KR20190089534A - Nanoparticle for drug delivery in inner ear - Google Patents

Abstract

The present invention relates to a nanoparticle for drug delivery in an inner ear, which provides an inner ear-specific drug delivery nanoparticle containing a drug for treating or preventing inner ear diseases based on phospholipid nanoemulsions. The nanoparticle of the present invention can deliver drugs into the inner ear, thereby being expected to be remarkably useful for preventing and treating inner ear diseases.

Description

[0001] NANOPARTICLE FOR DRUG DELIVERY IN INNER EAR [0002]

The present invention relates to nanoparticles for drug delivery in inner ear.

Hearing problems can arise from various disorders, diseases or trauma of the inner ear and are an increasing problem in society today. The inner ear disease is often classified as a refractory disease, and one of the major reasons is that the drug or gene transfer is separated from the systemic circulation by the blood-labyrinthine barrier and is located deep in the cranium, to be. To overcome this problem, drugs are placed in the middle ear cavity through the eardrum for effective drug delivery to the inner ear, and drugs are transferred through the round window membrane of the cochlea from the middle ear to the inner ear. Local delivery is under study.

On the other hand, nanoparticles have been used as promising tools in biomedical fields such as drug delivery, gene transfer, intracellular imaging, and phototherapy. In particular, gold nanomaterials have been used as a means of synthesis, ease of action, chemical stability, , And adjustable optical and electrical properties. Therefore, drug delivery using nanoparticles has been studied mainly in cancer cell lines for cancer treatment and has recently been tried in inner ear disease. However, in the case of local delivery, it has not yet been possible to effectively transfer a sufficient amount of drug It is true.

In order to overcome this problem, a drug delivery system capable of synthesizing nanoparticles based on poly (2-hydroxyethyl L-aspartamide) and octaarginine to co-transfer drugs into the inner ear has been developed, (Yoon, Ji Young, et al., "Intratympanic delivery of oligoarginine-conjugated nanoparticles as a gene (or drug) carrier to the inner ear." Biomaterials 73 (2015): 243-253; Kim, Dong-Kee, et al., "Drug delivery 22.3 (2015): 367-374).

Accordingly, there is a need to develop a novel drug delivery system that can be used for the prevention and treatment of inner ear diseases.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a nano-specific drug delivery nanoparticle comprising a drug for treating or preventing inner ear disease based on phospholipid nanoemulsion as an active ingredient And to provide the above-mentioned objects.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, the particular features, shapes, and compositions may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Hereinafter, the present invention will be described in detail.

In one embodiment of the present invention, "inner ear disease" means a disease that can be caused by various disorders, diseases, or trauma, and is one of diseases classified as intractable diseases because the delivery of drugs or genes is not easy. The inner ear disease may be classified into two types: vertigo such as tinnitus, BPPV, hearing loss, sensory nerve impairment, tinnitus, chronic otitis media, external lymphatic fistula, secondary endolymphatic carcinoma, myelitis and vestibular neuritis, But are not limited to, inner ear disease (AIED) and Meniere's disease. Specifically, it refers to sensorineural hearing loss. More specifically, it refers to sensorineural hearing loss, which is caused by damage to hair cells, spiral ganglion neurons (SGN), cortical organs, etc. in inner ear cochlea It means hearing loss.

In one embodiment of the present invention, the term "nanoparticle" means particles of various materials having a diameter of nano unit, and the nanoparticles are not particularly limited as long as they are nanoparticles. In medicine, when the particle size of the therapeutic agent is made into nanoparticles, there are the following advantages. First, at the time of administration, the particle size can be more absorbed than in the case of large, and consequently, the biological efficiency of the therapeutic agent can be increased. In addition, the form of the therapeutic agent formulation can be varied, for example, by administering the drug, which was only possible orally administered, to the patient in an inhaled form. In addition, since the release rate of the therapeutic agent is a very important factor in the formulation of the sustained-release therapeutic agent, the release rate of the therapeutic agent can be predicted as the size of the therapeutic agent particle becomes nanoparticles, An effective therapeutic agent can be produced. The nanoparticles used in the present invention are based on a phospholipid nano emulsion form prepared by an ordinary water-in-oil (O / W) method in an aqueous medium and have neutral, anionic, cationic charge on the surface, An additional charge of polyethylene glycol (PEG) may be added to the charge.

