WO2016163718A1 - Sirna-based nanomedicine having sustained efficacy for retinal diseases and method for preparing same - Google Patents

Sirna-based nanomedicine having sustained efficacy for retinal diseases and method for preparing same Download PDF

Info

Publication number
WO2016163718A1
WO2016163718A1 PCT/KR2016/003523 KR2016003523W WO2016163718A1 WO 2016163718 A1 WO2016163718 A1 WO 2016163718A1 KR 2016003523 W KR2016003523 W KR 2016003523W WO 2016163718 A1 WO2016163718 A1 WO 2016163718A1
Authority
WO
WIPO (PCT)
Prior art keywords
core
particle
sirna
nucleic acid
hyaluronic acid
Prior art date
Application number
PCT/KR2016/003523
Other languages
French (fr)
Korean (ko)
Inventor
김현철
장인석
이종범
박규형
우세준
홍혜경
Original Assignee
서강대학교 산학협력단
서울시립대학교 산학협력단
서울대학교병원 (분사무소)
서울대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 서강대학교 산학협력단, 서울시립대학교 산학협력단, 서울대학교병원 (분사무소), 서울대학교산학협력단 filed Critical 서강대학교 산학협력단
Publication of WO2016163718A1 publication Critical patent/WO2016163718A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a particle for the delivery of a dielectric drug and a method for manufacturing the same, and more particularly, a self-assembled condensed high molecular weight siRNA core particles and the outside is sequentially coated with a cationic polymer and anionic hyaluronic acid to retina
  • the present invention relates to a particle and a method for producing the same, which effectively transfer the siRNA.
  • Age-related macular degeneration is the leading cause of adult blindness and is an ocular disease with increasing prevalence and increasing prevalence. It is characterized by an increase in intraocular vascular endodthelial growth factor (VEGF) and subsequent choroidal neovascularization (CNV).
  • VEGF intraocular vascular endodthelial growth factor
  • CNV choroidal neovascularization
  • photodynamic therapy involves injecting a light sensitizer (photosensitizer) that reacts to the light, Shooting selectively selectively accumulates light on diseased cells and has a therapeutic effect.
  • the drug is injected intravenously and activated by the laser in the eye to prevent the formation of neovascularization.
  • anti-VEGF treatment is usually performed using Avastin, Lucentis, and fusion protein Eylea, which are anti-VEGF antibodies.
  • Photodynamic therapy has been shown to inhibit CNV activity but have no improvement in visual acuity.
  • intraocular anti-VEGF injections have been found to be most effective for improving vision and have become the standard of care for AMD.
  • Intraocular injections of anti-VEGF agents have been proven to be effective in a variety of VEGF-related retinal diseases, including AMD, diabetic retinopathy, retinal vein occlusion, and myopic macular degeneration.
  • Intraocularly injected anti-VEGF preparations have a half-life of 3-7 days and require monthly intraocular administration to sustain their efficacy.
  • RNA and DNA can be transcribed into several proteins
  • gene therapy using RNA and DNA may be an effective treatment method.
  • siRNA Small interfering RNA
  • mRNA messenger RNA
  • RISC messenger RNA induced silencing complex
  • siRNA-based drugs in the treatment of retinal diseases is that the duration of drug efficacy is shorter than that of antibody-based protein drugs, requiring more frequent intravitreal injection.
  • development of various carriers and conjugation of various molecules to siRNA itself have been studied.
  • cationic polymers or lipid molecules and peptides are coupled to anionic siRNAs by electrical attraction to form nanoscale complexes with cationic properties as a whole.
  • the study of increasing the intracellular delivery efficiency by protecting and improving the interaction with the anion cell membrane is representative.
