KR20170050485A - Nanoparticle for drug delivery in inner ear - Google Patents

Abstract

The present invention relates to a PHEA-based nanoparticle for the drug delivery in the inner ear, and provides a PHEA-g-C18-Arg8 nanoparticle, a novel drug carrier, among PHEA-based nanoparticles. The nanoparticle of the present invention can deliver drugs into the inner ear, and therefore, can be remarkably effective in preventing and treating diseases of the inner ear.

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. To overcome this problem, PHEA nanoparticles among recent technologies related to drug delivery have been developed for intracellular drug delivery in various cancer cell lines, and it has been confirmed that the PHEA nanoparticles are positioned in the middle ear cavity to cause internalization Drug Deliv 2015 May; 22 (3): 367-74).

The present invention relates to a method for attaching oligoarginine, a cell penetrating peptide (CPP), to an outer wall of a PHEA nanoparticle identified in a prior study to enhance permeability to a window and inner ear cells, It was confirmed that PHEA-g-C18-Arg8, a novel drug delivery with decylamine (C18), is capable of simultaneous or separate delivery of drugs and genes, and possible endogenous transfer through the intrathecal route. It is expected to be useful for prevention and treatment of inner ear disease.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned conventional problems, and it is an object of the present invention to provide a novel PHEA- It is another object of the present invention to provide nanoparticles for treating inner ear diseases, which contain the PHEA-based particles as an active ingredient.

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 is caused by a variety of symptoms including tinnitus, vertigo such as BPPV, hearing loss, chronic otitis externa, extrinsic fistula, secondary endolymphatic carcinoma, myosinitis and vestibular neuritis, auditory neuropathy, inner ear toxicity, autoimmune inner ear disease But not limited to, Meniere's disease, but specifically refers to sensorineural hearing loss. More specifically, the inner ear's hair cells and spiral ganglion neurons (SGN) Of the hearing impaired.

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. Also, since the release rate of the therapeutic agent is a very important factor in the formulation of the sustained-release therapeutic agent, if the size of the therapeutic agent particle is set to be nanoparticles, the release rate of the therapeutic agent becomes more predictable as the size becomes relatively uniform, A therapeutic agent can be produced. The nanoparticles used in the present invention include nanoparticles based on poly (amino acid) (2-hydroxyethyl L-aspartamide: PHEA), and PHEA-g-C18, PHEA- The sizes of the g-C18-Arg8 particles are 35.1 nm, 1.25 탆 and 103.1 nm, respectively.

In one embodiment of the present invention, "PHEA-based particle" means a newly bonded nanoparticle based on PHEA nanoparticles, and the three types of PHEA-based nanoparticles used in the present invention are PHEA-g-C18 , PHEA-g-Arg8 and PHEA-g-C18-Arg8. Here, PHEA-g-C18 is used as a control, and PHEA refers to nanoparticles developed for intracellular drug delivery in various cancer cell lines in the existing studies. The nanoparticles were placed in the middle ear cavity and tested for possible inner ear transmission. C18 is octadecylamine, which means a substance that can be easily filled with hydrophobic drugs inside the PHEA nanoparticle. Arg8 means a cell permeable peptide that promotes cell uptake and has excellent potential as a transfer vector There are features. In the present invention, Arg8 was conjugated to the outer wall of the PHEA nanoparticles and used for increasing the permeability to the window and inner ear cells. The present invention aims at a novel nanoparticle PHEA-g-C18-Arg8 which is used for drug delivery in the inner ear. The nanoparticles bind CPAP and C18 to PHEA to deliver drugs and genes simultaneously or individually Means a nanoparticle made possible.

