KR20160120028A

KR20160120028A - Nanoparticles for genes drug delivery with siRNA for the long -term treatment of retinal disorders and method for preparing the same

Info

Publication number: KR20160120028A
Application number: KR1020150048982A
Authority: KR
Inventors: 김현철; 이지황; 이종범; 박규형; 우세준; 홍혜경
Original assignee: 서강대학교산학협력단; 서울시립대학교 산학협력단; 서울대학교산학협력단; 서울대학교병원 (분사무소)
Priority date: 2015-04-07
Filing date: 2015-04-07
Publication date: 2016-10-17
Also published as: KR101880790B1; WO2016163718A1

Abstract

The present invention relates to a self-assembled and condensed high molecular weight siRNA core particle and a particle for efficiently delivering a large amount of siRNA into the inside of the retina by coating the outside with a cationic polymer and hyaluronic acid, and a method for producing the same.
Since the core of the present invention contains at least 1 x 10 ⁶ small interfering RNA (siRNA) or antisense nucleic acids, it can deliver a larger amount of siRNA than the conventional one.
By coating the outer shell with (-) charged hyaluronic acid, it is possible to prevent aggregation with the vitreous meshwork structural materials having (-) charge and reach the inner limiting membrane of the retina. In addition, hyaluronic acid binds to the CD44 receptor of Muller cells, allowing the structure of the retina to pass through the sub-retinal space through the transcytosis mechanism. Some of them inhibit the translation of target gene mRNA in Muller cells, and many of them bind to the CD44 receptor present in the retinal pigment epithelial (RPE) cell membrane and enter the cell to analyze the mRNA of the target gene in RPE translation.

Description

[0001] The present invention relates to a long-term siRNA-based nano-medison and a method for preparing the same,

The present invention relates to a dielectric drug delivery particle and a method for producing the same, and more particularly, to a method for manufacturing a dielectric drug delivery particle and a method for preparing the same, The present invention relates to a particle for efficiently transferring the siRNA into the inside thereof and a method for producing the same.

Age-related macular degeneration (AMD) is the leading cause of blindness in adults and is an age-related ocular disease with an increased prevalence. It is characterized by increased vascular endothelial growth factor (VEGF) and subsequent choroidal neovascularization (CNV). The number of AMD patients is increasing with the aging society, but the complete treatment of AMD has yet to be developed. Currently, photodynamic therapy and anti-VEGF (vascular endothelial growth factor) therapy are used. Photodynamic therapy is the treatment of light with a light-sensitive photopheresis agent (photosensitizer) , Light is selectively accumulated only in diseased cells and has a therapeutic effect. In order to treat CNV associated with macular degeneration, the drug is injected intravenously and activated in the eye by a laser to prevent the formation of new blood vessels. And anti-VEGF therapy usually treats CNV using Avastin, Lucentis and Eylea, which are anti-VEGF antibody bases. It is known that the broad-spectrum method can inhibit the activity of CNV, but does not have the effect of improving vision. Currently, intravenous anti-VEGF injections have been shown to be the most effective for improving vision and have been established as the standard treatment for AMD. Intraocular injection of anti-VEGF agents has been proven to be effective in various retinal diseases related to VEGF, such as diabetic retinopathy, retinal vein occlusion, and myopathic macular degeneration, as well as AMD. The half-life of anti-VEGF injected intraocular injections is about 3-7 days.

Recently, gene therapy has been studied for the treatment of macular degeneration. Since mRNA and DNA can be transcribed into several proteins, gene therapy using RNA and DNA can be an effective treatment method.

Small interfering RNA (siRNA) is a short RNA of double helix structure composed of 19-22 nucleic acids. When it binds to the same messenger RNA (messenger RNA, mRNA) with its sense strand, (Elbashir, SM et al., 2001, Nature 411, 494-8) by degrading the mRNA associated with the RNA induced silencing complex (RISC). Various possibilities have been studied for the treatment of gene related diseases by utilizing the property of such siRNA which suppresses effective and precise expression of the same mRNA with a small amount, and the market for the therapeutic drug development using siRNA has been remarkably increased year by year .