  • DOTAP 2-dioeloyl-3-trimethylammonium
  • PEI poly ethylenimine
  • Chitosan groups and poly L-lysine (PLL) are positively charged and have high RNA acceptability and cell penetration efficiency. It has a well-ordered, three-dimensional network structure consisting of collagen fibrils linked to proteoglycan filaments with 98% water in the vitreous steel but 1-2% with anions. Injecting RNA nanocomposites with cationic properties into the vitreous cavity is known to bind to the vitreous cavity structure and fail to reach the retina.
  • the retina is a tissue composed of several cell layers
  • the innermost inner membrane is composed of fine three-dimensional meshwork structure with an average 13.43 nm pore structure, and the external limiting membrane pore between the outer plexiform layer and the photorecepotr layer.
  • the present inventors have proposed a linear PEI-coated carrier in which a siRNA is cloned by RCT and coated with self-assembled siRNA-microsponges (self-assembled RNA interference microsponges for efficient siRNA delivery (nature material, published on Nov. 26, 2012)
  • siRNA-microsponge coated with linear PEI showed cationic and in vivo toxicity, making it difficult to inject into the retina.
  • modified cationic polymers such as glycol chitosan and branched PEI have been used to solve the cytotoxicity problem by lowering the strength of positive charges, but reducing the strength of positive charges reduces the binding force between RNA and the carriers, thereby reducing the amount of It causes another problem that is easily degraded by enzymes within.
  • the present invention effectively overcomes various diseases including retinal pigment eipthelium cells by overcoming the barriers of delivery by intravitreal and retinal tissues in large amounts of siRNA, a gene therapy, for the treatment of various retinal diseases including senile macular degeneration associated with choroidal neovascularization. It provides a particle for gene drug delivery for the treatment of retinal diseases that can be delivered to the relevant retinal cells.
  • the present invention provides a particle for the delivery of a genome drug having high cytotoxicity and low cytotoxicity.
  • the present invention provides a siRNA hydrogel-based genetic material produced by the RCT process, which is capable of reaching a cell in the retina without being degraded by an enzyme and penetrating the retina along with the vitreous structure.
  • One aspect of the invention provides a core comprising one or more nucleic acid molecules, self-assembled, that target a gene;
  • dielectric drug delivery particles comprising an anionic hyaluronic acid outer layer coated on the outer surface of the cationic polymer layer.
  • the present invention provides a method for producing a plurality of double-stranded RNA by amplifying a cyclic DNA including antisense RNA and a sense RNA complementary thereto, wherein the plurality of double-stranded RNAs are self-assembled to form a spherical core;
  • It relates to a method for producing a particle for dielectric drug delivery comprising coating an outer layer of the anionic hyaluronic acid with an electrostatic attraction on the outer surface of the condensed core.
  • the core of the present invention includes at least 1 ⁇ 10 6 or more of these small interfering RNAs (siRNAs) or antisense nucleic acids, so that a larger amount of siRNAs can be delivered than in the prior art, and siRNAs can be released over a long period of time. It can be very long lasting efficacy.
  • siRNAs small interfering RNAs
  • antisense nucleic acids so that a larger amount of siRNAs can be delivered than in the prior art, and siRNAs can be released over a long period of time. It can be very long lasting efficacy.
  • the present invention prevents aggregation with the vitreous meshwork structures having negative charges and reaches the inner limiting membrane of the retina by coating the outer shell with a negative charge hyaluronic acid.
  • hyaluronic acid binds to the CD44 receptor of Muller cells and passes the retinal structure to the sub-retinal space through the transcytosis mechanism, and then effectively enters the retinal pigment epithelium cells by the CD44 receptor-mediated endocytosis mechanism. Block the translation mechanism of mRNA.
  • Figure 1 shows a conceptual diagram of the nano-location for dielectric delivery according to an embodiment of the present invention.
  • Figure 2 shows a method of making a core (siRNA-microsponge) of the present invention.
  • Figure 3 shows the siRNA-microsponge core, siRNA-microsponge core-branched PEI (b-PEI), and finally the size distribution (A) of the nanoball of the present invention coated with the hyaluronic acid outer layer and TEM image of the nanoball ( B) is shown.