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. The concentration of nile red was calibrated to a concentration in the range of 0.1-1.67 μg / mL and the loading efficiency of Nile Red was calculated as follows:

Nile Red Injection Efficiency = (Nile Red in micelle / Nile Red added early) ⅹ 100

In one embodiment of the present invention, "transfection" refers to a phenomenon in which a nucleic acid is chemically isolated from a virus, purified, introduced into a host cell of the virus and expressed in a host cell, is a compound word formed from transformation and infection. DNA or RNA is isolated and purified from viral particles and introduced into the original host cell to induce viral infection. This method is the same as transformation in that the nucleic acid is introduced into the cell and expresses the genetic information, It is also distinguished from transformation in that it is formed.

In one embodiment of the present invention, "EGFP " means an enhanced green fluorescent protein, which means a protein used as an index of gene transfer through the expression of the protein, and also an additional reporter of transfection .

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 to transmit medicines to the parental or spinal ganglion cells, which are the main obstacles to hearing loss, through 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 are excellent in permeability by attaching a cell permeable peptide, Arg8, in order to increase permeability to inner ear cells, 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. The maximal safe dose determined by the above experiment was 0.25 mg / mL for PHEA-g-C18, and PHEA-g- 0.08 mg / mL for Arg8 and 0.025 mg / mL for PHEA-g-C18-Arg8.

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 from the artificial external lymph fluid was measured with a UV spectrometer.

In one embodiment of the present 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.

In one embodiment of the present invention, inner-specific drug delivery nanoparticles are provided, wherein oligoarginine, polyhydroxyaspartamide and octadecylamine are bound. In this embodiment, the oligorhagenin has an outer shell-bound form, wherein the polyhydroxyaspartamide and the octadecylamine are present in the interior of the cabinet. Specific drug-transferring nanoparticles, wherein the nanoparticles are represented by the following formula (1): wherein n Is an integer of 10 to 18, and m is an integer of 1 to 8.

Formula 1

Polyhydroxyethylaspartamide- (CH) _n- NH- (Arg) _m

In this embodiment, the polyhydroxyethylaspartamide is poly (2-hydroxyethyl) -L-aspartamide, wherein the nanotubes for inner-specific drug delivery are provided, Wherein the particles are grafted with octadecylamine and oligoarginine to polyhydroxyethylaspartamide, wherein the nanoparticles have an average particle size of from 90 to 110 nm < RTI ID = 0.0 > Specific drug delivery nanoparticles, wherein the nanoparticles have a maximum safety level of 0.025 mg / mL, wherein the inner-specific drug delivery nanoparticles have a size of In the above embodiment, the nanoparticles provide nanostructures for internal-specific drug delivery, which permeate through the window and deliver the drug to the cell or spinal ganglion cells.

In one embodiment of the present invention, there is provided a process for preparing polyhydroxyethylaspartamide, comprising: a first step of binding octadecylamine to polyhydroxyethylaspartamide; And a second step of further binding oligoarginine to the conjugate. The present invention also provides a method for producing inner-specific drug delivery nanoparticles. In this embodiment, the nanoparticles are provided by the following formula (1): wherein n is an integer of 10 to 18 and m is an integer of 1 to 8.

Formula 1

Polyhydroxyethylaspartamide- (CH) _n- NH- (Arg) _m

In this embodiment, the first step is carried out in a hydrophobic inclusion reaction, wherein the second step is carried out under conditions of pH 7.4 Specific drug delivery nanoparticles, wherein the nanoparticles have a size of 90 to 110 nm, wherein the inner-specific drug delivery nanoparticles have a size of from 90 to 110 nm. Wherein said nanoparticles have a maximum safety level of 0.025 mg / mL. In this embodiment, the nanoparticles are in the form of a gill window Specific drug-transferring nanoparticles, wherein the medicament is delivered to the parent cell or the spinal ganglion cell by permeation of the drug.

