Porous silica nanoparticles and peptide delivery method using the same.

Abstract

The present invention aims to provide a porous silica nanoparticle composite capable of continuously delivering an active substance to the vitreous humor within the retina. The porous silica nanoparticle composite is delivered to the anionic vitreous humor within the retina, and the active substance loaded inside the nanoparticles can be released under oxidative stress. Specifically, it can continuously release humanin peptides in the vitreous humor within the retina, and accordingly, it can have a therapeutic effect on neovascular retinal disease in an in vivo oxygen-induced retinopathy mouse model.

Description

Technology Field

[0001] The present invention relates to porous silica nanoparticles and a pharmaceutical composition for treating eye diseases containing the same. Background Technology

[0002] Neovascular retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinal vein occlusion (RVO), are known to be serious conditions that can lead to visual impairment and blindness. Age-related macular degeneration is a major cause of vision loss in the elderly and statistically accounts for approximately 9% of all causes of blindness worldwide. Meanwhile, diabetic retinopathy affects diabetic patients and can occur at any age, with the risk increasing with the duration of diabetes and aging. The underlying cause of these neovascular retinal diseases is known to be the overproduction of reactive oxygen species (ROS) in the eye. This leads to chronic oxidative stress, which damages mitochondrial DNA and creates a vicious cycle of ROS and organelle dysregulation, ultimately resulting in apoptosis. This overproduction of ROS is also caused by various cytoplasmic enzymes, which induces oxidative damage to macromolecules, inflammation, and abnormal vascular growth in the retina.

[0003] To treat the aforementioned neovascular retinal disease, conventional methods have utilized protein therapies targeting vascular endothelial growth factor (VEGF), such as the injection of monoclonal antibodies like ranibizumab or aflibercept. However, these treatment methods require high costs because they necessitate multiple injections over a long period. Furthermore, while the aforementioned conventional treatments can alleviate the patient's symptoms, they cannot fundamentally cure the disease, which presents a problem in that efficacy can vary significantly from patient to patient.

[0004] To overcome these limitations, humanin peptides (HN) are being actively researched. Humanin peptides are mitochondrial peptides composed of 24 amino acids found in the occipital lobes of Alzheimer's disease patients, and their protective effects against various mechanisms such as oxidative stress, endoplasmic reticulum stress, serum starvation, and hypoxia have been demonstrated. Humanin peptides bind to Bax, a pro-apoptotic protein, to activate the STAT-3 pathway and reduce apoptosis induced by caspases 3 and 7. In ocular tissues, humanin peptides protect neurons and may have an alleviating effect on age-related macular degeneration (AMD) by activating chaperone-mediated autophagy (CMA), which can be reduced by continuous oxidative stress in retinal pigment epithelial (RPE) cells. In this case, humanin peptides directly activate chaperone-mediated autophagy by promoting substrate binding and translocation to lysosomes, thereby reversing the pathology of age-related macular degeneration. As such, the multifaceted mechanism of humanin peptide is gaining attention as a very promising treatment for neovascular retinal disease, for which there is currently no cure.

[0005] However, despite these pathological effects, successfully delivering drugs containing humanin peptides to the eye remains a challenging task. Local administration of peptides is less effective due to the instability of the peptides and the barrier effects of the cornea and conjunctiva, while systemic administration is also limited in efficacy due to the blood-retinal barrier (BRB). Consequently, intravitreal injection is currently used, but this method presents the problem of requiring multiple injections to maintain drug activity and carries associated side effects. Furthermore, although various long-term ocular drug delivery systems, such as polymer nanocarriers, hydrogels, and implants, have been developed to date, they cannot adjust drug release according to the patient's condition, which may result in limited therapeutic effects or unwanted side effects. Accordingly, new methods for delivering humanin peptides are required. The problem to be solved

[0006] The object of the present invention is to provide a porous silica nanoparticle composite capable of continuously delivering an active substance to the vitreous humor within the retina.

[0007] Specifically, the objective of the present invention is to provide a porous silica nanoparticle composite capable of controlling the drug release rate of humanin peptide.

[0008] The objective of the present invention is to provide a pharmaceutical composition for treating ocular diseases that can treat and prevent neovascular retinal diseases by inhibiting apoptosis. means of solving the problem

[0009] The porous silica nanoparticle composite of the present disclosure may comprise: porous silica nanoparticles; arginine bonded to the surface of the nanoparticles; an active oxygen degrading linker having one end bonded to the surface of the nanoparticles; a water-soluble polymer bonded to the other end of the active oxygen degrading linker; and an active substance supported inside the nanoparticles.

[0010] The bonding area of ​​arginine and water-soluble polymer on the surface of the nanoparticles may be 1:1 to 1:10.

[0011] The average pore size of the above nanoparticles may be 1 to 10 nm.

[0012] The pore volume of the above nanoparticles is 0.1 to 4 cm 3 It can be / g.

[0013] The above nanoparticles may have a positive surface charge.

[0014] The above nanoparticles may have a negative surface charge under active oxygen conditions.