  • FIG. 4 is a photograph of the nanoball solution of the present invention coated with siRNA-microsponge solution and hyaluronic acid.
  • Figure 5 shows the test results delivered to the retinal cells under in vitro conditions of the prepared nanoparticles and PCR results that the anti-VEGF-A siRNA delivered by the nanoparticles selectively inhibit VEGF-A expression.
  • Figure 6 is a result of confirming the drug efficacy persistence for 2 weeks and 6 weeks by injecting the prepared nanoparticles into the vitreous cavity of the CNV-induced mouse using a laser.
  • the injected nanoparticles are delivered to the choroid through intraretinal cells, limiting VEGF-A expression, inhibiting CNV.
  • FIG. 1 is a conceptual diagram of a particle for dielectric delivery according to one embodiment of the present invention.
  • the dielectric nanoparticle for delivery includes a core 10, a cationic polymer layer 20, and an outer layer 30 coated with hyaluronic acid.
  • the core portion 10 includes one or more molecules of self-assembled nucleic acid 11 that target mRNAs of a particular gene.
  • the specific gene may be any mRNA that causes retinal disease.
  • the core part includes a nucleic acid molecule that targets a gene causing retinal diseases such as age-related macular degeneration (AMD) and glaucoma.
  • AMD age-related macular degeneration
  • the core includes a plurality of nucleic acid molecules, the nucleic acid molecules may all have the same nucleic acid sequence or at least one consensus 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 include a nucleotide sequence having a double stranded or triple stranded structure.
  • the nucleic acid molecule in the core may include a nucleotide sequence having two or more complementary units.
  • Nucleic acid molecules in the core may comprise a stem loop (STEM-LOOP) or a linear structure.
  • the complementary unit can have one or more stem-loop (eg, hairpin structure) structures.
  • the nucleic acid molecule in the core may include one or more nucleotide sequences remaining in a single strand.
  • the nucleic acid 11 molecule may be a nucleic acid intended for delivery into a cell, such as small indirect RNA (siRNA), antisense nucleic acids or microRNA (microRNA, miRNA).
  • siRNAs can inhibit the expression of certain proteins by binding to messenger RNAs (RNAs) that have the same bases as their sensor strands and break down mRNAs bound to RNA-induced silencing complexes (RISCs). Can be.
  • RNAs messenger RNAs
  • RISCs RNA-induced silencing complexes
  • the nucleic acid molecule 11 may include anti-sensor RNA that targets VEGF of the retina and double-stranded RNA combined with a sense RNA complementary thereto, but is not limited thereto.
  • Nucleic acid molecules in the core can be folded into a stable three-dimensional structure to be self-assembled. Typically, different parts in a nucleic acid can self assemble by noncovalent interactions.
  • the nucleic acid molecules in the core may be arranged in a crystal structure having 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 ⁇ 10 5 g / mol or more.
  • the core may be formed by aggregation of nucleic acids having a plurality of copies of small interfering RNA (siRNA) unit sequences or antisense nucleic acid unit sequences.
  • siRNA small interfering RNA
  • the core can carry a very large amount of nucleic acid. That is, a small interfering RNA (siRNA) unit sequence or antisense nucleic acid unit sequence may be mounted on the core at least 1 ⁇ 10 3 or more, and 1 ⁇ 10 3 to 1 ⁇ 10 10 or more ranges may be replicated and mounted. Can be.
  • siRNA small interfering RNA
  • the nucleic acid molecule can be broken down into small interfering RNAs (siRNAs) or antisense nucleic acids that target specific genes that lead to retinal disease (eg, retinal vascular endothelial growth factor (VEGF)).
  • siRNAs small interfering RNAs
  • VEGF retinal vascular endothelial growth factor
  • the nucleic acid molecule may comprise at least one cleavage site.
  • 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 condenses or compresses while surrounding the core of the negative charge so that the core portion may be in the form of nanoparticles. A cationic polymer layer having a strong (+) charge is coated on the outer surface of the core.