In one embodiment of the present invention, there is provided a pharmaceutical composition for treating inner ear diseases, which comprises the nanoparticles and contains a drug for treating inner ear disease as an active ingredient. In this embodiment, the inner ear disease is selected from the group consisting of tinnitus, vertigo such as paraplegia, deafness, chronic otitis, external lymphoid fistula, secondary endolymphatic carcinoma, myosinitis and vestibular neuritis, auditory neurotoxicity, Meniere's disease, wherein the inner ear disease is characterized by sensory nerve impairment, wherein the inner ear disease is characterized by comprising a pharmaceutical composition for the treatment of inner ear disease Wherein the pharmaceutical composition is selected from the group consisting of steroids, diuretics, vasodilators, beta histidine, antispasmodics, dextran, mannitol or heparin, , The nanoparticles are transmitted through a window to transmit the drug to the parent cell or spiral ganglion cell, And a pharmaceutical composition for treating inner ear disease.

The present invention relates to nanoparticles for drug delivery in the inner ear, and it is possible to deliver drugs and genes at the same time or separately with PHEA-g-C18-Arg8, a novel drug delivery among PHEA-based nanoparticles, And it is expected to be used for the prevention and treatment of inner ear disease.

1 shows the morphology of PHEA-g-C18, PHEA-g-Arg8 and PHEA-g-C18-Arg8 according to an embodiment of the present invention.
FIG. 2 shows release of Nile Red for each PHEA particle according to time in an artificial external lymph fluid according to an embodiment of the present invention.
Figure 3 shows the cytotoxicity of PHEA-based particles in HEI-OC1 and HMEEC cell lines according to one embodiment of the present invention.
FIG. 4 shows absorption of nanoparticles in the HEI-OC1 cell line according to an embodiment of the present invention.
Figure 5 shows the absorption of PHEA-based particles in an organotypic culture according to one embodiment of the present invention.
FIG. 6 shows a comparison of zeta potential and particle size of PHEA-g-C18-Arg8 according to an embodiment of the present invention before and after injection of EGFP.
FIG. 7 shows an in vivo confocal microscope image and gene expression of inner ear tissue according to an embodiment of the present invention.
Figure 8 shows nuclear uptake in HEI-OC1 cells for each of the PHEA-based nanoparticle types according to one embodiment of the present invention.

Example  One PHEA -Based drug delivery particles

Example  1-1: Materials

Octadecylamine (C18), ethanolamine, 1,6-hexanediamine, and 4- (N - maleimidomethyl) cyclohexane-carboxylic acid N - hydroxysuccinimide Sigma imide ester (SMCC) - Aldrich (St. Louis, MO) . Arg8 conjugated with oligo arginine peptide (Arg8) and fluorescein isothiocyanate (FITC) was purchased from Peptron (Daejeon, Korea). N , N -dimethylformamide (DMF) and dimethylsulfoxide (DMSO) solvents and fluorescent dyes Nil red (NR) and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich. Polysuccinimide (PSI) was used as a precursor for three types of nanoparticles.

Example  1-2: PHEA -g-C18 synthesis

PSI was dissolved in DMF at a concentration of 138.6 mg / mL and octadecylamine was dissolved in DMF at 70 < 0 > C. The octadecylamine solution was added dropwise to the PSI solution and subjected to a hydrophobic conjugation reaction at 70 ° C for 24 hours. The remaining succinimide chain was converted into poly (2-hydroxyethyl L-aspartamide) (PHEA) by dropwise addition of ethanolamine dropwise to the PHEA-g-C18 solution.

Example  1-3: PHEA -g- Arg8  synthesis

Ethanolamine was added dropwise to the PSI solution at room temperature and reacted for 24 hours. It was added and the mixture DMF solution of 1,6-hexanediamine-containing mixture was stirred at 70 ℃ for 24 hours (PHEA-NH _2). PHEA (PHEA-M) containing a maleimide group in its side chain was prepared through the reaction of PHEA-NH ₂ with SMCC. PHEA-M mixture and Arg8 peptide powder were mixed in PBS buffer (pH 7.4) and cultured at room temperature for 24 hours.

Example  1-4: PHEA -g-C18- Arg8  synthesis

PHEA-g-C18-Arg8 was synthesized through a combination of the methods described for PHEA-g-C18 and PHEA-g-Arg8. After free chemical residues were dialyzed, the product was freeze-dried. Nile red dyes were used to investigate drug release patterns from each drug delivery particle in place of direct drug delivery. Arg8-FITC was also used to confirm co-localization of nuclear particles in each drug particle and inner nuclear cells.