[0015] The above-mentioned water-soluble polymer is one or more selected from polyethylene glycol, acrylic acid-based polymers, methacrylic acid-based polymers, polyamidoamines, poly(2-hydroxypropyl methacrylamide)-based polymers, water-soluble polypeptides, water-soluble polysaccharides, and polyglycerols, forming a porous silica nanoparticle composite.

[0016] The molecular weight of the above water-soluble polymer may be 1,000 to 10,000 g / mol.

[0017] The above active oxygen degrading linker may be any one selected from thio-ether, selenium, thioketal, aminoacrylate, boronic acid-ester, peroxalate-ester, and polyproline.

[0018] The above active substance may be a humanin peptide.

[0019] The above humanin peptide may be loaded at a concentration of 200 to 1000 mg / g.

[0020] Humanin peptides may be released as the above-mentioned reactive oxygen species-degrading link is cleaved.

[0021] The above porous silica nanoparticle composite may be used for the treatment of one or more of age-related macular degeneration, neovascular retinal disease, diabetic retinopathy, and retinal vein occlusion.

[0022] The present disclosure may provide a method for delivering humanin peptides comprising the steps of: injecting a porous silica nanoparticle composite according to the present disclosure into a retina; fixing the composite to the vitreous humor within the retina; cleaving a degradable link within the composite under reactive oxygen species so that the surface charge of the composite is reversed to a negative charge; and releasing a humanin peptide due to the reversed charge.

[0023] The pharmaceutical composition for treating ocular diseases of the present disclosure may comprise a porous silica nanoparticle composite according to one embodiment of the present disclosure.

[0024] The above pharmaceutical composition may be for ophthalmic administration. Effects of the invention

[0025] A porous silica nanoparticle composite according to one embodiment of the present invention is delivered to the vitreous humor within the anionic retina, and an active substance supported inside the nanoparticles can be released under oxidative stress.

[0026] A porous silica nanoparticle composite according to one embodiment of the present invention can continuously release humanin peptides in the vitreous humor within the retina, and accordingly, can have a therapeutic effect on neovascular retinal disease in an in vivo oxygen-induced retinopathy mouse model. Brief explanation of the drawing

[0027] Figure 1 shows the results of the C1 peak analysis of Ar-MSNs-TK-PEG and Ar-MSNs-TK measured by X-ray photoelectron spectroscopy. Figure 2 shows the results of cytotoxicity analysis of Ar-MSNs-TK-PEG and Ar-MSNs-TK according to treatment concentrations. Figure 3 shows the results of measuring the zeta potential of MSNs-TK and MSNs-TK-PEG after tBH treatment. Figure 4 shows the distribution of (a) Ar-MSNs-TK, (b) Ar-MSNs-TK-PEG, and (c) Ar-MSNs-TK-PEG after tBH treatment and injection into the small vitreous lumen. Figure 5 shows the appearance of Arpe-19 cells treated with MSNs-TK-PEG before and after co-culture with tBH (80 μM), measured by confocal immunofluorescence microscopy. Figure 6 shows the cumulative release results of humanin peptides from Ar-MSNs-TK and Ar-MSNs-TK-PEG under tBH treatment at various concentrations. Figure 7 shows the release of humanin peptide from Arpe-19 cells per culture time under tBH (110 μM) treatment, measured by confocal immunofluorescence microscopy. Figure 8 shows the co-localization of Ar-MSNs-TK-PEG-rhodamin (green) and HN-FITC b (red) as average Pearson's r coefficient, Mander's M1 coefficient, and M2 (n = 9) over time. Figure 9 shows the average percentage of apoptotic cells (TUNEL positive) calculated under tBH treatment. Figure 10 shows the average percentage of apoptotic cells (activated caspase-3) calculated under tBH treatment. Figure 11 shows the Western blot results of Ar-MSNs-TK-PEG and Ar-MSNs-TK-PEG loaded with humanin peptide. Figure 12 shows the neovascularization inhibitory effect of humanin peptide alone or AR-MSNs-TK-PEG loaded with humanin peptide on the retina. Figure 13 shows the uptake of Ar-MSNs-TK-PEG in Arpe-19 cells per various culture times, measured by confocal immunofluorescence microscopy. Specific details for implementing the invention

[0028] The present invention will be described in detail below. Unless otherwise defined, terms used in this specification should be interpreted as generally understood by those skilled in the art. The drawings and embodiments of this specification are intended to enable those skilled in the art to easily understand and practice the present invention; therefore, details that may obscure the essence of the invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments.

[0029] The singular form used in this specification may be intended to include the plural form unless specifically indicated otherwise in the context.

[0030] Furthermore, the numerical range used in this invention includes lower and upper limits and all values ​​within the range, increments logically derived from the form and width of the defined range, all of the specified values, and all possible combinations of upper and lower limits of the numerical range defined in different forms. Unless otherwise specifically defined in the specification of this invention, values ​​outside the numerical range that may occur due to experimental error or rounding are also included in the defined numerical range.

[0031] In this specification, terms such as "include," "have," and "have" mean that the features or components described in the specification are present, and unless specifically limited, this does not preclude the possibility that one or more other features or components may be added.

[0032] The present disclosure provides a porous silica nanoparticle composite that is delivered to the anionic vitreous humor within the retina and can continuously release an active substance supported inside the nanoparticles under oxidative stress.