  • the cationic polymer 20 may be any one or more selected from the group consisting of polyethyleneimine, polyamine, and polyvinylamine, and preferably branched PEI having relatively low or no toxicity may be used.
  • the cationic polymer was injected into the core in a weight ratio of 1: 0.05 to 1, preferably 1: 0.1 to 0.5, and more preferably 1: 0.2 to 0.25, thereby forming a cationic polymer layer.
  • the outer layer 30 is a hyaluronic acid layer coated on the outer surface of the cationic polymer layer 20.
  • the hyaluronic acid outer layer 30 is coated with an electrostatic attraction on the outer surface of the cationic polymer layer 20.
  • the outer layer of hyaluronic acid exhibits a negative charge, and therefore, the particles including the core of the present invention also exhibit a negative charge on the outside thereof.
  • hyaluronic acid Various types are possible for the purposes of the present invention.
  • a large molecular weight and a small hyaluronic acid can be used, preferably 5-10K, more preferably 5K hyaluronic acid can be used.
  • Hyaluronic acid is a large complex oligosaccharide consisting of up to 50,000 pairs of disaccharide glucuronic acid- ⁇ (1-3) N-acetylglucose-amine ⁇ (1-4) as the 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 about 50 nm in diameter.
  • the intravitreal vitreous space has a well-ordered, 3-dimensional network structure consisting of collagen fibrils linked to proteoglycan filaments with 98% water but 1-2% anions. Particles coated with only PEI branched in these spaces do not pass through the vitreous structure by the mutual attraction of the positive and negative charges and are aggregated in the vitreous space. Since the nanoparticles of the present invention are coated with the hyaluronic acid as an outer layer and thus have a (-) charge as a whole, the particles may be prevented from moving together due to the aggregation of the vitreous forming material.
  • the retina is composed of several cell layers, the innermost of which is called the inner limiting membrane, consisting of a fine three-dimensional meshwork structure with an average of 13.43 nm pore structure, and the pore size of the external limiting membrane between the outer plexiform layer and the photorecepotr layer.
  • the physical barriers have a tight junction structure of 3-3.6 nm.
  • the particle of the present invention has a size of 50 to 350 nm, preferably 200 to 300 nm, more preferably 250 to 260 nm.
  • the nanoparticles cannot overcome the physical barriers of the retina by considering their size, but they react with the CD44 receptors of Muller cells by transluctosis through the retina by hyaluronic acid coated on the outer shell of the nanoparticles. can do. That is, the nanoparticles of the present invention can efficiently reach the outer retinal by having a hyaluronic acid outer layer.
  • the nanoparticles remain in some Muller cells during transcytosis and prevent the translation of target gene mRNA in Muller cells to prevent the expression of target genes, and the nanoparticles that reach the outer retina are retinal pigment epithelium. Reacts with the CD44 receptor intracellularly, endocytosis enters the cell and interferes with the translation of the mRNA of the target gene, thereby preventing target gene expression.
  • the invention in another aspect, relates to a method of making nanoparticles for dielectric drug delivery.
  • the method includes a core forming step, a core condensation step and an outer layer coating step.
  • the core forming step is to amplify a circular DNA containing antisense RNA and a sense RNA complementary thereto to prepare a plurality of double stranded RNA, and the plurality of double stranded RNAs are self-assembled to form a spherical core.
  • the circular DNA may be formed using a rolling cycle amplification (RCA) or a rolling circle transcription (RCT) to form a plurality of double stranded RNAs.
  • FIG. 2 shows a method of making the core.
  • a long linear single-stranded DNA encoding the antisense and complementary sense sequence of the anti-VEGF siRNA is prepared. Since the ends of the linear DNA are partially complementary to the T7 promoter sequence, the long linear strand hybridizes with the short DNA strand containing the T7 promoter sequence to form a cyclic DNA.