In one embodiment of the present invention, "nile red" is used as a fluorescent indicator and a hydrophobic drug model as a hydrophobic fluorescent dye encapsulated in nanoparticles in order to examine the possibility of drug delivery of nanoparticles to various tissues of the inner ear . Nile red is used in a variety of drug delivery, drug release and cell uptake assays.

In one embodiment of the present invention, "drug delivery" means that a drug is selectively administered to a treatment site in an amount necessary for minimizing adverse effects and maximizing efficacy and effectiveness, so that a healthy tissue is not exposed to the drug, It means to deliver efficiently so as to have excellent therapeutic effect. The drug delivery according to the present invention is intended for local delivery for delivering drugs to hairy cells, cortical or spiral ganglion cells, which are the main obstacles to hearing loss through permeation of the window.

In the present invention, "permeation" refers to the ability of nanoparticles to penetrate through the window to effectively transport medicines, and the drug is placed in the middle ear through the tympanic membrane, It means to penetrate the window. The nanoparticles of the present invention bind to polyethylene glycol (PEG) on the surface of nanoparticles in order to increase the permeability to inner ear cells, so that they are excellent in permeability and thus can be usefully used as materials for drug delivery in inner ear.

In one embodiment of the present invention, "cytotoxicity" means an effect of inhibiting or reducing the function of cells to cause cell destruction. In the cytotoxicity test according to the present invention, cell viability was determined using MTT assay, and the maximum safe dose determined through the experiment was 0.25 mg / mL to 0.5 mg / mL.

In one embodiment of the present invention, "cell uptake" can be confirmed through intracellular absorbed nile red and measured by comparison of fluorescence intensity with fluorescence microscope or confocal microscope.

In one embodiment of the invention, "drug release" can be divided into sustained release or controlled release, continuous release means continuous release of the drug or therapeutic agent or any combination thereof over a period of time, Controlled release refers to the modulation of the rate and / or amount of drug or therapeutic agent delivered. The controlled emission may be continuous or discontinuous and / or linear or nonlinear. This may be accomplished using one or more types of polymer compositions, drug loading, excipients or degradation-improving agents, or other modifying agents, alone, in combination, or sequentially to provide the desired effect. In the present invention, the amount of nile red released in the artificial external lymph fluid was measured with a UV spectrometer.

In one embodiment of the invention, the term "zeta potential" refers to an unmixed powder attached to the surface of a charged particle and an electrodynamic potential difference resulting from the difference in the positive charge density in the diffusion double layer of mobile water, . It may also be expressed as an electric potential difference or zeta potential between the cell surface and the surrounding culture liquid.

In one embodiment of the invention, "treatment" refers to any activity that is clinically intervened to alter the natural course of an individual or cell to be treated, including, but not limited to, . The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, to alleviate the symptoms, to reduce all direct or indirect pathological consequences of the disease, to prevent metastasis, to reduce the rate of disease progression, Or temporarily alleviating, or improving the prognosis. In addition, the term " treatment " used in the present invention means all actions that improve or ameliorate symptoms of inner ear disease upon administration of nanoparticles.

In one embodiment of the present invention, "prevention" means all actions that inhibit or delay the inner ear disease by administration of the nanoparticles.

The present invention relates to a phospholipid nanoemulsion comprising, as an active ingredient, a drug for the treatment or prevention of inner ear disease, wherein 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocerine, sodium salt (DPPS); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (DPPE-PEG2000); And inner ear-specific drug delivery nanoparticles comprising at least one member selected from the group consisting of 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) to provide.

In one embodiment of the present invention, the nanoparticles may further comprise flaxseed oil or soybean oil. In case of phospholipid, liposome is formed and most of the inner space is mainly hydrophilic aqueous solution environment. However, when flax seed oil or soybean oil is added to the inner space, the inner part becomes a hydrophobic oil layer, and a space for containing hydrophobic drugs such as dexamethasone It is possible to secure sufficiently. Flaxseed oil or soybean oil is most preferable for producing a hydrophobic oil layer, but it is preferable to use flaxseed oil or soybean oil to produce a hydrophobic oil layer. However, flaxseed oil or soybean oil is most preferable, It may contain almond oil, avocado oil, olive oil, camellia oil, rice bran oil, cottonseed oil, peanut oil, hoso oil, rape oil, rice bran oil, sesame oil, soybean oil, castor oil, cocoa butter, palm kernel oil, Or in combination. However, the present invention is not limited to the above listed kinds.