[0033] A porous silica nanoparticle composite according to one embodiment of the present disclosure may comprise porous silica nanoparticles; arginine bonded to the surface of the nanoparticles; an active oxygen degrading linker having one end bonded to the surface of the nanoparticles; a water-soluble polymer bonded to the other end of the active oxygen degrading linker; and an active material supported inside the nanoparticles.

[0034] Conventionally, methods involving the injection of active substances such as ranibizumam or aflibercept via injection have been used for intravitreal drug administration. However, such methods have limitations in that the drug release rate cannot be controlled according to the patient's condition and multiple drug injections are required to achieve a therapeutic effect. Consequently, excessive treatment costs are incurred for patients, and the therapeutic effect is also limited due to the immediate drug release following injection. The porous silica nanoparticle composite of the present disclosure, due to the aforementioned composition, can maximize the therapeutic effect through the delayed release of the drug embedded within it depending on specific conditions, and can achieve a long-term therapeutic effect with fewer injections than conventional methods.

[0035] The porous silica nanoparticle composite of the present disclosure has a partially positive surface charge due to the binding of arginine, a peptide that carries a positive charge on the surface. Additionally, as a water-soluble polymer is bound to the reactive oxygen species degrading linker, the negative charge of the degrading linker is neutralized, and consequently, the porous silica nanoparticle composite acquires a positive surface charge. Accordingly, the porous silica nanoparticles can successfully aggregate within a negatively charged vitreous network.

[0036] In addition, as the active oxygen-degrading linker is cleaved in an oxidative stress environment, the water-soluble polymer is separated from the surface, and the surface charge of the porous silica nanoparticle composite is reversed to a negative charge, causing the nanoparticles to disperse, thereby controlling the release rate of the active substance supported thereon.

[0037] The porous silica nanoparticle composite of the present disclosure can have different active substance release rates depending on the concentration of active oxygen under oxidative stress.

[0038] Specifically, the active substance may be one or more selected from the group consisting of peptide or protein drugs, antimicrobial agents, anticancer agents, and anti-inflammatory agents, but according to one embodiment, the active substance may be a humanin peptide.

[0039] According to one embodiment, the bonding area of ​​the arginine and the water-soluble polymer on the surface of the nanoparticle may be 1:1 to 1:10, specifically 1:2 to 1:8, and more specifically 1:3 to 1:5.

[0040] According to one embodiment, the average diameter of the nanoparticles may be 50 to 200 nm, specifically 60 to 160 nm, more specifically 70 to 140 nm, or 70 to 120 nm, most preferably 70 to 100 nm.

[0041] According to one embodiment, the average pore size of the nanoparticle may be 1 to 10 nm, specifically 2 to 10 nm, more specifically 3 to 10 nm, or 4 to 10 nm, most preferably 5 to 9 nm.

[0042] According to one embodiment, the pore volume of the nanoparticle is 0.1 to 4 cm. 3 / g, specifically 0.5 to 3.5 cm 3 / g, more specifically 1 to 3.0 cm 3 / g can be.

[0043] According to one embodiment, the nanoparticles may have a positive surface charge. Specifically, the nanoparticles may have a surface charge of 0.1 to 10 mV, 0.5 to 8 mV, or 1 to 5 mV, and more specifically, 1 to 3 mV.

[0044] According to one embodiment, the nanoparticles may have a negative surface charge under active oxygen conditions. Specifically, the nanoparticles may have a surface charge of -0.1 to 50 mV or -5 to 45 mV, more specifically -10 to 40 mV.

[0045] The above water-soluble polymer may be one or more selected from polyethylene glycol, acrylic acid-based polymers, methacrylic acid-based polymers, polyamidoamines, poly(2-hydroxypropyl methacrylamide)-based polymers, water-soluble polypeptides, water-soluble polysaccharides, and polyglycerols, and specifically may be polyethylene glycol, and preferably may be methoxypolyethylene glycol (mPEG).

[0046] According to one embodiment, the molecular weight of the water-soluble polymer may be 1,000 to 10,000 g / mol, specifically 2,000 to 8,000 g / mol, and more specifically 3,000 to 7,000 g / mol.

[0047] The above active oxygen decomposition linker may be any one selected from thio-ether, selenium, thioketal, aminoacrylate, boronic acid-ester, peroxalate-ester, and polyproline, and specifically may be thioketal.

[0048] According to one embodiment, the active material supported inside the porous silica nanoparticle composite of the present disclosure may be a humanin peptide.

[0049] Humanin peptide is a mitochondrial peptide composed of 24 amino acids found in the occipital lobe of Alzheimer's disease patients, and has been proven to have protective effects against various mechanisms such as oxidative stress, endoplasmic reticulum stress, serum starvation, and hypoxia. Humanin peptide binds to Bax, a protein that promotes apoptosis, to activate the STAT-3 pathway and reduces apoptosis induced by caspases 3 and 7. In ocular tissues, humanin peptide may have an effect on mitigating age-related macular degeneration (AMD) by protecting neurons and activating chaperone-mediated autophagy (CMA), which can be reduced by continuous oxidative stress in retinal pigment epithelial (RPE) cells.