  • the nick of the circular DNA is chemically closed with a T4 DNA ligase. Closed circular DNA is used to produce RNA transcripts via RCT.
  • RCT is used to generate an antisense and sense hair strand RNA structure of the anti-angiogenic factor siRNA from the circular DNA in repeat units.
  • the plurality of hairpin structures may be self-assembled to form a sponge-shaped core. That is, the present invention can produce spherical cores having a large number of small interfering RNA (siRNA) unit sequences or antisense unit sequences, for example, at least 1 ⁇ 10 3 or more using R
  • the condensation step is a step of condensing the core by ion interaction by mixing a cationic polymer in the spherical core.
  • 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 reacted at neutral pH 6.5 ⁇ 7.5 conditions at room temperature.
  • the coating step is a step of coating the outer layer of hyaluronic acid on the cationic polymer outer surface.
  • the method may coat the outer layer with an electrostatic attraction between the positive charge of the cationic polymer and the negative charge of the hyaluronic acid.
  • the hyaluronic acid may be 1: 0.1 to 0.5 (core: hyaluronic acid), preferably 1: 0.2 to 0.25 in a weight ratio to the core.
  • the coating reaction is carried out at neutral pH (6.5 ⁇ 7.5) conditions at room temperature.
  • For the coating step may refer to the above-described content.
  • VEGF-A rat vascular endothelial growth factoer A
  • antisense 5'-UUCUUUGGUCUGCAUUCA CAU TT-3'.
  • Figure 2 a long linear single-stranded DNA encoding the sense and antisense was prepared.
  • Ligase-typed circular DNA templates were incubated in a reaction buffer (8 mM Tris-HCl, 0.4 mM spermidine, 1.2 mM MgCl 2 , and 2 mM dithiothreitol) for 20 hours at 37 ° C. like T7 RNA polymerase.
  • the resulting solution was pipetted several times and sonicated for 5 minutes to decompose the particles.
  • the solution was centrifuged at 6,000 rpm for 6 minutes and the supernatant was removed.
  • RNase-free water was then added to wash the particles.
  • the solution was sonicated again for 1 minute and centrifuged. The washing step was repeated three more times to remove the RCT reagent to obtain an RNA-microsponge core.
  • RNA-microsponge cores The concentration of RNA-microsponge cores was measured using Quant-iT RNA BR assay kits (Invitrogen). Core preparation was referred to the inventor's prior papers. (Self-assembled RNA interference microsponges for efficient siRNA delivery (Nature Material, Nov. 26, 2012 publication))
  • siRNA nanoballs (particles) from siRNA microsponges cores the synthesized 1 mg of siRNA microsponges were dissolved in 100 microliters of nuclease-free water, which was then branched to a PEI (bPEI) solution (1 mg bPEI / ml of nuclease-). After vortexing in free water, the cells were incubated at room temperature for 10 minutes. Subsequently, hyaluronic acid (HA) solution (1 mg HA / ml of nuclease-free water) was added to the solution, followed by incubation for 5 minutes. The solution was centrifuged at 13,200 rpm to obtain nanoballs (particles).
  • bPEI PEI
  • HA hyaluronic acid
  • the siRNA-microsponge core is 659.92 ⁇ 45.79nm (FIG. 3A, black)
  • the siRNA-microsponge-bPEI is 141.68 ⁇ 19.38nm (FIG. 3A, red)
  • the nanoballs (particles) of the present invention are 259.82 ⁇ 33.35nm (FIG. 3A). , Blue).
  • 3B is an electron micrograph showing that the finally prepared nanoballs (particles) are spherical.
  • the siRNA-microsponge solution has a high turbidity because it is a sub-micro or micro size, but the nanoball solution of the present invention is very transparent because it is nanosize.
  • the nanoball solution of the present invention is very transparent. This is the effect of the coating, the size of the particles is condense to increase the transparency can be indirectly confirmed to reduce the particle size by the coating.