In one embodiment of the invention, the nanoparticles may contain 3 to 10 mg of DPPC, preferably 4 to 8 mg, and most preferably 6 mg. The surface of the nanoparticles containing it represents a neutral charge. Nanoparticles with neutral charge can penetrate through the mucosa and be effectively absorbed into the cell.

In one embodiment of the present invention, the nanoparticles contain DPPC and DPPS in a ratio of 1: 1 to 1: 4, preferably 1: 2. The surface of the nanoparticles containing it exhibits anionic charge and is effective in drug delivery in the inner ear.

In one embodiment of the present invention, the nanoparticles contain DPPC and DPTAP in a ratio of 1: 1 to 1: 4, preferably 1: 2. The surface of the nanoparticles containing it represents a cationic charge. Nanoparticles with cationic charge have excellent absorption effect in the cell.

In one embodiment of the present invention, the nanoparticles contain DPPE-PEG2000 and DPTAP in a ratio of 1: 1 to 1: 4, preferably 1: 2. The surface of the nanoparticles containing it exhibits a cationic charge and has a form in which polyethylene glycol (PEG) is bonded to the surface. By binding PEG to the surface of the nanoparticles, the permeability to the inner cell can be increased and the circulation of the drug can be induced longer in the outer lymph.

In one embodiment of the present invention, the nanoparticles have a size of 150 to 250 nm, preferably about 250 nm.

In one embodiment of the invention, the nanoparticles pass through a round window membrane to deliver drug to the hair cells or cortical organs.

In one embodiment of the present invention, the medicament for treating or preventing the inner ear disease is selected from the group consisting of dexamethasone, steroids, diuretics, vasodilators, beta histidine, vestibulant, dextran, mannitol or heparin, There is no limit to drugs that can be prevented.

In one embodiment of the invention, the inner ear disease is selected from the group consisting of tinnitus, dizziness such as BPPV, hearing loss, sensory nerve impairment, tinnitus, chronic migraine, external lymphatic fistula, secondary endolymphaticoma, , Aural carcinoma, inner ear toxicity, autoimmune inner ear disease (AIED), and Meniere's disease.

The present invention is a nano-specific drug delivery nanoparticle comprising a drug for treating or preventing inner ear disease based on a phospholipid nanoemulsion as an active ingredient, , And has excellent effects in terms of stability and efficacy. It is also expected to be useful for the prevention and treatment of inner ear disease.

Figure 1 shows a schematic diagram of intimal drug delivery.
Figure 2 shows the size and zeta potential of nanoparticles.
Figure 3 shows the results of testing the cytotoxicity of nanoparticles.
Fig. 4 shows the results of the cell uptake test of nanoparticles.
Fig. 5 shows the penetration rate of nanoparticles into the hair cells in the artificial inner ear window model.
Fig. 6 shows the results of analysis of toxicity and penetration of the in vitro nanoparticles.
Figure 7 shows the release profile of dexamethasone loaded in cationic-PEG.
Fig. 8 shows the results of comparing the distribution efficiencies of CAT-PEG-Dex and dexamethasone.
FIG. 9 shows the recovery results of antibiotic-induced hearing loss mice by CAT-PEG-Dex.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Manufacturing example : Manufacture of nanoparticles

material

(DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocerine, sodium salt (DPPS) were dissolved in Echelon Biosciences Salt Lake City, UT). (Pentaethylene glycol) -2000] (DPPE-PEG2000) and 1,2-dipalmitoyl-3-trimethylammonium -Propane (DPTAP) was purchased from Avanti Polar lipids (Birmingham, AL). Flaxseed oil and nile red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dexamethasone (Dex) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Dexamethasone fluorescein (Dex-FITC) was purchased from Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide, 99.0% (methylsulfoxide, DMSO) and Triton X-100 were purchased from Samchun (Korea). Phosphate buffered saline (PBS, pH 7.4) was purchased from Gibco ^® . Pre-wetted RC tubing (MWCO: 25KD) was purchased from Spectrum labs (Rancho Dominguez, California, USA).