[0050] The present disclosure provides a porous silica nanoparticle composite that overcomes the problems of local administration of peptides caused by the instability of conventional peptides and the barrier effects of the cornea and conjunctiva, thereby enabling long-term drug delivery and maximizing therapeutic effects.

[0051] According to one embodiment, the porous silica nanoparticle composite may be loaded with humanin peptide at a concentration of 200 to 1000 mg / g, specifically 300 to 800 mg / g, or 400 to 800 mg / g, more specifically 500 to 800 mg / g.

[0052] According to one embodiment, the porous silica nanoparticle composite may release a humanine peptide as the reactive oxygen species degrading link is cleaved. Specifically, in an oxidative stress environment, the reactive oxygen species degrading link is cleaved, and the water-soluble polymer connected to the reactive oxygen species degrading link is separated from the nanoparticle, causing a change in the surface charge of the nanoparticle. Accordingly, the nanoparticle, which was previously aggregated in a negatively charged glass body, diffuses, and the release rate of the humanine peptide supported thereon can be controlled.

[0053] The above porous silica nanoparticle composite may be used for the treatment of one or more of neovascular retinal diseases, specifically age-related macular degeneration, neovascular retinal disease, diabetic retinopathy, and retinal vein occlusion.

[0054] The present disclosure provides a method for delivering humanin peptides, comprising the steps of: injecting a porous silica nanoparticle complex into a retina; fixing the complex to the vitreous humor within the retina; cleaving a degradable link within the complex under reactive oxygen species so that the surface charge of the complex is reversed to a negative charge; and releasing a humanin peptide due to the reversed charge.

[0055] It is believed that the above-described humanin peptide delivery method can overcome the low peptide delivery efficiency and peptide instability caused by the barrier effects of the cornea and conjunctiva in the past, thereby enabling a high peptide delivery effect with fewer cycles, and can maximize the action of the peptide by controlling the release of the peptide.

[0056] The present disclosure provides a pharmaceutical composition for treating ocular diseases comprising a porous silica nanoparticle composite according to one embodiment of the present disclosure.

[0057] The pharmaceutical composition according to the present invention is not limited but may additionally include a pharmaceutically acceptable carrier. In the present invention, the term “pharmaceutically acceptable carrier” means a carrier or diluent that does not impair the biological activity and properties of the porous silica nanoparticle composite. For example, acceptable pharmaceutical carriers for a composition formulated as a liquid solution may include saline solution, sterile water, Ringer’s solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, as they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0058] The above pharmaceutical composition for treating eye diseases is administered in a pharmaceutically effective amount.

[0059] The above "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art.

[0060] The above pharmaceutical composition may be administered to an individual via various routes. The mode of administration may be, for example, taken orally or administered via various routes. It may be administered subcutaneously, ophthalmologically, intravenously, intramuscularly, intraperitoneally, intrauterinely, or intracerebrovascularly. The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease.

[0061] The above pharmaceutical composition may be in a solid or liquid form, and the method of administration may be oral, parenteral, intranasal, oral, sublingual, airway spray, or rectal, but is not limited thereto, and according to the results of one embodiment of the present invention, it may have a form of administration via injection, etc. during parenteral administration.

[0062] The formulation of the above pharmaceutical composition is not particularly limited, but may be a formulation such as a capsule, pill, tablet, solution, suspension, spray, foaming agent, patch, or paste, and according to the results of one embodiment of the present invention, it may have a form such as a tablet.

[0063] Preferably, the above pharmaceutical composition may be for ophthalmic administration.

[0064] The above ophthalmic administration includes local injection within the eye, including intravitreal injection or conjunctival injection, or administration by direct instillation into the conjunctival sac of the eye in the form of eye drops. In one embodiment, the pharmaceutical composition has the form of eye drops.

[0065] The porous silica nanoparticle composite according to the present invention will be described in more detail below through specific examples. However, the following examples are merely references for the detailed explanation of the present invention and do not limit the invention to such examples, and the invention may be implemented in various forms. Furthermore, the terms used in the description of the present invention are intended only to effectively describe specific examples and are not intended to limit the invention.

[0067] <Materials and Where to Buy>

[0068] N-cetyltrimethylammonium chloride (CTAC), triethylamine (TEA), tetraethyl orthosilicate (TEOS, 98%), decahydronaphthalene, ammonium nitrate (NH4NO3), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCl), (3-aminopropyl)triethoxysilane (APTES), hydrazine monohydrate, tert-butyl hydroperoxide, methoxypolyethylene glycol 5,000 propionic acid and Nα-acetyl-L-arginine were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification.

[0069] FITC (98%) conjugated humanin peptide (HN) and HN terminal amine were purchased from Biomatik (Cambridge, Canada).

[0070] Propane-2,2-diylbis(thio)diacetic acid (95%) was purchased from BLDpharm (China).

[0071] Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), CCK-8 assay, trypsin, fluorescein isothiocyanate (FITC), and other cell-related reagents were purchased from Invitrogen (Carlsbad, CA, USA).