Example  1-5: PHEA -g-C18- Arg8 / CMV - EGFP  synthesis:

The CMV-EGFP DNA fragment (1762 bp) was eluted through an enzymatic cleavage from pEGFP-C2 plasmid (Invitrogen, Calif.) To Apa I and ApaL I (Takara, Japan). PHEA-g-C18-Arg8 nanoparticles were diluted in HEPES solution (20 mM, pH 7.4) at a concentration of 1 mg / mL and sonicated. After PHEA-g-C18-Arg8-GFP was obtained, it was immediately vortexed for 30 seconds and then incubated at a specific weight ratio at room temperature for the next 5 minutes. The newly prepared complexes were used in the following experiments. Zeta potential and particle size were measured through a dynamic light scattering measurement (Zetasizer Nano ZS90; Malvern, Worcestershire, UK).

Example  2 Characterization and confirmation of nanoparticle delivery

Example  2-1: Size of nanoparticles and Morphology

Arg8 was confirmed by 400 MHz Fourier transform nuclear magnetic resonance spectroscopy (FT-NMR) in a DMSO-d6 solvent. Zeta potential and particle size were measured with a dynamic light scattering meter (Zetasizer Nano ZS90; Malvern, Worcestershire, UK). The sizes of PHEA-g-C18 and PHEA-g-C18-Arg8 nanoparticles were found to be 35.1 nm and 103.1 nm, respectively, and PHEA-g-Arg8 particles were 1.25 μm due to weak gelation due to hydrophilic properties. The measurement results of zeta potential and particle size of PHEA-g-C18 and PHEA-g-C18-GFP nanoparticles synthesized in Examples 1-4 and 1-5 are shown in FIG.

Example  2-2: Preparation of nanoparticles Morphology

 The morphology of each particle dyed with phosphotungstic acid was measured by a 300 kV TEM. The morphology of PHEA-g-C18, PHEAN-g-Arg8 and PHEA-g-C18-Arg8 measured by TEM is shown in Fig. The amount of nile red injected into each nanosource was determined by fluorescence spectroscopy (LS50B; Perkin Elmer, Waltham, Mass.). That is, a 10 mg Nile Red-injected nanotransporter sample was dissolved in 3 mL DMSO and mixed for several minutes to disintegrate all particles and release Nile Red into solution. The fluorescence excitation and emission wavelengths are 517 nm and 584 nm, respectively. The amount of Nile Red in DMSO was calibrated to a concentration ranging from 0.1 to 1.67 μg / mL and the loading efficiency of Nile Red was calculated as follows:

Nile Red Injection Efficiency = (Nile Red in micelle / Nile Red added early) ⅹ 100

Example  2-3: Drug release

Nile Red release in a simulated perilymph environment was tested with three drug nanotransporters. The simulated other lymphoid environment is an aqueous solution in 137mM NaCl, 5mM KCl, 2mM CaCl 2, 1mM MgCl 2 And it was done with 1mM NaHCO _3. 5 mg of Nile Red-injected drug delivery vehicle Aliquot was added to 1 mL of the artificial external lymph solution and incubated for a defined period of time at 37 DEG C with stirring at 100 rpm. Whole medium was replaced with fresh artificial external lymph at each time point. Each sample solution was collected and 1 mL of 50% ethanol was added, and the amount of Nile Red released into the UV spectrometer at 510 nm was quantified 3 times. Due to the stable hydrophobic interaction between octadecylamine and nile red, the release rate was sustained in the C18-bonded nanoparticles, whereas in PHEA-g-Arg8, a large amount of nile red was released due to unstable interactions with the carrier particles (Fig. 2).