Manufacture of nanoparticles

The nanoparticles were prepared in an aqueous medium by the conventional water-in-oil (O / W) emulsion method. (6 mg), DPPC (2 mg) / DPPS (4 mg), DPPC (2 mg) / DPTAP (4 mg) and DPPE-PEG2000 (2 mg) / DPTAP (4 mg) for neutral, anionic, cationic and cationic PEG nanoemulsions, (4 mg) was used. Nile red (0.3 mg) or Dex (10 mg) was dissolved in 20 ul DMSO and mixed with 50 mg of flaxseed oil. Then, flaxseed oil (containing Nile Red or Dex) and phospholipid were dispersed by ultrasonication in 2% glycerol water (lipid concentration 2.4 mg / ml). Unloaded Nile Red or Dex was removed by dialysis (MWCO: 25KD) using distilled water for 1 hour. After completely destroying the liposomes by DMSO and Triton X-100, the amount of nile red in the nanoparticles was evaluated based on Nile Red fluorescence (510/605 nm).

The hydrodynamic radius and zeta potential of the nanoparticles in PBS (pH 7.2) were measured at 25 ° C with a Zetasizer (Zetasizer Nano ZS90, Malvern Instruments, Malvern, UK) using DTA (Data Transfer Support) software. To confirm the morphology of the nanoparticles, negative staining was performed with a 2% (w / v) uranyl acetate solution and observed with a TEM (transmission electron microscope).

Dex-FITC was used to measure Dex emission of nanoparticles over time. Dex (Dex: Dex-FITC = 49: 1 weight ratio) -loaded nanoparticles were placed in a dialysis membrane in PBS (pH 7.2) at room temperature. Then, the fluorescence (485/530 nm) of Dex-FITC in the external solution was measured at appropriate time points.

Example  1: Screening of candidate nanoparticle carriers

One. HEI -OC1 in vitro cell culture

Cellular uptake and cytotoxicity of CPP Were evaluated using immortalized mice of the cortical organ cell line HEI-OC1 provided by MK Park (Seoul National University, College of Medicine, Seoul, Korea). HEK-OC1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50 U / mL recombinant mouse interferon-gamma and subcultured and cultured at 33 < 0 > C in a 10% CO ₂ humidity environment.

2. Cytotoxicity test

The cytotoxicity of four candidate nanoparticle carriers to HEI-OC1 cells was investigated as follows. HEI-OC1 cells applied to a 96-well plate at a density of 1x10 ⁴ number / well in complete growth medium 0.1mL, and the value was 80% growth for 24 hours. Cells were treated with four candidate nanoparticle carriers for 24 hours. (MTT) which is commercially available according to the protocol of the manufacturer (EZ-Cytox, Daisetsu Laboratories, Seoul, Korea) was added to a solution of 3- (4,5-dimethylthiazol- To determine cell viability. After incubation, the optical density of the sample was determined at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, Calif., USA).

3. Cellular absorption test

HEI-OC1 cells were applied to the Lab-Tek II Chamber Slide System (Nunc, Roskilde, Denmark) and treated with the candidate nanoparticle carrier conjugated with nile red for 4 hours at the maximum safety dose determined in the cytotoxicity study. Cells were fixed with 4% paraformaldehyde solution and sealed with Vectashield Mounting Medium and DAPI (Vector Laboratories, Burlingame, CA, USA). The treated cells were observed with a confocal microscope LSM5 Live Configuration Variable VRGB, Zeiss, Jena, Germany) and a fluorescence-activated cell sorter (FACS, BD FACS Canto II; Becton Dickinson and Company, Franklin Lakes, NJ, USA).

4. In vitro drug screening model using artificial mucosa

To mimic the three-dimensional executable and reconstruct human mucosal layer of RWM was purchased neo bonus ^{Neoderm ® -OD (Tego science, Seoul} , South Korea). The epithelial cells consist of human oral mucosal epithelial cells, and the stroma is composed of fibroblasts of the collagen matrix. Its thickness is about 100 탆, which is similar to the thickness of human RWM of about 70 탆. Neoderm ^® -OD was transferred to the bottom of the plate with HEI-OC1-6-month. HEI-OC1 cells filled the same culture medium for incubation in a 6-well plate until, as shown in FIG contact with Neoderm ^® -OD 1.