[0073] <Apparatus and Analysis Method>

[0074] The mesoporous silica nanoparticles (MSNs) of the present disclosure were modified by surface functionalization, and their size and morphology were analyzed using a transmission electron microscope (TEM, Tecnai G2 F30). To achieve this, MSNs were dispersed in ethanol at a concentration of 0.2 mg / mL, and a small amount of the suspension (10 microliters) was dropped onto a carbon-coated copper grid and dried at room temperature. Additionally, the N2 adsorption-desorption isotherms of the MSNs were obtained using a Micromeritics (ASAP 2420) instrument at liquid N2 temperature (77K) after degassing at 110°C for 12 hours prior to measurement.

[0075] X-ray photoelectron spectroscopy (XPS) data and variations of MSN were acquired using an ESCALAB 250 spectrometer equipped with a monochromatic X-ray source of Al anode Kα radiation (1486.6 eV).

[0076] The binding energy was corrected to the C1s line of carbon at 284.6 eV during actual measurements. Finally, the data could be analyzed more comprehensively by using XPSPEAK41 software in the form of Gaussian-Lorentzian lines to separate individual spectral peaks.

[0077] In addition, various state-of-the-art technologies were used to comprehensively analyze the properties of mesoporous silica nanoparticles (MSNs). The pore size distribution and surface area of ​​modified MSNs were evaluated using Barrett-Joyner-Halenda (BJH) and Brunauer-Emmett-Teller (BET) methods, and the dynamic light scattering (DLS) magnitude and zeta potential values ​​were measured using a Malvern Zetasizer Nano ZS instrument. The structure of mPEGs terminated with hydrazide groups was confirmed using a Proton Nuclear Magnetic Resonance 1H NMR spectrometer (AC-80, Bruker Biospin, Germany).

[0078] UV-vis spectrophotometric measurements were performed using a Perkin Elmer Lambda 35 spectrophotometer to quantify the absorbance of the peaks, and fluorescence spectra were recorded using a JASCO FP-6300 spectrophotometer according to the principles of the Beer-Lambert law.

[0079] A confocal laser scanning microscope (CLSM, Zeiss LSM 700, Zurich, Switzerland) was used for in vitro cell imaging and in vitro analysis.

[0081] [Example 1] Preparation of porous nano-silica particles

[0082] Preparation of Mesoporous Silica Nanoparticles (MSN)

[0083] Mesoporous silica nanoparticles (MSNs) were prepared using a one-pot biphase stratification method. 6 mL of CTAC solution (25 wt%), 0.045 g of TEA, and 9 mL of water were placed in a 25 mL round-bottom flask and gently stirred at 60 °C for 1 hour. Next, 5 mL of TEOS dissolved in decahydronaphthalene (at concentrations of 10%, 20%, and 30%) was added to the solution, and the mixture was heated in an oil bath at 60 °C for 12 hours under magnetic stirring. The resulting product was collected by centrifugation, washed with ethanol to remove residual reactants, dried under vacuum, and stored at 4 °C. To prevent blocking or reducing the pore size of the mesoporous silica nanoparticles, the outer surface was modified with amine groups, and the template was removed.

[0084] Functionalization of the outer surface of MSNs using amine groups (N-MSN)

[0085] To prepare modified mesoporous silica nanoparticles (MSN), a solution of 0.5 g of MSN dissolved in 30 mL of dry toluene was sonicated for 5 minutes, followed by the addition of 1.8 g of (3-aminopropyl)triethoxysilane. The resulting mixture was refluxed under a nitrogen atmosphere for 12 hours. After centrifugation and ethanol washing, the CTAC template was removed by extraction twice with a 0.6 wt% ammonium nitrate (NH4NO3) ethanol solution at 60°C for 6 hours. Finally, the product was freeze-dried to obtain a fine powder for subsequent experiments.

[0086] Post-hoc modification of the remaining amine group in Ar-MSN to acetyl-L-arginine

[0087] N-MSN (45 mg) with an amine content of 0.626 mmol / L was suspended in 10 mL of acetonitrile (ACN) in a 20 mL glass tube and sonicated for 10 minutes. In a separate 20 mL glass tube, propane-2,2-diylbis(thio)diacetic acid (0.501 mmol / L) and acetyl-L-arginine (0.125 mmol / L) were dissolved in ACN, followed by the addition of EDC.HCl (0.235 mmol / L) and NHS (0.261 mmol / L), and the mixture was stirred for 15 minutes. Subsequently, the resulting mixture was added dropwise to the N-MSN suspension, and the pH was raised to 10. The resulting solution was stirred at room temperature under nitrogen gas for 48 hours. The product was collected by centrifugation and purified by washing three times each with acetone and water. The purified product was dried under vacuum at room temperature for 24 hours.