However, enzyme instability and poor cellular permeability of siRNA have been a major obstacle to the practical application of clinical therapies. A major disadvantage of siRNA-based agents in the treatment of retinal disease is that the duration of the drug efficacy is shorter than that of antibody-based protein drugs and needs to be injected into a more frequent and repetitive vitreous cavity. To solve this problem, various transporters have been developed and various molecules are bonded to siRNA itself. In order to prevent direct exposure to the enzyme and increase cell permeability, a cationic polymer or a lipid molecule and a peptide are electrostatically bound to an anionic siRNA to form a nano-sized complex having a cationic property as a whole, And at the same time, the interaction with the anion-like cell membrane is improved to increase the intracellular delivery efficiency. More specifically, 2-dioeloyl-3-trimethylammonium (DOTAP), polyethylenimine (PEI), chitosan groups, and poly L-lysine (PLL) have positive charge characteristics and high RNA solubility and cell penetration efficiency. It has a well-ordered, 3-dimensional network structure consisting of collagen fibrils with 98% of the vitreous water but proteoglycan filaments with 1-2% anion. It is known that injection of cationic RNA nanocomposite into the vitreous binds to the vitreous body structure and does not reach the retina. In addition, the retina is composed of several layers of cells. Inner limiting membrane is composed of a fine three-dimensional meshwork structure having an average pore structure of 13.43 nm. The outer limiting layer is composed of pore The size of the tight junction structure is 3-3.6 nm. In general, nanotransporters (generally ~ 100 nm or more) pass through the diffusion mechanism to reach the retinal pigment epithelium layer and the choroid to treat the choroidal neovascularization It is a structure that can not be done.

The present inventors have proposed siRNA-microsponges coated with linear PEI by self-assembling siRNA replicated with RCT (self-assembled RNA interference microsponges for efficient siRNA delivery (published on Nov. 26, 2012) . However, siRNA-microsponge coated with linear PEI was cationic and toxic in vivo, which was difficult to inject into the retina.

Recently, modified cationic polymers such as glycol chitosan and branched PEI have been used to solve the cytotoxicity problem by lowering the positive charge intensity. However, decreasing the positive charge intensity has decreased the binding force between the RNA and the carrier, Lt; RTI ID = 0.0 > enzymes. &Lt; / RTI >

[Reference patents]

Korean Patent Publication No. 10-2011-83919

Korean Patent No. 10-806088

The present invention overcomes a number of siRNAs, gene therapy agents, for the treatment of various retinal diseases including senile macular degeneration associated with choroidal neovascularization, thereby effectively preventing retinal pigment epithelium cells And to provide gene drug delivery particles for treating retinal disease that can be delivered to the relevant retinal cells.

The present invention provides a dielectric drug delivery particle having a high efficiency of gene drug delivery into the retina and a low cytotoxicity.

The present invention provides a dielectric transporting particle in which a siRNA hydrogel-based gene material produced by an RCT process is not degraded by an enzyme but can also penetrate the retinal structure together with the vitreous structure to reach the cells in the retina.

One aspect of the present invention is directed to a core comprising one or more self-assembled nucleic acid molecules targeting a gene;

A cationic polymer layer surrounding and concentrating the core; And

And an anionic hyaluronic acid outer layer coated on the outer surface of the cationic polymer layer.

In another aspect, the present invention provides a method of amplifying a double-stranded RNA comprising the steps of amplifying a circular DNA comprising antisense RNA and a complementary sense RNA to produce a plurality of double-stranded RNAs, and self-

Mixing the spherical core with a cationic polymer to condense the core by ionic interaction; And

And coating an outer layer of an anionic hyaluronic acid with an electrostatic attraction on the outer surface of the condensed core part.

Since the core of the present invention contains at least 1 x 10 ⁶ small interfering RNA (siRNA) or antisense nucleic acids, it is capable of delivering a larger amount of siRNA than conventional ones, And the persistence of the effect is very large.