Neoderm ^® four candidate nanoparticle carrier with four days of red is attached via an -OD was applied for 40 hours in the same capacity as Nile Red 1ug of each of the nanoparticles carriers, ball absorption of Nile Red in the HEI-OC1 at the bottom And examined with focus microscopy and FACS.

Example 2 : Application of nanoparticle carriers to inner drug delivery

According to the results of the screening test, one nanoparticle carrier was selected, Nile red was loaded in the in vitro test, and dexamethasone was loaded in the in vivo test. In the in vitro test, Nile red absorption pattern was observed in the organ culture for efficiency evaluation, and damage of the cilia and microstructure of cortical organ of the hair cell were observed for stability evaluation.

In in vivo studies, dexamethasone or dexamethasone sodium phosphate loaded nanoparticle carriers currently used in clinical practice were administered into the middle ear cavity of normal or immunostimulatory animals. Thereafter, the drug was compared to the amount of absorption in the inner ear tissue between the two normal animal groups and the amount of absorption in the inner ear tissue was compared, and the degree of hearing protection between inner ear neurotoxic animal groups and how the damage to the cochlea was reduced to some extent Respectively.

One. In vitro  exam

In vitro transplantation of the primary cochlea was prepared from C57 / BL6 mice 3 days after birth (P3). Incised cortical organs were cultured in high-glucose DMEM containing 5% FBS, 5% horse serum and 10 ng / mL ampicillin in a humidified incubator at 37 ° C and 5% CO ₂ . The nanoparticle carrier loaded with Nile Red was treated for 24 hours at the maximum stable dose and half its volume. After treatment, the cells were fixed with 4% paraformaldehyde solution, permeabilized with acetone, applied with Alexa Fluor 488 Phalloidin (Molecular Probes, Eugene, OR) conjugated antibody, and transferred to Vecta shield mounting medium with DAPI (Vector Laboratories) Lt; / RTI > The absorption of nanoemulsions was investigated using a high-fidelity microscope for high-magnification images (Nikon, Tokyo, Japan and Eclipse TE300 Microscope for low magnification images.) The specimens were stained with DAPI and FITC-labeled paloides, The damage to the tissue structure was evaluated.

2. In vivo  Drug distribution

2.1 Animal Surgery

One month old male C57 / BL6 mice were used for the experiments. Prior to surgery, mice were anesthetized with a mixture of 30 mg / kg Zoletile (Virbac, Carros, France) and 10 mg / kg Rompun (Bayer, Leverkusen, Germany). The mouse was laid flat on a thermally controlled warming pad, midline incision was made, and the left bulb was exposed. A micro-forceps was used for the bump to puncture. Then dexamethasone (Sigma-Aldrich, St. Louis, MO) loaded with nanoparticles was loaded with dexamethasone sodium phosphate (Sigma-Aldrich, St. Louis, Mo.) or 5 mg / ml loaded with maximal stable capacity (nanoparticle concentration 0.5 mg / ml, Dex concentration 0.14 mg / Sigma-Aldrich, St. Louis, Mo.) was administered into the middle ear cavity until the middle ear was completely filled. To reflect more clinical symptoms, we did not use a gel foam that could hold medication in the middle ear for a long period of time. The hole was blocked with bone wax (B. Braun, Melsungen, Germany) and the wound was sutured. After surgery, pain was relieved by injection of limadill (1.0 mg / kg; Pfizer, UK, Surrey, UK). Baytrill (10 mg / kg, Orion, Hamburg, Germany) was intraperitoneally injected once a day as a preventive against middle ear infection.

2.2 Evaluation of drug distribution

Animals were sacrificed at 1, 24, and 72 hours after surgery and the cochlea was harvested. The separated cochlea was rinsed with tap water for one minute to remove free particles remaining on the outer surface and stored in a solution of 4% paraformaldehyde (Merck, Darmstadt, Germany) for 24 hours. Fixed overnight, decalcified with 5% EDTA (0.3M, pH 6.5) and embedded in paraffin. A 5 mu m wax section was obtained. Wow axis sections were applied according to the manufacturer's procedures of the Vectastain Elite ABC HRP Kit and Vector NovaRED Peroxidase Substrate (Vector Labs, CA). Anti-dexamethasone antibody (Abcam, UK) was used to target dexamethasone or dexamethasone sodium phosphate in the cochlea.