  • B16F10 murine melanoma cells were maintained in DMEM media (Welgene, Dalseogu, Daegu, South Korea) (10% FBS and 1% penicillin / streptomycin mixed) in a 37% 5% carbon dioxide incubator.
  • Human retinal pigment epithelial (ARPE-19) cells (American Type Culture Collection, Manassas, VA) were prepared using Dulbecco's modified Eagle's medium F-12 (DMEM / F-12, Life Technologies Korea, Seoul, South Korea) in a 37 ° C 5% carbon dioxide incubator. ) (10% FBS and 1% penicillin / streptomycin mixed).
  • 5C is data obtained by quantitative analysis while amplified by real-time PCR.
  • mono and siRNA-microsponge inhibit VEGF-A expression by 26.5% compared with almost no degradation of VEGF-A mRNA.
  • CNV phenomenon occurs in the eyes of rats irradiated with laser.
  • nanoballs are injected into the vitreous cavity of the eye with a Hamilton syringe to see the therapeutic effect.
  • dextran-FITC was injected into the heart of rats to stain blood vessels, and the extracted eye was flat-mounted and examined under a light microscope.
  • nanoballs were injected into the vitreous cavity of the eye with a Hamilton syringe and 4 weeks later, laser was irradiated to induce CNV in the rat eye.
  • dextran-FITC was injected into the heart of the rat to stain blood vessels, and the mounted eye was flat mounted for observation by fluorescence microscopy.
  • a representative image is observed in FIG. 6A by observing the CNV portion generated in the 2nd and 6th sample.
  • 6B is a result of CNV area analysis measurement using Image J program.
  • the flat mount CNV image of FIG. 6A shows that the CNV group was significantly reduced in area compared to the control group. This can be seen. All major blood vessels causing CNV were confirmed to be reduced.
  • the CNV area was reduced to about 1/4 compared to the control (CTL).
  • CTL control
  • the CNV area is reduced by about 1/2 compared to the control (CTL), indicating that the efficacy of the nano-ball lasts at least 6 weeks.
  • Oxygen-induced retiopathy OIR Vascular suppression experiments and quantification
  • mice Pregnant C57BL / 6 mice were used. Newborn rats are kept in 75% Oxygen tanks from 7 to 12 days after birth. Subsequently, the newborn rats are anesthetized with anesthesia and acid, and then the nanoballs are injected into the vitreous cavity of the eye with a Hamilton syringe and bred in a normal animal breeding room. After injection of nanoballs, eyes were extracted on day 5 (P17) and day 12 (P24), stained and flat-mounted using Lectin-FITC to stain blood vessels, and observed by fluorescence microscopy. 7 is shown.
  • the group injecting the nanoballs suppressed blood vessel generation, thereby decreasing the thickness and density of blood vessels and decreasing NA.
  • FIG. 8 is a quantified data of FIG. 7, through which the group corresponding to the 5th and 12th days after the nanoball injection was inhibited from angiogenesis and suppressed NW compared to the control group (OIR CTL).
  • MTT assay Dissolve Thiazolyl Blue Tetrazolium Bromide (MTT) in PBS at 5mg / ml to make solution. The MTT solution reacts within the mitochondria in the cell to produce a chromophore that absorbs a specific wavelength band.
  • the human ARPE-19 cell line is one of the target cells to be delivered by genomic particles to treat macular degeneration through CNV inhibition. 2 ⁇ 10 4 ARPE-19 cells are seeded in 96 well plate and incubated for 24 hours.
  • the cells were treated with serum free medium and nanoballs, and then washed with DPBS after 24, 48, and 72 h, treated with MTT reagent, and treated with 100 ⁇ L of DMSO after 6 hours to measure cell viability with a plate reader.