[0088] Preparation of α-methoxy w-hydrazide polyethylene glycol

[0089] Methoxypolyethylene glycol propionic acid with a molecular weight of 5 kDa (1.5 g) was dissolved in 50 mL of methanol while stirring for 15 minutes. Subsequently, THF was added once, followed by the addition of 15 mL of 1 M hydrazine solution (10 eq.), and the mixture was refluxed overnight under a nitrogen atmosphere. The reaction progress was monitored using TLC containing 14% MeOH in DCM as the eluent and ninhydrin as the stain. The concentrated mixture was dissolved in 50 mL of dichloromethane and stirred while washing three times with 1 M HCl solution, water, and brine. The organic layer was collected, and the product was dried with MgSO4. After concentration, the product was precipitated in diethyl ether to obtain a powder with a 95% yield. The NMR spectrum results are as follows. (400 MHz, CDCl3) δ3.7-3.5 (m, PEG backbone), 3.3 (s, 3H, CH3-O-), 2.5 (t, 2H, J = 6.0 Hz, -CH2-C(O)O-)

[0090] Preparation of Ar-MSNs-TK-PEG loaded with humanin (HN) peptide

[0091] Ar-MSNs-TK was prepared by conjugating the TK-carboxyl groups of surface-modified MSNs, using Ar and TK groups in a 1:5 ratio, with the methoxy PEG prepared above.

[0092] Specifically, Ar-MSNs-TK (5 mg) was suspended in 5 mL of water at pH 5.5 and sonicated for 10 minutes to obtain a homogeneous suspension. EDC.HCl (5 mg) and NHS (3 mg) were added to the suspension, and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was centrifuged, and the resulting pellet was washed three times with ethanol. HN peptide (7.5 mg) was dissolved in water at pH 5.5 in a separate vial and added to the MSN suspension while stirring at 4°C for 4 hours and sonicating every 15 minutes. Subsequently, hydrazide-terminated mPEG (0.02 g) was added to the suspension, and the reaction was continued with stirring for an additional 4 hours. The resulting product (HN-MSNs-TK-PEG) was purified by washing it three times with ethanol and three times with water to remove unreacted mPEG and other reagents. Finally, the product was freeze-dried for use in subsequent experiments.

[0094] To investigate the optimal loading dose of HN peptide, Ar-MSNs-TK with three different pore sizes were prepared as described in Table 1 below, and their respective characteristics were investigated. The MSN with the largest pore size was designated as Ar-MSNs-TK-L, the medium size as Ar-MSNs-TK-M, and the smallest size as Ar-MSNs-TK-S.

[0095] As shown in Table 1 below, the hydrodynamic diameters of Ar-MSNs-TK with three different pore sizes were similar with an average size of 80 ± 10 nm, and it was confirmed that Ar-MSNs-TK-S, with the smallest pore size of 2.8 nm and the highest negative surface charge (-39±4), had the highest HN peptide loading capacity of 64.4%.

[0096] This is thought to be due to the fact that small HN peptides can easily enter the small pore size of Ar-MSNs-TK-S, and due to the strong electrostatic attraction between negatively charged Ar-MSNs-TK-S and positively charged HN peptides (pKa = 10.5).

[0097] Average pore size (nm) surface area (m 2 / g) Pore ​​volume (cm 3 / g) surface charge (mV) HN Loading Capacity (%) MSN diameter (nm) Ar-MSNs-TK-S 2.8 567 0.81 -39 ± 4 64.4 80 ± 10 Ar-MSNs-TK-M 4.5 411 1.2 -32 ± 4 32 Ar-MSNs-TK-L 7.5 1035 2.5 -25 ± 4 18

[0098] In addition, to confirm whether methoxy PEG was well conjugated to the TK-carboxyl group in Ar-MSNs-TK-PEG prepared according to the above manufacturing method, the results of the C1 peak analysis of the XPS spectra of Ar-MSNs-TK-PEG and Ar-MSNs-TK are shown in Figure 1.

[0099] According to Fig. 1B, the Ar-MSNs-TK-PEG showed an increase in COC peak intensity at approximately 286 eV, which appears to be due to the ethoxy repeating unit of PEG. Furthermore, the disappearance of the OC=O peak associated with the carboxyl group at the TK molecule terminal was observed, indicating that PEG was successfully conjugated.

[0101] [Evaluation Example 1] Cell stability of Ar-MSNs-TK-PEG

[0102] To measure the viability of Arpe-19 cells for 24 hours after treatment with Ar-MSNs-TK and Ar-MSNs-TK-PEG, the results quantified using CCK-8 assay are shown in Figure 2.

[0103] According to Figure 2, it can be seen that there is no cytotoxicity for both Ar-MSNs-TK and Ar-MSNs-TK-PEG even at high concentrations (500 μg / ml). This may be attributed to the high amorphous properties and low degree of crosslinking of the mesoporous silica framework prepared using a two-phase stratification method. Additionally, TK combined with MSN can act as a ROS scavenger capable of alleviating oxidative stress to some extent.

[0104] In addition, at very high concentrations (1000 μg / mL), Ar-MSNs-TK-PEG exhibited lower toxicity (35% apoptosis) than Ar-MSN-TK (65% apoptosis), which is thought to be due to the hydrophilic cloning shell formed around it. This reduces the strength of the interaction between MSN and the cell membrane by PEG.

[0106] [Evaluation Example 2] Ar-MSNs-TK uptake in Arpe-19 cells

[0107] Figure 13 shows the results of confocal immunofluorescence imaging after culturing FITC-conjugated Ar-MSNs-TK with Arpe-19 cells for 4, 8, 12, and 24 hours, respectively. According to Figure 13, it can be seen that the fluorescence intensity of MSNs increases with increasing culture time, and it was confirmed that the fluorescence intensity of MSNs reaches equilibrium after 12 hours and increases slightly after 24 hours. Accordingly, it can be seen that the optimal culture time for MSN uptake is 12 hours.