3. Dexamethasone transfer to the inner ear in the innate neurotoxic mouse model

One month old male C57 / BL6 mice were divided into 4 groups and the experiment was performed. The control group did not undergo surgery or treatment, and the other three groups received the drug in the middle ear as described above 2 hours before inducing endogenous toxicity, and each of the three groups received dexamethasone, dexamethasone sodium Phosphate or saline-loaded nanoparticles were administered.

Myocardial toxicity was assessed by intravenous infusion of furosemide (120 mg / kg; Sigma-Aldrich, St. Louis, Mo.) 30 minutes after subcutaneous injection of kanamycin (500 mg / kg; Sigma-Aldrich, St. Louis, Mo.) Lt; / RTI > Forty hours after the induction of innate neurotoxicity, some mice were sacrificed for RT-PCT, and one week later, the remaining mice were subjected to ABR.

3.1 ABR

Auditory brainstem responses (ABR) were obtained 7 days after induction of myotoxicity by System III Evoked Potential Workstation (Tucker-Davis Technologies, Alachua, FL, USA). That is, the mice were anesthetized with a mixture of 30 mg / kg Zoletil (Virbac, Carros, France) and 10 mg / kg rumpun (Bayer, Leverkusen, Germany) and placed on the heating pad inside the soundproofed acoustic chamber. In the ABR test, click and tone burst stimuli (4, 8, 16 and 32 kHz) were presented with a MF1 magnetic speaker (TDT) of 90 to 20 dB SPL at 5 to 10 dB SPL steps. The click stimulus lasted 0.1ms and the tone burst stimulus was 5ms (2.5ms for both rise and fall without congestion). The critical response was defined as the maximum amplitude (latency, 2.5-7.5 ms) of the induced response at two or more standard deviations above the mean background activity (15-20 ms) before stimulation. The threshold difference between groups of mice was statistically determined. After ABR measurement, mice were sacrificed for inclusion in slides.

3.2 RT- PCR

Total RNA was extracted from the cochlea using the easy-BLUE total RNA extraction kit (iNtRON Biotechnology, WA). CDNA was synthesized using Reverse Transcription Enzyme Premix (Elpis Biotech, Korea) and amplified using a Power SYBR ^TM with a gene-specific primer pair Amplified by Green polymerase chain reaction (PCR) Master Mix (Applied Biosystems, CA, USA); IL1b forward, 5'-GCC CAT CCT CTG TGA CTC AT-3 ', IL1b reverse, 5'-AGG CCA CAG GTA TTT TGT CG-3', IL6 forward, 5'- CCG GAG AGG AGA CTT CAC AG- , IL6 reverse direction, 5'-CCG GAG AGG AGA CTT CAC AG-3, TNFa forward, 5'-ACG GCA TGG ATC TCA AAG AC-3 ', TNFa reverse, 5'- GTG GGT GAG GAG CAC GTA GT- , IFNr forward, 5'-GCT ACA CAC TGC ATC TTG GCT TT-3 ', IFNr reverse, 5'- AAT GAC TGT GCC GTG GCA GTA A-3'. Quantitative real time PCR was performed on an ABI 7500 FAST instrument (Applied Biosystems). The expression level of mRNA was normalized by the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We used the ΔΔCt method using an Excel spreadsheet containing the algorithms provided by Microsoft for data analysis. The change values were then recorded and expressed as log-normalized ratios of values from the saline application group.

3.3 Corti  Agency( OC ) Slide production

After ABR measurement, mice were sacrificed to extract cochlea, and the extracted cochlea was fixed and decalcified by the method described above. The cortical organ (OC) was exposed by carefully removing the bone and membrane labyrinth and tectorial membrane using angled, sharp microsomes. OC is divided into two parts, one part containing the vertex and intermediate rotation of the cochlea and the other part containing the base rotation of the cochlea, placed on a plate (Nunclon Microwell Plates, Sigma Aldrich) filled with PBS 4 < / RTI > Samples were permeabilized with a shaker at room temperature for 15 minutes with 0.5% Triton X-100 (Sigma Aldrich) and then incubated with 1: 1000 phalloidin (R Alexa Fluor 488 phalloidin, Invitrogen) in PBS for 1 hour. After washing three times with PBS, the sample was transferred to a slide and fixed to a Vectashield Mounting Medium containing DAPI (Vector Laboratories).