Example  3: HEI -OC1 and HMEEC  Cell culture of cells

Example  3-1: HEI -OC1 and HMEEC  Cell In vitro (in vitro) cell culture

Immortalized mouse Corti organ cell line HEI-OC1 and human midgut cells (HMEEC) MK Park (Seoul National University College of Medicine, Seoul, Korea). HEI-OC1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) filled with 10% fetal bovine serum (FBS) and 50 U / mL recombinant mouse interferon-y. The cultivation was carried out at 33 캜 in a humidified 10% CO ₂ environment. HMEEC cells were maintained in a mixture of DMEM and BEBM (1: 1) filled with growth factors. The growth factors were: hEGF (0.5 ng / mL), epinephrine (5 μg / mL), bovine pituitary extract (0.13 mg / mL), hydrocortisone (0.5 μg / mL) (5 μg / mL), triiodothyronine (6.5 ng / mL), transferrin (10 μg / mL), retinoic acid (0.1 nM), gentamycin (50 μg / mL) and amphotericin B ). The sheep were run at 37 ° C in a humidified 5% CO ₂ environment.

Example  3-2: Cytotoxicity test

The cytotoxicity of each drug delivery vehicle to HEI-OC1 and HMEEC cells was tested as follows. Move the HEI-OC1 and HMEEC cells in 96-well plates at a density of 0.1mL complete growth medium per well in × 10 ⁴ cells to 80% confluence (confluence) and incubated for 24 hours. The cells were then treated with PHEA-based particles (PHEA-g-C18, PHEA-g-Arg8 and PHEA-g-C18-Arg8) and DMSO as the vehicle for 30 or 60 hours. (EZ-Cytox; Daeil Lab, Seoul, Korea) using a commercially available 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay ) To determine cell viability. After incubation, 10 세포 of cell viability assay reagent was added to each well and the optical density of the sample was determined at 450 nm with a microplate reader (Bio-Rad Laboratories, Hercules, Calif.). The cytotoxicity of each of HEI-OC1 and HMEEC cells was found to be decreased in the order of PHEA-g-C18-Arg8 and PHEA-g-C18 in the order of PHEA-g-Arg8 decrease (FIG. 3).

Example  3-3: Cell Absorption Test

G-C18, PHEA-g-Arg8 and PHEA-g-C18-Arg8 at 1, 15 and 30 hours after treatment of the cell uptake of the nanoparticles with a confocal microscope (LSM5 Live Configuration Variable VRGB ). HEI-OC1 cells were placed on a Lab-Tek II chamber slide system (Snow, Roskilde, Denmark) and then treated with nanoparticles in the culture medium with the maximum safety dose determined in the cytotoxicity test. The maximum safe amount determined in Example 3-2 was 0.25 mg / mL for PHEA-g-C18, 0.01 mg / mL for PHEA-g-Arg8 and 0.025 mg / mL for PHEA-g-C18- to be. Cells were fixed with 4% paraformaldehyde solution and fixed with Vectashield fixed medium (Vector Laboratories, Burlingame, Calif.) With DAPI. The treated cells were observed with a confocal microscope at 415-480 nm band pass filters for DAPI signals, 500-525 nm band pass filters for FITC and EGFP and 550 nm long wavelength pass filters for Nile Red. Untreated cells were used as negative control and Lipofectamine 3000 (Invitrogen, Carlsbad, Calif.) Was used as positive control. As shown in Fig. 4, PHEA-g-C18-Arg8 nanoparticles showed superior cell uptake and co-localization in red and green signals. PHEA-g-C18 nanoparticles showed the weakest cell uptake, while PHEA-g-Arg8 showed better absorption of intracellular nile red, while co-localization with FITC was low. The fluorescence intensities of nile red and FITC with Arg8 in PHEA-g-Arg8 and PHEA-g-C18-Arg8 were measured to confirm co-localization degree between Nile red and Arg8 conjugated particles in HEI-OC1 cells. Where green represents FITC-conjugated Arg8, red for nile red and blue for DAPI stained nuclei. The relative fluorescence intensities of PHEA-g-C18-Arg8 particles were highest for each of the PHEA-based nanoparticle types in FIG. 8, and the absorption of HEI-OC1 cells was the highest Respectively.