By coating the outer shell with (-) charged hyaluronic acid, it is possible to prevent aggregation with the vitreous meshwork structural materials having (-) charge and reach the inner limiting membrane of the retina. In addition, hyaluronic acid binds to the CD44 receptor of the Mueller cells to pass the structure of the retina into the sub-retinal space through a transcytosis mechanism, and then to enter the retinal pigment epithelium cell by CD44 receptor-mediated endocytosis block the translation mechanism of mRNA.

FIG. 1 is a conceptual diagram of a nano location for dielectric transfer according to an embodiment of the present invention.
Figure 2 shows a method for producing the core (siRNA-microsponge) of the present invention.
Figure 3 shows the size distribution (A) of the nanoballs of the present invention coated with siRNA-microsponge core, siRNA-microsponge core -branched PEI (b-PEI) and finally outer layer with hyaluronic acid, B).
FIG. 4 is a photograph of a nano-ball solution of the present invention coated with siRNA-microsponge solution and hyaluronic acid.
FIG. 5 shows the results of a test in which the produced nanoparticles are delivered into retinal cells under in vitro conditions, and a PCR in which anti-VEGF-A siRNA delivered by nanoparticles selectively inhibits VEGF-A expression.
FIG. 6 shows the results of confirming the efficacy of CNV-induced intramuscular injection of CNV-induced nanoparticles using a laser for 2 weeks and 6 weeks. The injected nanoparticles are transferred to the choroid through the retina cells to inhibit VEGF-A expression and inhibit CNV.
FIGS. 7 and 8 show that the produced nanoparticles inhibit angiogenesis by inhibiting VEGF-A expression in retinal cells by intravitreal injection of an oxygen-induced retinopathy model.
FIG. 9 shows the result of toxicity test using the prepared nanoparticles.

The present invention can be all accomplished by the following description. The following description should be understood to describe preferred embodiments of the present invention, but the present invention is not necessarily limited thereto.

1 is a conceptual diagram of a dielectric-transmitting particle according to an embodiment of the present invention.

Referring to FIG. 1, the dielectric nanoparticles of the present invention include a core 10, a cationic polymer layer 20, and an outer layer 30 coated with hyaluronic acid.

The core portion 10 comprises one or more self-assembled nucleic acid (11) molecules targeting mRNAs of a specific gene. The specific gene may be any mRNA that causes retinal disease. For example, the core moiety includes nucleic acid molecules that target genes that cause retinal disease, such as age-related macular degeneration (AMD), glaucoma, and the like.

The core may comprise a plurality of nucleic acid molecules, and the nucleic acid molecules may all have the same nucleic acid sequence or may have at least one common nucleic acid sequence. The nucleic acid molecule may have a nucleotide sequence including one or more sequence elements. The nucleic acid molecule in the core may comprise a nucleotide sequence having a double-stranded or triple-stranded structure. The nucleic acid molecule in the core may comprise a nucleotide sequence having two or more complementary units. The nucleic acid molecule in the core may comprise a STEM-LOOP or a linear structure. For example, the complementary unit may have one or more STEM-LOOP (e.g., hairpin structure) structures. The nucleic acid molecule in the core may also comprise one or more nucleotide sequences that remain as a single strand.

The nucleic acid (11) molecule may be a nucleic acid such as small indirect RNA (siRNA), antisense nucleic acids, or microRNA (miRNA) for the purpose of delivery into cells. siRNA binds to the same messenger RNA (mRNA) as the sense strand of its sense strand to inhibit the expression of a specific protein by degrading the mRNA bound to the RNA induced silencing complex (RISC) .

For example, the nucleic acid molecule 11 may include, but is not limited to, anti-sense RNAs targeting vascular endothelial growth factor (VEGF) of the retina and double-stranded RNAs complementary thereto.

