin staining using TRITC-labeled phalloidin (ex: 540-545, em: 570-573).

4. In vitro distribution of nanocarriers and nanocarriers/nucleotides in pig eyes

Fresh pig eyes were purchased from a slaughterhouse and used for in vitro experiments. As previously reported, the fluorescent dye cy5.5 was combined into a nanocarrier (Cy5.5-NC) by reacting the nanocarrier with the amine group of Cy5.5-NHS. Nanocarrier and nanocarrier/nucleotide solution (100 μl) consisting of Cy5.5 labeled nanocarrier (12 mg) and 100 mM ATP as an exothermic reagent and GTP as a non-thermogenic reagent were instilled in the pig eye using a 31 gauge syringe. It was injected into the vitreous humor. After injection, porcine eye samples in PBS were cultured in a humidified cell incubator at 37°C for 24 hours. Afterwards, an incision was made in the middle part of the eye to observe the cross section of the eye. The distribution of Cy5.5-NC within the eye was observed using an in-vivo diagnostic imaging device (FOBI, Korea).

5. Maintenance of encapsulation rate of nanocarriers and proteins in the eye and penetration of encapsulated proteins into mouse blood vessels through RPE in vivo Experiment

BSA was labeled with FITC (FITC-BSA) for penetration analysis or RITC (RITC-BSA) for serum level analysis, and isocyanate was used for analysis using RITC (Research Institute of Tuberculosis and Cancer) or FITC (fluorescent antibody). This was achieved by reacting with technique. Briefly, 2 mg/mL of BSA was prepared in 0.1 M sodium carbonate (pH 9). 100 μl of fresh FITC solution (1 mg/mL) was slowly added to the BSA protein solution. The next day, BSA was purified using a PD-10 column using PBS buffer as a buffer solution.

FITC-BSA was encapsulated in cy5.5-labeled NC and NC/NTP nucleotides using the temperature-dependent size change of the pluronic nanocarrier. Briefly, FITC-BSA (10 μg) was added to cy5.5-NC (1 mg) and ct5.5-NC/NTP (1 mg, 0.5 mg nucleotides, 0.1 mL deionized water) and the mixture was incubated at 4°C. Incubated for 2 hours on a rotary shaker. The temperature of the mixed solution was increased to 37°C, and FITC-BSA was encapsulated by size change for 20 minutes.

Afterwards, unencapsulated FITC-BSA (4000 rpm, 10 minutes, 2 times) was removed by centrifugation using a nanocentrifuge (MWCO 300kDa). The FITC-BSA encapsulation ratio of cy5.5-nanocarrier and cy5.5-nanocarrier/nucleotide was measured by absorbance at 490 nm. For in vivo experiments, 8-week-old BALB/C male mice from Orient Bio Co., Ltd. (Seongnam, Korea) were used.

All experiments were conducted in accordance with the guidelines of the Gwangju Institute of Science and Technology (GIST) Animal Care and Use Committee (GIST-2018-068). Rats were randomly distributed into 6 groups with 3 rats per group (n=3). They were completely anesthetized with isoflurane and their corneas were dilated with phenylephrine and atropine. Approximately 2 μl of the nanocarrier solution was injected into the vitreous humor of the eye using a 31G syringe, and the rats in each group were sacrificed after 90 minutes of injection. The eye was removed and fixed in 4% formalin solution for 1 hour. The fixed eye was embedded in OCT (Optimal Cutting Temperature) compound, frozen at -30°C, and cryosections were obtained at 5 μm thickness. Segmented eye samples were stained with DAPI to identify retinal structures. Then, scanning microscopy was performed to determine the distribution of cy5.5-NC, cy5.5-NC/NTP, and FITC-BSA in the retinal area.

6. In vivo Transmission inspection

After administering cy5.5-nanocarrier and cy5.5-nanocarrier/nucleotide, blood was collected from the treated rat's eye (Retro-Orbital plexus) for 90 minutes. The collected blood was centrifuged at 4°C and 3000 rpm for 5 minutes to obtain serum. The fluorescence of serum was measured by a microplate reader (Thermo fisher).

7. Biodistribution of nanocarriers and nanocarriers/nucleotides in mouse eyes

Cy5.5 conjugated nanocarriers and NC/NTP were injected into the vitreous humor of BALB/C rats using a 31G syringe. Six hours after injection, the mice were sacrificed and the eyes were removed. The isolated eye was embedded in optimal OCT, frozen at -20°C, and cryosectioned at 6 μm thickness to determine the distribution of cy5.5-NC, cy5.5-NC/NTP, and FITC-BSA in the retina area. Scanning microscopy was performed for the purpose.