[0109] [Evaluation Example 3] HN peptide release effect of Ar-MSNs-TK-PEG

[0110] Surface charge reversal

[0111] In this study, the distribution and diffusion of Ar-MSNs-TK and Ar-MSNs-TK-PEG in the bovine vitreous humor were evaluated at 0.5 mg / mL, which was determined to be the maximum non-toxic dose of MSN for Arpe-.

[0112] First, the zeta potentials for Ar-MSNs-TK, Ar-MSNs-TK-PEG, Ar-MSNs-TK-PEG loaded with HN peptide, and Ar-MSNs-TK-PEG loaded with tBH-treated HN peptide are shown in Figure 3 below.

[0113] Figure 4 shows the appearance of (a) Ar-MSNs-TK, (b) Ar-MSNs-TK-PEG, and (c) Ar-MSNs-TK-PEG after tBH treatment as measured by confocal immunofluorescence imaging, and Figure 5 shows Arpe-19 cells treated with MSNs-TK-PEG before and after co-culture with tBH (80 μM) in vitro to investigate the charge reversal that occurs when Ar-MSNs-TK-PEG responds to ROS in Arpe-19 cells.

[0114] As shown in Figure 4, Ar-MSNs-TK with a zeta potential of -40±4 mV was uniformly distributed and easily diffused in the vitreous humor, whereas Ar-MSNs-TK-PEG with a zeta potential of 2±0.1 mV was significantly aggregated in the vitreous humor. However, after treatment with tBH at a 50% equimolar concentration of the bound PEG amount, the aggregated Ar-MSNs-TK-PEG separated and dissociated, and it was confirmed that Ar-MSNs-thiol with a zeta potential of -25±8 mV was dispersed.

[0115] As shown in Figure 5, Ar-MSNs-TK-PEG exhibited initial mitochondrial targeting ability due to the electrostatic attraction between the positive surface charge of MSNs-TK-PEG and the negative surface charge of mitochondria. However, after the addition of tBH, it was observed that the loss of the TK-PEG linker caused the MSN surface to become negatively charged, resulting in repulsion with mitochondria. Accordingly, it appears that Ar-MSNs-TK-PEG undergoes a charge inversion reaction through ROS.

[0116] ROS-sensitive HN-peptide release

[0117] To confirm the release of HN peptides from Ar-MSNs-TK-PEG in the presence of ROS, HN-MSNs-TK-PEG loaded with HN peptides was produced. For comparison, MSNs-TK loaded with HN peptides was used as a control. According to Figure 6, even without tBH treatment, MSNs-TK explosively released HN peptides during the first 24 hours, whereas MSNs-TK-PEG released almost no peptides under the same reaction conditions. On the other hand, when treated with tBH, MSN-TK-PEG began to release HN peptides. The release of HN peptides increased with increasing tBH concentration, which is because PEG was detached from the surface of MSNs-TK-PEG upon tBH treatment.

[0118] In vitro HN-peptide release

[0119] To observe the release of HN peptide from Ar-MSNs-TK-PEG in vitro, Ar-MSNs-TK-PEG loaded with HN peptide was cultured with Arpe19 cells under tBH (110 μM) treatment for 12, 24, 48, and 72 hours, and the results of the confocal fluorescence imaging are shown in Figure 7.

[0120] According to Figure 7, after 12 hours of cell culture, the red fluorescence signal (FITC-HN) appeared mainly in the cytoplasm of Arpe-19 cells, with minimal co-localization with mitochondria. The rate of HN peptide release fluctuated during the first 12 hours, and as shown in Figure 8, the co-localization coefficients of HN-FITC b (red) and Ar-MSNs-TK-PEG-FITC (green) ranged from 0.85 to 0.75 (Mander's M1). This behavior appears to be due to the rapid release of HN, which can be localized on the outer pore surface of MSNs. However, HN peptide release gradually increased after 24, 48, and 72 hours. Mander's M1 dropped from 0.85 to 0.65 after 72 hours of culture, indicating successful and sustained in vitro release of HN peptides.

[0122] [Evaluation Example 4] Improvement of neovascular retinal disease by Ar-MSNs-TK-PEG

[0123] inhibition of apoptosis

[0124] Since oxidative stress is an important causative factor of neovascular degenerative disease, the results of investigating the in vitro anti-apoptotic effect (TUNEL staining and activated caspase-3) of Ar-MSNs-TK-PEG loaded with HN peptide are shown in Figures 9 and 10.

[0125] According to Figures 9 and 10, it can be confirmed that apoptosis was significantly inhibited in the case of Ar-MSNs-TK-PEG loaded with HN peptide. Specifically, when treated with tBH, the number of apoptotic cells was approximately 25% of the total, but when treated with Ar-MSNs-TK-PEG, the degree of apoptosis was approximately 21%, showing a slight reduction in apoptosis. Furthermore, the reduction in apoptosis in cells treated with MSNs-TK-PEG loaded with HN peptide was approximately 3%, which can be confirmed to be substantially superior to that of MSNs-TK-PEG and the control group.