Example  4: Corti  Absorption of nanoparticles into the organ

Example  4-1: In vitro (ex vivo ) exam

Primary cochlear explants were prepared from C57 / BL6 mice 3 days postnatally (P3). The dissected cortical organ was incubated with high-glucose DMEM containing 5% FBS, 5% horse serum and 10 ng / mL ampicillin at 37 ° C in 5% CO ₂ in an incubator incubated at 37 ° C. After 4 and 15 hours of treatment, PHEA-based particles were treated with the maximum safe amount. The cells were fixed with 4% paraformaldehyde solution, permeabilized with acetone, conjugated with Alexa Fluor 488 Phalloidin (Molecular Probes, Eugene, USA), and fixed with Vectashield fixed medium containing DAPI Vector reverberator). Fluorescence microscopy (Eclipse TE300 microscope; Nikon, Tokyo, Japan) was used for low-magnification images and nanoparticle absorption was investigated through a confocal microscope for high-magnification images. The specimens were stained with DAPI and FITC-labeled phalloidin to assess the intactness of the hair cell cilia and tissue structure. In Fig. 5, red particles indicate intracellular absorption and show absorption of main nanoparticles in the spiral limb, while weak absorption in the parental cells (green: Paloidine, red: nile red, blue : DAPI staining nuclei).

Example  4-2: In vivo (in vivo ) exam

Surgical procedures for one month old male C57 / BL6 mice were performed as follows. Prior to surgery, mice were anesthetized with a mixture of 30 mg / kg zolethyl (Kerberos, France) and 10 mg / kg Rompun (Bayer, Level Cusen, Germany). The mice were placed in a supine position on a temperature-regulated thermal pad, and the ventral side of the neck was shaved and sterilized with 70% ethanol. A midline incision was made and the thymus was dissected to expose the posterior belly of the left-side digastric muscle. Then, the above-described two-fingered muscle was removed and the left bulla was exposed. The puncture was made with a forceps and the hole was widened until the round window niche was exposed. The diameter of the hole made in the blister is approximately 2 mm. A small portion of a gelatin sponge (Johnson and Johnson, New Brunswick, NJ) absorbing 20 μl of the nanoparticle solution was placed in the niche. The hole was then covered with the wax (Beck Brown, Mel, Germany) and the wound site was closed. After surgery, limadill (1.0 mg / kg; Pfizer, Frost, UK) was injected for pain relief. Baytril (10 mg / kg; Orion, Hamburg, Germany) was intraperitoneally administered once daily as a prophylaxis against middle ear infection. Twenty-four hours after surgery, the blisters were fixed with cardiac perfusion containing cardiac perfusion 4% paraformaldehyde (Merck, Darmstadt, Germany). The isolated cochlea was washed with tap water for 1 minute and stored in fixative for 2 hours to remove free particles that might remain on the outer surface. After washing with PBS, the isolated cochlea was dissected under a stereoscopic microscope, and a cortical organ, a lateral wall and a basal turn modiolus were harvested. Dyeing and image capture were performed in the same manner as described except for the fixing method. Specimens were observed with a confocal microscope (Fig. 7).

Example  4-3: In vivo  Gene expression

The same surgical procedure as described was carried out with PHEA-g-C18-Arg8 / EGFP DNA. After 48 hours of operation, the isolated cochlea was dissected, fixed in 4% paraformaldehyde for 2 hours, and decalcified in 5% EDTA for 6 hours. The cortical organ, sidewall and base-wow axis were harvested. The tissues were stained with GFP-antibody (Life Technologies, Oregon, USA) for GFP expression and the Corti organ was stained with Alexa Fluo 594 Paloidin (Molecular Probe, Eugene, USA). Thereafter, it was observed through a confocal microscope. To visually validate the gene delivery system, EGFP was used as an additional reporter of gene transfection. As shown in FIG. 7, PHEA-g-C18-Arg8-GFP showed the most abundant EGFP expression in the wax axis and low expression in the sidewall but no expression in the parental cells (PHEA-g-C18-Arg8-NR Group: Green: Paloidine, Red-Nile Red; PHEA-g-C18-Arg8-GFP Group: Green: GFP, Red: Paloidine, Blue: DAPI staining nucleus).