8. Increased immunity of retinal tissue

Tight junction fluorescent antibody analysis was performed on the retinal pigment epithelium (RPE). Eyes injected with BSA-nanocarriers were opened by a circumferential incision in the surgical limbus. The prefrontal cortex and lens were removed. The posterior segment was placed on filter paper and fixed with 4% formalin solution for 2 hours. After fixation, it was possible to separate the posterior segment from the filter paper while maintaining a flat structure. These flat structures were stained with the primary antibody of ZO-1 diluted at 1% in BSA blocking solution (1:100). Stained sections were visualized with Alexa 633 secondary antibody and observed under a fluorescence microscope.

9. Results

1) Biocompatibility of chitosan functionalization and rearrangement of pluronic-based nanocarriers (NCs) on retinal pigmented epithelial cells

The biocompatibility of chitosan functionalization was evaluated at different concentrations (0, 12.5, 25, and 50 μg/mL) in ARPE 19, human retinal pigment epithelial layer. There was no significant difference between the nanocarrier treated group and the control group in terms of cell proliferation detection by WST test. Nucleotides (NTPs) are biocompatible substances, but extracellular nucleotides can be cytotoxic in vitro and in vitro. Additionally, extracellular adenine nucleotides are directly linked to growth inhibitors of several mammalian cells, such as breast cancer cells, skin cancer cells, pancreatic cancer cells, and leukemia cells. Therefore, confirmation of appropriate ATP concentrations in retina-related cell lines is required.

To apply the nanocarrier system, the concentration of ATP was determined to be 10 mM and ARPE19 cells were treated with ATP. After 30 minutes, ARPE19 cells were stained with AO/PI. In this state, no toxicity occurred in ARPE19 cells, but rearrangement of the retinal cell layer was confirmed (Figure 4). Interestingly, nucleotides are active ingredients in tears, aqueous humor, inner eye, and vitreous humor. These nucleotides affect not only the function of eye structures but also pharmacological treatments for dry eye, glaucoma, and retinal detachment.

2) Distribution of nanocarriers and nanocarriers/nucleotides in pig eyes.

The pig eye is the in vitro animal model often used in vision science research because it most closely resembles the human eye. To determine the distribution of nanocarriers/nucleotides throughout the eye, nanocarriers/nucleotides were injected into the vitreous humor (Figure 5). After incubation at 37°C for 24 hours, the eyes were dissected by cutting the cornea, glass, retina, and choroid/sclera horizontally. The fluorescence signals of cy5.5-NC and cy5.5-NC/NTP were measured using fluorescence imaging equipment (FOBI). The results in Figure 6 show that the nanocarrier/ATP is a nonpyrogenic nucleotide that accumulated the most fluorescence signal in the retina area compared to NC and NC/GTP when administered 24 hours later.

3) Retention of nanocarriers and nanocarriers/nucleotides and penetration rate of nanocarriers and protein drugs

After administering the nanocarrier/nucleotide to the mouse eye, the fluorescence of the cy5.5-labeled nanocarrier was detected by fluorescence imaging equipment (FOBI) over time. More than 38% of the nanocarrier remained in the eye for 90 minutes after injection. However, the nucleotide mixed nanocarrier/nucleotide case was only about 28% (NC/ATP) and 45% (NC/GTP), which was consistent with the results in Figure 6 (A). In the vitreous humor, nanocarriers can penetrate into the choroid/sclera by rearranging the retinal pigment epithelial cell layer with the P2Y2 receptor, which means that the choroid flows out into the blood more quickly.

To measure the serum concentration of nanocarrier and BSA as a protein model, FITC-BSA@cy5.5 labeled nanocarrier/nucleotide was administered into the vitreous humor and serum was collected 6 hours later. In Figure 6 (B) and (C), the nanocarrier/ATP nanocarrier had the highest fluorescence for both cy5.5-nanocarrier and FITC-BSA. Through this, it was found that the nanocarrier/ATP nanocarrier can quickly reach the retina from the vitreous humor due to the neutralizing surface effect and penetrate under the RPE layer by the P2Y2 receptor.