The nucleic acid molecules in the core can be self-assembled by folding into a stable three-dimensional structure. Typically, different portions within the nucleic acid can be self-assembled by non-covalent interactions. The nucleic acid molecules in the core may be arranged in a crystal structure comprising a lamellar sheet.

The core may consist of one or more entangled nucleic acid molecules.

The nucleic acid molecule of the core may have a molecular weight of 1 x 10 < ⁵ > g / mol or more.

The core may be formed by aggregating a small interfering RNA (siRNA) unit sequence or a nucleic acid having a plurality of copies of antisense nucleic acid unit sequences.

A very large amount of nucleic acid can be loaded into the core. That is, the core may be loaded with at least 1 × 10 ³ small interfering RNA (siRNA) unit or antisense nucleic acid unit sequences, and may be replicated in a range of 1 × 10 ³ to 1 × 10 ¹⁰ or more, .

For example, the nucleic acid molecule may be degraded into small interfering RNA (siRNA) or antisense nucleic acids targeting a specific gene (e.g., retinal vascular endothelial growth factor (VEGF)) that induces retinal disease .

The nucleic acid molecule may comprise at least one cleavage site. For example, the cleavable site can be separated by enzymes (nuclease, dicer, DNAase, RNAase).

The cationic polymer 20 surrounds the core and condenses the core by ionic interaction. More specifically, the branched PEI having a strong positive charge may be condensed or compressed while surrounding a negative charge core, so that the core portion can be in a nanoparticle form. A cationic polymer layer having a strong (+) charge is coated on the outer surface of the core.

The cationic polymer 20 may be at least one selected from the group consisting of polyethyleneimine, polyamine and polyvinylamine, and preferably branched PEI having a relatively low or no toxicity.

The cationic polymer was injected into the core at a weight ratio of 1: 0.05 to 1, preferably 1: 0.1 to 0.5, more preferably 1: 0.2 to 0.25, to form a cationic polymer layer.

The outer layer 30 is a layer of hyaluronic acid coated on the outer surface of the cationic polymer layer 20.

The hyaluronic acid outer layer 30 is coated on the outer surface of the cationic polymer layer 20 with electrostatic attraction.

The hyaluronic acid outer layer represents a negative charge, and therefore the particles including the core of the present invention also exhibit negative charges.

Various forms of hyaluronic acid are possible for the purposes of the present invention. Particularly, a hyaluronic acid having a large molecular weight and a small hyaluronic acid may be used, preferably 5 to 10 K, more preferably 5 K hyaluronic acid.

Hyaluronic acid (HA) is a large complex oligosaccharide consisting of up to 50,000 pairs of disaccharide glucuronate-β (1-3) N-acetylglucose-amine β (1-4) as a basic unit. It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is in the form of a random coil with a diameter of about 50 nm.

The vitreous space in the eye has a well-ordered, 3-dimensional network structure consisting of collagen fibrils with 98% water but proteoglycan filaments with 1-2% anions. Particles coated only with branched PEI in this space can not pass through the vitreous structure due to the mutual attractive force of (+) charge and (-) charge and aggregate in the vitreous space. The nanoparticles of the present invention are coated with the outer layer of hyaluronic acid so that the particles are entirely negatively charged, thereby preventing the particles from clumping with the vitreous material to prevent them from being disturbed.

The retina is composed of several cell layers. The innermost layer is composed of a fine three-dimensional meshwork structure with an average pore size of 13.43 nm. The pore size of the external limiting membrane between the outer plexiform layer and the photoreceptor layer And has physical barriers of a structure having a tight junction structure of 3-3.6 nm.

The particles of the present invention have a size of 50 to 350 nm, preferably 200 to 300 nm, and more preferably 250 to 260 nm.

Although the nanoparticles can not overcome the physical barriers of the retina in consideration of their size, they react with the CD44 receptor of Muller cells by hyaluronic acid coated on the outer shell of the nanoparticles to transcytosis and pass through the retina can do. That is, the nanoparticles of the present invention can reach the outer retina efficiently with the outer layer of hyaluronic acid.