Example  5: Statistical analysis

All data were expressed as mean ± standard deviation of three measurements. One-way ANOVA and two-tailed independent t-test for multiple groups were used to confirm differences between groups. In all analyzes, p <0.05 was considered statistically significant. All experiments were conducted according to the National Ethical Guidelines. This study was approved by the Institutional Review Board of the hospital (CMCDJ-AP-2014-004, DC15TISE0011).

Claims (21)

Specific examples include inner ear-specific drug delivery nanoparticles, wherein oligoadenosine, polyhydroxyaspartamide and octadecylamine are bound. The method according to claim 1,
Wherein said oligorhagenin is bound to the outer shell. The method according to claim 1,
Wherein said polyhydroxyaspartamide and octadecylamine have the form of being bound in a micelle structure to the interior of a core. The method according to claim 1,
Wherein the nanoparticles are represented by the following Formula 1:
Formula 1
(Arg) _m - polyhydroxy ethyl aspartic amide - (CH) _n -NH ₂
here,
n is an integer from 10 to 18,
m is an integer of 1 to 8; The method according to claim 1,
Wherein said polyhydroxyethylaspartamide is poly (2-hydroxyethyl) -L-aspartamide. The method according to claim 1,
Wherein the nanoparticles are grafted with polyhydroxyethylaspartamide with octadecylamine and oligoarginine. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; The method according to claim 1,
Wherein the nanoparticles have a size of 90 to 110 nm. The method according to claim 1,
Wherein said nanoparticles have a maximal safe dose of 0.025 mg / mL. The method according to claim 1,
Wherein the nanoparticle transmits a drug to a parent cell or a spiral ganglion neuron by passing through a round window membrane. A first step of binding octadecylamine to polyhydroxyethylaspartamide; And
And a second step of further binding oligoarginine to the conjugate. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; 11. The method of claim 10,
Wherein the nanoparticles are represented by the following Formula 1:
Formula 1
Polyhydroxyethylaspartamide- (CH) _n- NH- (Arg) _m
here,
n is an integer from 10 to 18,
m is an integer of 1 to 8; 11. The method of claim 10,
Wherein the first step is carried out by a hydrophobic conjugation reaction. &Lt; Desc / Clms Page number 19 &gt; 11. The method of claim 10,
Wherein the second step is carried out at a pH of 7.4. 11. The method of claim 10,
Wherein the nanoparticles have a size of 90 to 110 nm. 11. The method of claim 10,
Wherein said nanoparticles have a maximum safety of 0.025 mg / mL. 11. The method of claim 10,
Wherein the nanoparticles transmit the drug to the parent cell or spiral ganglion cell through a window. 10. A pharmaceutical composition for the treatment of inner ear diseases, which comprises a nanoparticle according to any one of claims 1 to 10 and contains a medicament for treating inner ear disease as an effective ingredient. 18. The method of claim 17,
The inner ear disease may be selected from the group consisting of tinnitus, vertigo such as BPPV, hearing loss, chronic otitis externa, external lymphoid folliculitis, secondary endolymphatic carcinoma, myosinitis and vestibular neuritis, auditory neurotoxicity, And Meniere's disease. The pharmaceutical composition for treating inner ear disease according to claim 1, 19. The method of claim 18,
Wherein said inner ear disease is characterized by sensorineural hearing loss. 19. The method of claim 18,
Wherein the pharmaceutical composition is selected from the group consisting of steroids, diuretics, vasodilators, beta histidine, antispasmodics, dextran, mannitol or heparin. 18. The method of claim 17,
Wherein the nanoparticles are permeable to a gill window or a spinal ganglion cell.

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