4) Distribution of nanocarriers and nanocarriers/nucleotides in the retina

Figure 7 shows the retinal tissue distribution of nanocarriers and nanocarriers/nucleotides 6 hours after injection. Cy5.5-labeled nanocarriers (red), BSA were stained with FITC (green), and retinal cell nuclei were stained with DAPI (blue). Six hours after NC and NC/NTP were injected into the eye, a red signal was detected throughout the retina only from the NC/NTP in the retinal layer, indicating that the actually administered NC/NTP had diffused well through the vitreous humor tube and reached the retinal layer. it means. The vitreous humor is composed of anionic proteoglycan and collagen fibers, which can limit the movement of cations after vitreous humor injection. Therefore, this means that the NC/NTP system can penetrate well because the systemic nanocarrier system has been neutralized with nucleotides.

In particular, NC/ATP, the pyrogenic nucleotide case, remains as a detectable red fluorescence in deeper retinal layers, resulting in relatively greater penetration through the P2Y2 receptor response. Supporting information 2 shows FITC-BSA as modeling medicine spreads further into the choroidal/scleral tissue region, below the RPE.

5) Tight junction whole mount after nanocarrier and nanocarrier/nucleotide treatment

Immunocytochemical analysis was performed on rat retinal tissue to detect the tight junction between ZO-1 and RPE (Figure 8). 24 hours after injection of nanocarrier and nanocarrier/nucleotide in the rat eye, eyes were collected, the entire eye was dissected, and stained as a flat mount. As can be observed in Figure 8, while ZO-1 labeling appeared in whole-mount retinal tissue, the applied NC/ATP interfered with ZO-1 labeling. However, NC and NC/GTP did not. It was found that NC/ATP, or exothermic nucleotide, reacting with the P2Y2 receptor induces RPE layer rearrangement.

10. Conclusion

In the present invention, it was demonstrated that a nanocarrier system mixed with nucleotides diffuses rapidly through the vitreous humor and binds to the P2Y2 receptor of the retinal pigment epithelium (RPE), leading to drug penetration under the RPE layer after in vivo injection. In the RPE layer, in the case of retinal diseases, the choroid, which is composed of blood vessels, may be the main site associated with abnormal blood vessels, so this drug delivery nanocarrier system with biocompatible nucleotides has great potential for treating retinal inflammatory disorders and retinal degeneration.

Claims (9)

A temperature-sensitive polymer in which glycidyl methacrylated chitosan is photocrosslinked to diacrylated pluronic; and a nucleotide that binds to the P2Y2 receptor,
The nucleotide is one or more selected from the group consisting of ATP (adenosine triphosphate), UTP (uridine triphosphate), ADP (adenosine diphosphate), and UDP (uridine diphosphate), or UDP-glucose and Ap4A (P1,P4-Di(adenosine- 5') tetraphosphate) and one or more nucleotide variants selected from the group consisting of
A retina-targeted carrier, wherein the chitosan exhibits a positive charge, the nucleotides exhibit a negative charge, and the nucleotides are distributed on the surface of the temperature-sensitive polymer by ionic interaction of positive and negative charges.
delete delete The retina-targeted carrier according to claim 1, wherein the carrier contains a protein, nucleic acid molecule, nanoparticle, or fluorescent substance as a target material.
(a) photo-crosslinking glycidyl methacrylated chitosan to diacrylated pluronic to prepare a temperature-sensitive polymer;
(b) cooling the temperature-sensitive polymer obtained in step (a) to form a solution; and
(c) mixing a nucleotide that binds to the P2Y2 receptor with the solutionized temperature-sensitive polymer obtained in step (b), comprising:
The nucleotide is one or more selected from the group consisting of ATP (adenosine triphosphate), UTP (uridine triphosphate), ADP (adenosine diphosphate), and UDP (uridine diphosphate), or UDP-glucose and Ap4A (P1,P4-Di(adenosine- A method for producing a retina-targeted carrier, comprising one or more nucleotide variants selected from the group consisting of 5') tetraphosphate).
The method of claim 5, wherein the temperature sensitive polymer of step (a) is,
A method for producing a retina-targeted carrier, characterized in that it is manufactured by adding a photoinitiator to a solution in which chitosan and pluronic having a photo-crosslinkable functional group are dispersed and curing the solution.
delete The method of claim 5, further comprising: preparing a mixture by mixing the material to be transported with the carrier; And a method for producing a retina-targeted carrier, characterized in that it additionally comprises the step of incubating the mixture to encapsulate the carrier material inside the carrier.
The method of claim 8, wherein the delivery target material is a protein, nucleic acid molecule, nanoparticle, or fluorescent substance.