The nanoparticles inhibit the translation of target gene mRNA in Muller cells to remain in Muller cells during the transcytosis process and prevent the expression of the target gene. In addition, the nanoparticles reaching the outer retina are again transported to the retinal pigment epithelium It reacts with the CD44 receptor in the cell and enters the cell by endocytosis, which interferes with the translation of the target gene mRNA and blocks the target gene expression.

In another aspect, the invention relates to a method of making a nanoparticle for delivering a dielectric drug. The method includes a core forming step, a core condensing step and an outer layer coating step.

The core forming step is a step of amplifying a circular DNA containing antisense RNA and a sense RNA complementary thereto to prepare a plurality of double strand RNAs, and self-assembling the plurality of double strand RNAs to form a spherical core. The circular DNA can be formed into a plurality of double stranded RNAs by using Rolling Cycle Amplification (RCA) or Rolling Cycle Transcription (RCT).

Figure 2 shows a method of making the core. Referring to FIG. 2, a long linear single-stranded DNA encoding an antisense of anti-VEGF siRNA and a sense sequence complementary thereto is prepared. Because the ends of the linear DNA are partially complementary to the T7 promoter sequence, the long linear strand is hybridized with short DNA strands containing the T7 promoter sequence to form cyclic DNA. The nick of circular DNA is chemically closed with T4 DNA ligase. Closed circular DNA is used to produce RNA transcripts through RCT. RCT is used to generate the antisense, antisense, anti-angiogenic factor siRNA-encoded hairpin RNA structure from the circular DNA as repeating units. The plurality of hairpin structures may be self-assembled to form a sponge-like core. That is, the present invention can produce a large number of spherical cores having a small interfering RNA (siRNA) unit sequence or antisense unit sequence of at least 1 × 10 ³ or more using RCT from the circular DNA.

The condensing step is a step of mixing the cationic polymer to the spherical core to condense the core by ionic interaction.

The weight ratio of the core and the cationic polymer may be 1: 0.05 to 1, preferably 1: 0.1 to 0.5, more preferably 1: 0.2 to 0.25.

The condensation step is carried out at a room temperature and a neutral pH of 6.5 to 7.5.

The coating step is a step of coating a hyaluronic acid outer layer on the outer surface of the cationic polymer. Preferably, the method may coat the outer layer with an electrostatic attraction between the (+) charge of the cationic polymer and the (-) charge of the hyaluronic acid.

The weight ratio of hyaluronic acid to the core may be 1: 0.1-0.5 (core: hyaluronic acid), preferably 1: 0.2-0.25.

The coating reaction is carried out at room temperature and at neutral pH (6.5 to 7.5).

As for the coating step, the above description can be referred to.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following embodiments are provided for the purpose of easier understanding of the present invention, but the present invention is not limited thereto.

Example One

Synthesis of core

The sense of vascular endothelial growth factor (VEGF-A) in rats is 5'-AUGUGAAUGCAGACCAAAGAA TT-3 'and the antisense is 5'-UUCUUUGGUCUGCAUUCA CAU TT-3'. As shown in Fig. 2, a long linear single-stranded DNA encoding the sense and antisense was prepared.

The ligated circular DNA template was incubated with the reaction buffer (8 mM Tris-HCl, 0.4 mM spermidine, 1.2 mM MgCl ₂ , and 2 mM dithiothreitol) at 37 ° C for 20 hours as in T7 RNA polymerase. The resulting solution was pipetted several times and sonicated for 5 minutes to disintegrate the particles. The solution was centrifuged at 6,000 rpm for 6 minutes and the supernatant was removed. The particles were then washed with RNase-free water. The solution was again sonicated for 1 minute and centrifuged. The washing step was repeated three more times to remove the RCT reagent to obtain RNA-microsponge cores. Concentrations of RNA-microsponge cores were determined using Quant-iT RNA BR assay kits (Invitrogen). The self-assembled RNA interference microsponges for efficient siRNA delivery (nature material, published on Nov. 26, 2012) were used for core preparation.