[0126] STAT3 phosphorylation induction

[0127] Previous studies have shown that HN peptides exert various anti-apoptotic functions by binding to the trimeric receptors CNTF, WSX1, and GP130 and inducing the STAT3 phosphorylation and activation pathways. To investigate the STAT3 signaling activation pathway, cells were pre-cultured with a STAT3 inhibitor (SINH) (0.5 μM) and then co-cultured with Ar-MSNs-TK-PEG loaded with HN peptides. Western blot results for Phospho-STAT3, STAT3, and B-Actin are shown in Figure 11. According to Figure 11, it can be confirmed that the expression level of Phospho-STAT3 is higher in Ar-MSNs-TK-PEG loaded with HN peptides compared to Ar-MSNs-TK-PEG without HN peptides and control samples. Accordingly, it can be seen that Ar-MSNs-TK-PEG loaded with HN peptide stimulates STAT3 phosphorylation induction through the release of HN peptide, and these results suggest that the STAT3 pathway is actively involved in protecting against apoptosis.

[0128] Improvement of neovascular retinal disease

[0129] In neovascular retinal disease, VEGF expression generally increases, leading to the formation of new blood vessels in the retina. Accordingly, we investigated whether HN peptide alone or Ar-MSNs-TK-PEG loaded with HN peptide could act as an anti-VEGF agent for the improvement of neovascular retinal disease in vivo. HN peptide or Ar-MSNs-TK-PEG loaded with HN peptide (1.5 μL at 0.5 mg / mL) was injected into the vitreous humor of oxygen-induced retinopathy (OIR) model mice. Subsequently, eyes were collected, and neovascularization in the retina was visualized using a confocal microscope. The area of ​​neovascularization was calculated and is shown in Figure 12.

[0130] According to Figure 12, it can be seen that both the experimental group injected with HN peptide alone and the experimental group injected with Ar-MSNs-TK-PEG loaded with HN peptide inhibited angiogenesis, and in the case of the experimental group injected with Ar-MSNs-TK-PEG loaded with HN peptide, the formation of vascular bundles was completely inhibited. Accordingly, it can be seen that Ar-MSNs-TK-PEG can increase the half-life of HN and is a promising drug delivery system for the treatment of angiogenic diseases.

[0131] As described above, the present invention has been explained by specific details, limited embodiments, and comparative examples; however, these are provided merely to aid in a more comprehensive understanding of the invention, and the invention is not limited to the above embodiments. Those skilled in the art can make various modifications and variations from this description.

[0132] Accordingly, the scope of the present invention is not limited to the described embodiments, and all things equivalent to or having equivalent variations to the claims set forth below, as well as the claims set forth below, shall be considered to fall within the scope of the concept of the present invention.

Claims

Claim 1 A porous silica nanoparticle composite comprising: porous silica nanoparticles; arginine bonded to the surface of the nanoparticles; an active oxygen degrading linker connecting a water-soluble polymer to the surface of the porous silica nanoparticles; and an active substance supported inside the nanoparticles, wherein the active oxygen degrading linker is a thioketal and the active substance is a humanin peptide. Claim 2 A porous silica nanoparticle composite according to claim 1, wherein the bonding area of ​​arginine and a water-soluble polymer on the surface of the nanoparticles is 1:1 to 1:

10. Claim 3 A porous silica nanoparticle composite according to claim 1, wherein the average pore size of the nanoparticles is 1 to 10 nm. Claim 4 In claim 1, the pore volume of the nanoparticle is 0.1 to 4 cm 3 / g, porous silica nanoparticle composite. Claim 5 In claim 1, the nanoparticles are porous silica nanoparticle composites having a positive surface charge. Claim 6 In claim 1, the nanoparticles are porous silica nanoparticle composites that carry a negative surface charge under active oxygen conditions. Claim 7 A porous silica nanoparticle composite according to claim 1, wherein the water-soluble polymer is one or more selected from polyethylene glycol, acrylic acid-based polymer, methacrylic acid-based polymer, polyamidoamine, poly(2-hydroxypropyl methacrylamide)-based polymer, water-soluble polypeptide, water-soluble polysaccharide, and polyglycerol. Claim 8 A porous silica nanoparticle composite according to claim 1, wherein the molecular weight of the water-soluble polymer is 1,000 to 10,000 g / mol. Claim 9 delete Claim 10 delete Claim 11 A porous silica nanoparticle composite according to claim 1, wherein the humanin peptide is supported at a concentration of 200 to 1000 mg / g. Claim 12 A porous silica nanoparticle composite according to claim 1, wherein a humanin peptide is released as the active oxygen decomposition link is cleaved. Claim 13 In claim 1, the porous silica nanoparticle composite is a porous silica nanoparticle composite used for the treatment of one or more of age-related macular degeneration, neovascular retinal disease, diabetic retinopathy, and retinal vein occlusion. Claim 14 delete Claim 15 A pharmaceutical composition for treating neovascular retinal disease comprising a porous silica nanoparticle composite according to any one of claims 1 to 8 and claims 11 to 13. Claim 16 In claim 15, the above pharmaceutical composition is a pharmaceutical composition for treating neovascular retinal disease, intended for ophthalmic administration.

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