Statistical analysis

All data were expressed as mean ± standard error of three measurements. One-way analysis of variance (ANOVA) was performed on the various groups to identify differences between the groups. In all analyzes, P <0.05 represents statistical significance.

Experiment result

Preparation and Characterization of Nanoparticles

As described in the above Preparation Example, the nanoparticles were prepared by a conventional O / W emulsion method based on self-assembly. The core of the nanoparticles is filled with linseed oil and the shell is composed of phospholipid. Nile red and dexamethasone were loaded into the cores of the nanoparticles. The size of the prepared nanoparticles was about 200 nm, and the transmission electron microscope (TEM) image had a spherical shape under an aqueous condition (FIG. 2). The zeta potential values for neutral, anionic, cationic and cationic-PEG nanoparticles are -4.32, +25.8, -26.0, and -0.28, respectively. These values show that the surface charge is appropriately controlled according to the composition of the phospholipid while maintaining a similar size.

Screening result of candidate nanoparticle carrier

As described in Example 1, cell viability and cell uptake were confirmed. After the candidate nanoparticle treatment, cell viability was measured by MTT assay. The safety capacity of each nanoparticle carrier was 0.5 mg / ml for anionic, 0.25 mg / ml for cationic and 0.5 mg / ml for CAT-PEG, compared to untreated control cells. In addition, the cytotoxicity of neutral nanoparticle carriers was not detected at a test dose of 2.5 mg / ml (FIG. 3).

Cellular uptake of Nile Red-loaded nanoparticles in HEI-OC1 cells cultured under normal culture conditions was tested. Cells were treated with 0.5 mg / ml of each nanoparticle for 4 hours, then analyzed by FACS and confirmed by confocal microscopy. The cationic nanoparticles showed the strongest Nylon absorption of HEI-OC1 in both FACS and confocal microscopes, followed by Cat-PEG, neutral and anionic nanoparticle carriers (FIG. 4).

Next, the intensity of Nile Red fluorescence in HEI-OC1 cells transduced through the nanoparticle carrier through an artificial mucosa and culture medium was observed using an in vitro RWM model. Cat-PAEG nanoparticles showed the strongest fluorescence in FACS and confocal microscopy, followed by neutral, cationic and anionic nanoparticles (FIG. 5). Neutral nanoparticles showed similar efficiencies to Cat-PEG, thus selecting Cat-PEG nanoparticles to confirm the drug delivery confirmation and therapeutic results in the innate-toxic mouse model.

Using cat-PEG nanoparticles in inner ear In vitro  And In vivo  Drug delivery

As shown in Example 2, in vitro and in vivo drug delivery was confirmed using Cat-PEG nanoparticles. As a result of the screening test, cultured cortical organs were treated with a stable dose of Cat-PEG. After 24 h of treatment with 0.25 and 0.5 mg / ml CAT-PEG, no fluorescence and confocal microscopic examination showed structural damage to the hairy ciliated cells and cortical tracts, and Nyl red was 0.25 mg / ml Than in the 0.5 mg / ml group. Both groups showed a high uptake of Nile Red in the medial side of the tracheal ganglia and spiral ganglia (Fig. 6).

Dex was then loaded onto Cat-PEG nanoparticles for treatment. The loading efficiency was about 93.1 ± 7.6% and the particle size was about 143.53 ± 21.18 nm, which was similar to that before drug loading. The loaded Dex was slowly released from Cat-PEG nanoparticles in PBS (pH 7.4) (Figure 7). About half of the loaded Dex was released after 72 hours and 90% of the drug was released after 10 days. After the administration of Cat-PEG-Dex or dexamethasone sodium phosphate (Dex-SP) to the middle ear, immunohistochemistry was performed on the anti-dexamethasone antibody to confirm the distribution of Dex or Dex-SP. As shown in FIG. 8, in both groups, strong absorption of Dex or Dex-SP was observed in the inner ear tissues from 1 hour to 24 hours after administration, and in particular, OCs were found in the inner part of the cochlea including the worm axis, spiral ganglia, And the strongest absorption than the sidewall. In the Cat-PEG-DEX treated group, the absorption of Dex was slightly increased compared to the Dex-SP group, but the levels were similar. After 72 hours of administration, Dex or Dex-SP uptake was still weak in both groups of spiral ganglia and inner hair cells of the cochlea (Fig. 8).