Nano ball (Particle) manufacturing

To prepare siRNA nanoballs (particles) from the siRNA microsponges core, 1 mg of siRNA microsponges synthesized were dissolved in 100 μl of nuclease-free water and dissolved in a branched PEI (bPEI) solution (1 mg bPEI / ml nuclease- free water), vortexed, and cultured at room temperature for 10 minutes. Then, hyaluronic acid (HA) solution (1 mg HA / ml nuclease-free water) was added to the solution, and the mixture was incubated for 5 minutes. The solution was centrifuged at 13,200 rpm to obtain nanoballs (particles).

Characterization of manufactured nanoparticles

The size distribution of the finally prepared nanoballs, siRNA-microsponge core, and siRNA-microsponge core-bPEI is shown in Fig. 3A.

(Fig. 3A, red), siRNA-microsponge core is 659.92 ± 45.79 nm (Fig. 3A, black), siRNA-microsponge-bPEI is 141.68 ± 19.38 nm , Blue). FIG. 3B is an electron micrograph showing that the finally prepared nanoballs (particles) are spherical.

FIG. 4 is a photograph of the siRNA-microsponge solution and finally the nanoball solution prepared. Referring to FIG. 4, since the siRNA-microsponge solution has a high turbidity due to its sub-micro or micro size, the nano-ball solution of the present invention is very transparent because it is nano-sized. The nanoball solution of the present invention is very transparent. This is due to the effect of the coating, as the size of the particles is condense to increase the transparency, which indirectly confirms the particle size reduction by the coating.

Cell culture

B16F10 murine melanoma cells were maintained in DMEM media (Welgene, Dalseogu, Daegu, South Korea) (mixed with 10% FBS and 1% penicillin / streptomycin) in a 37 ° C 5% CO2 incubator. Human retinal pigment epithelial (ARPE-19) cells (American Type Culture Collection, Manassas, Va.) Were cultured in Dulbecco's modified Eagle's medium F-12 (Life Technologies Korea, Seoul, South Korea ) (10% FBS mixed with 1% penicillin / streptomycin).

Cell adsorption test

Human ARPE-19 cells (2 × 10 ⁴ ) were seeded in a 12-well plate and incubated for 24 hours. In each well, SYBR® Green I dyed nanoballs were mixed with serum free medium and added at a concentration of 6 μg / μl. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) after 3 hours and 12 hours, and stained with DAPI. The cells were observed under a fluorescence microscope and are shown in FIG. 5A.

mRNA Expression inhibition test

3 × 10 ⁵ B16F10 mouse melanoma cells were seeded on a 6-well plate, and mono-siRNA (Mono), siRNA-microsponge (Sponge), no treatment (CTL, C), and commercialized transfection agents (lipofectamine, Lipo ), And the nanoparticles (NP) of the present invention were mixed with serum free medium. Afterwards, all cells were removed and mRNA was extracted and cDNA was prepared for mouse VEGF-A and mouse beta actin. The cDNA was amplified by PCR cycler and the level of mRNA was confirmed. For quantitative analysis, real-time PCR 5B and 5C, respectively.

Referring to FIG. 5A, it was confirmed that the nano-balls were not treated in the case of CTL, and no glare of red wavelength was detected at all. In the case of treatment with nanoballs, it was confirmed that nanoballs were introduced into the cells according to time, and they were located around the nucleus stained with DAPI (blue). After 12 hours, the emission of siRNA-microsponge from the nanoballs was reddish .

Referring to FIG. 5B, only Lipo and NanoBall (NP) groups, which are commercialized agents, show that mouse VEGF-A mRNA is degraded without being expressed. In other words, it can be seen that nanoball effectively inhibits the expression of mouse VEGF-A in mouse melanoma B16F10 cells. mouse beta actin is the data that confirms that the PCR experiment was carried out with the same cDNA concentration in each sample.

FIG. 5C is data obtained by quantitative analysis while amplifying by real-time PCR. In the case of nanoballs, mono and siRNA-microsponge inhibit VEGF-A expression up to 26.5% compared to the case that VEGF-A mRNA is not degraded.

CNV Inhibition test and quantification

6-8 weeks of C57BL / 6J mice were used. Anesthesia and shodong agents were administered to the eye and then irradiated to the RPE layer with a green Argon laser (532 nm) (Lumenis Selecta Duet SLT (Lumenis, Santa Clara, CA, USA).

The CNV in the eyes of the rats irradiated with laser was examined. After the laser irradiation, nanoballs were injected into the vitreous cavity of the eye with a Hamilton syringe. After 2 weeks, dextran-FITC was injected into the heart of the mouse to stain the blood vessels, and the extracted eyeballs were flat-mounted and observed with a light microscope. In addition, in order to investigate the sustained effect of the particles for 6 weeks, CNV was induced in the rat eye by irradiating the nanoball with intravitreal injection of the eyeball with Hamilton syringe and laser after 4 weeks. After 2 weeks, dextran-FITC was injected into the heart of the mouse to stain the blood vessels, and the extracted eye was flat-mounted and observed with a fluorescence microscope. A typical image is shown in FIG. 6A by observing the CNV portion generated in the second and sixth samples. 6B is a result of CNV area analysis measurement using Image J program.

The CNV image of FIG. 6A (control from the left, 2-sample after nano-ball injection, 6-sample after nano-ball injection) showed that CNV- . It was confirmed that all of the major blood vessels causing CNV were reduced.

Referring to FIG. 6B, it was confirmed that the CNV area was reduced to about 1/4 of that of the control (CTL) in the 2-week sample after CNV injection. In addition, the CNV area of the 6th sample after the injection of nanoballs was also reduced to about 1/2 of that of the control (CTL), indicating that the nanoball's effect lasted for at least 6 weeks.

Oxygen-induced retiopathy ( OIR ) Vessel Suppression Experiment and Quantification in Model

Pregnant C57BL / 6 mice were used. Newborn rats are housed in 75% Oxygen tanks from 7 to 12 days after birth. After that, the neonatal rats are anesthetized and injected into the eyeballs, and the nano balls are injected into the vitreous cavity of the eyeballs using Hamilton syringes and raised in an animal breeding room under normal conditions. The eyeballs were removed on day 5 (P17) and 12th day (P24) after nano-ball injection, stained with Lectin-FITC for staining blood vessels, and flat mounted and observed under a fluorescence microscope. 7.

In order to quantitate the observed images and to confirm the effect of the nanoballs, four points of 500 μm × 500 μm were randomly selected at a distance of 1 mm from the optic nerve, and the branched points were calculated per unit area, As shown in Fig. Also, the neovascularization area (NA) was calculated for each image and compared with each group, and this was indicated by the bar graph in FIG. 8B.

FIG. 7 shows that the angiogenesis is suppressed in the nano-ball injected group compared to the control group (OIR CTL), and the thickness and density of blood vessels are reduced, and NA is also reduced.

FIG. 8 is data obtained by quantifying the image of FIG. 7, showing that the group corresponding to the 5th day and the 12th day after nano-ball injection inhibited angiogenesis and inhibited NW compared to the control group (OIR CTL).

Toxicity experiment

MTT assay: Thiazolyl Blue Tetrazolium Bromide (MTT) is dissolved in PBS at 5 mg / ml to make a solution. The MTT solution reacts in the mitochondria within the cell to form a chromogenic material that absorbs a specific wavelength band, which is indirectly known by the number of cells. The human ARPE-19 cell line is one of the target cells that the dielectric particles must deliver to treat macular degeneration through CNV inhibition. 2 × 10 ⁴ ARPE-19 cells are seeded in a 96-well plate and incubated for 24 hours. When the cells were placed in the plate, they were washed with DPBS after 24, 48 and 72 h after treatment with serum free medium and nanoball, treated with MTT reagent, and treated with 100 μl of DMSO for 6 hours. Cell viability was measured with a plate reader.