In the neuroprotective mouse model, Cat-PEG- Dex  or Dex - SP  Therapeutic result

As shown in Example 2, the therapeutic results in the innate neurotoxic mouse model were confirmed. The ABR thresholds of the three groups were measured at day 7 and were measured at 4 kHz (p = 0.001), 8 kHz (p = 0.002), 16 kHz (p = 0.002), 32 kHz (p = 0.001), indicating significant differences between the three groups (Fig. 9). The Deaf-Cat-PEG-Dex group was significantly higher than the Deaf-Dex-SP group at 4 kHz (p = 0.008), 8 kHz (p = 0.008), 16 kHz (P = 0.012), 16 kHz (p = 0.012), and 32 kHz (p = 0.02) compared with the Deaf-saline group (p = 0.010), and CK (p = 0.012), respectively. However, there was no significant difference in all frequency between Deaf-Saline group and Deaf-Dex-SP group.

Antibiotic-induced inflammatory responses were quantified by PCR. Expression of proinflammatory cytokines, IFN gamma, IL-6, IL-12 and IL-1 beta was upregulated by antibiotics. The reverse reaction was revealed by Dex-SP and more by CAT-PEG-Dex. To examine the protective effect of Cat-PEG-Dex or Dex-SP on kanamycin and furosemide, the organs of whole mount Corti of each group stained with Paloidin were observed with a confocal microscope, Four specific areas were observed according to the place-frequency map. As shown in FIG. 9, severe loss of floating ciliate and HC was observed in all four frequency regions of the Deaf-Saline group, and Deaf-Cat-PEG-Dex group showed slight damage at the 16 kHz region, Well preserved. The Deaf-Dex-SP group showed intermediate results between the Deaf-saline group and the Deaf-Cat-PEG-Dex group.

Claims (13)

A phospholipid nanoemulsion comprising as an active ingredient a drug for treating or preventing inner ear disease,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocerine, sodium salt (DPPS); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (DPPE-PEG2000); And inner ear-specific drug delivery nanoparticles comprising at least one member selected from the group consisting of 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP). The method according to claim 1,
Wherein the nanoparticle further comprises flaxseed oil or soybean oil. The method according to claim 1,
Wherein the nanoparticles contain 3 to 10 mg of DPPC. The method according to claim 1,
Wherein the nanoparticles contain DPPC and DPPS in a ratio of 1: 1 to 1: 4. The method according to claim 1,
Wherein the nanoparticles contain DPPC and DPTAP in a ratio of 1: 1 to 1: 4. The method according to claim 1,
Wherein the nanoparticles contain DPPE-PEG2000 and DPTAP in a ratio of 1: 1 to 1: 4. The method according to claim 1,
Wherein the nanoparticle has neutral, anionic or cationic charge on its surface. The method according to claim 1,
Wherein said nanoparticles are cationic-PEG having polyethylene glycol bound to its surface. The method according to claim 1,
Wherein the nanoparticles have a size of 150 to 250 nm. The method according to claim 1,
Wherein the nanoparticle has a maximal safe dose of 0.25 mg / mL to 0.5 mg / mL. The method according to claim 1,
Wherein the nanoparticle transmits a drug to a hair cell or Corti organ through a round window membrane. The method according to claim 1,
The drug for treating or preventing the inner ear disease is selected from the group consisting of dexamethasone, a steroid, a diuretic, a vasodilator, a betahistidine, a vestibular suppressant, dextran, mannitol or heparin. The method according to claim 1,
The inner ear disease may be selected from the group consisting of tinnitus, vertigo such as BPPV, hearing loss, sensory nerve impairment, tinnitus, chronic otitis, external lymphoid fistula, secondary endolymphatic carcinoma, Immune inner ear disease (AIED), and Meniere's disease.

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