Hematoxylin and eosin stain: Hematoxylin and eosin stain (H & E stain)

A commonly used tissue analysis method is to put the tissue into paraffin, make a block, make slices of appropriate size, remove paraffin by xylene 3-5 times washing, and stain with hematoxylin (nuclei staining). Then remove the remaining hematoxylin with 1% HCL. It is then stained with Eosin (cytoplasmic stain). It was observed that each cell layer of the slice of the eyeball was changed when the particles were injected.

Referring to FIG. 9, the case where nano balls are injected into the mouse eye and the case where the nano ball is injected into the mouse eye. Compared to the negative (CTL) group, no cells were observed due to changes in the cell layer or macrophage immune responses.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

10: core part 20: cationic polymer layer
30: outer layer of hyaluronic acid

Claims

A core comprising at least one nucleic acid molecule self-assembled to target the gene;
A cationic polymer layer surrounding and concentrating the core; And
And a hyaluronic acid outer layer coated on the outer surface of the cationic polymer layer.

The dielectric drug delivery particle according to claim 1, wherein the particles have a size of 50 to 350 nm.

The dielectric drug delivery particle according to claim 1, wherein the core is shrunk to a diameter of 500 nm or less by the cationic polymer layer.

The dielectric drug delivery particle according to claim 1, wherein the core comprises a plurality of nucleic acid molecules, and the nucleic acid molecules all have the same nucleic acid sequence or have at least one common nucleic acid sequence.

[2] The particle for delivering a drug for drug delivery according to claim 1, wherein the nucleic acid molecule comprises an antisense RNA targeting mRNAs of a specific gene inducing a retinal disease and a double stranded RNA complementary to a sense RNA.

2. The particle for delivering a drug according to claim 1, wherein the nucleic acid molecule is decomposed into small interfering RNA (siRNA) or antisense nucleic acids targeting mRNA of a specific gene for inducing retinal disease.

The method of claim 6, wherein the core is a small interfering RNA (siRNA) unit sequence or antisense nucleic acids (antisense nucleic acids) unit sequence replication (copy) of 1 × 10 ⁶ or more wherein the small interfering RNA (siRNA) or antisense nucleic acid ( antisense nucleic acids. < / RTI >

2. The particle for delivering a dielectric drug according to claim 1, wherein the nucleic acid molecules in the core are arranged in a crystal structure having a lamellar sheet.

2. The particle for delivering a dielectric drug according to claim 1, wherein the nucleic acid molecule in the core comprises a STEM-LOOP or a linear structure.

The dielectric drug delivery particle according to claim 1, wherein the outer layer of hyaluronic acid has hyaluronic acid coated on the outer surface of the cationic polymer layer with an electrostatic attraction to show a negative charge.

2. The particle for dielectric drug delivery according to claim 1, wherein the particle represents a negative charge.

The dielectric drug delivery particle according to claim 1, wherein the cationic polymer is at least one selected from the group consisting of polyethyleneimine, polyamine, and polyvinylamine.

Amplifying a circular DNA comprising antisense RNA and sense RNA complementary thereto to prepare a plurality of double-stranded RNAs, and self-assembling the plurality of double-stranded RNAs to form a spherical core;
Mixing the spherical core with a cationic polymer to condense the core by ionic interaction; And
And coating the outer surface of the cationic polymer with an outer layer of hyaluronic acid by electrostatic attraction.

14. The method of claim 13, wherein the cyclic DNA forms a plurality of double stranded RNAs using rolling circle amplification (RCA) or rolling circle transfer (RCT).

14. The method of claim 13, wherein the weight ratio of the core to the cationic polymer is 1: 0.05 to 1.

14. The method of claim 13, wherein the hyaluronic acid is in a weight ratio of 1: 0.1 to 0.5 (core: hyaluronic acid) to the condensed core.

14. The method of claim 13, wherein the antisense RNA targets mRNA of a specific gene that induces retinal disease to inhibit its expression.