  • Hematoxylin and eosin stain Hematoxylin and eosin stain (H & E stain)
  • tissue analysis method the tissue is placed in a paraffin into blocks, and then slices are made into appropriate sizes, and the paraffin is removed by xylene 3-5 times washing and stained with hematoxylin (nucleus staining). Then remove the remaining Hematoxylin with 1% HCL. Then stain with Eosin (cytoplasmic staining). This was to observe whether each cell layer of the cross-sectional slice of the eye changes when the particles are injected.
  • the present invention contains large amounts of siRNA, a gene therapy that can be used for the treatment of various retinal diseases, including senile macular degeneration associated with choroidal neovascularization.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to particles for efficiently delivering a large amount of siRNA into the inside of the retina by sequentially coating siRNA core particles having high molecular weight, which are self-assembled and condensed, and the exterior of the particles with a cationic polymer and hyaluronic acid and a method for preparing same. Since the core of the present invention includes 1X106 or more of small interfering RNAs (siRNA) or antisense nucleic acids, the core can deliver more siRNA than the typical core. By coating the exterior (shell) with hyaluronic acid showing a (-) charge, the present invention can prevent aggregation with vitreous meshwork structural materials having a (-) charge, and can be delivered to an inner limiting membrane in a retina. Also, hyaluronic acid can pass through a structure of a retina to the sub-retinal space through transcytosis mechanism by being bound to a CD44 receptor of a Muller cell. Some of the core particles inhibit mRNA translation of a target gene in Muller cells and most of parts bind again to CD44 receptors in a cell membrane of retinal pigment epithelial (RPE), enter inside of the cells and also inhibit mRNA translation of the target gene in RPE.

Description

망막질병 치료를 위한 효과 지속성 ｓｉＲＮＡ 기반 나노메디슨 및 이의 제조방법Effect-Resistant SiRNA Based Nanomedicine for the Treatment of Retinal Diseases
본 발명은 유전체 약물 전달용 입자 및 이의 제조방법에 관한 것으로서, 보다 자세하게는, 자기조립되어 응축된 고분자량의 siRNA 코어입자와 외부를 양이온성 고분자와 음이온성 히알루론산을 이용하여 순차적으로 코팅하여 망막내부로 상기 siRNA를 효율적으로 전달하는 입자 및 이의 제조방법에 관한 것이다.The present invention relates to a particle for the delivery of a dielectric drug and a method for manufacturing the same, and more particularly, a self-assembled condensed high molecular weight siRNA core particles and the outside is sequentially coated with a cationic polymer and anionic hyaluronic acid to retina The present invention relates to a particle and a method for producing the same, which effectively transfer the siRNA.
나이관련 황반변성(age-related macular degeneration, AMD)은 성인 실명의 제일 원인으로서 나이가 들 수고 유병률이 증가하는 안과 질환이다. 안구내 VEGF (vascular endodthelial growth factor)의 증가와 이에 따른 맥락막 신생혈관 형성(Choroidal neovascularization(CNV))이 특징이다. AMD 환자수는 고령화 사회가 됨에 따라 더욱 증가하고 있으나 아직까지 완전한 AMD의 치료법은 개발되지 못하고 있는 실정이다. 현재는 광역학 치료(photodynamic therapy)와 anti-VEGF(혈관내피세포성장인자) 치료법이 사용되고 있는데, 광역학 치료는 피부에 빛에 반응하는 광감작제(광과민제)를 주입한 후 특정 파장의 빛을 쏘이면 질병세포에만 선택적으로 빛이 축적되어 치료적 효과를 갖는다. 이를 이용하여 황반변성에 동반된 CNV를 치료하기 위해 약을 정맥 내로 주입하고 레이저에 의해 눈 안에서 활성화되어 신생혈관의 생성을 막아준다. 그리고 anti-VEGF 치료법으로는 일반적으로 anti-VEGF 항체기반인 아바스틴(Avastin)과 루센티스(Lucentis) 및 융합 단백질인 아일리아 (Eylea)를 사용하여 CNV를 치료하고 있다. 광역학요법은 CNV의 활성도는 억제할 수 있으나 시력개선 효과가 없음이 알려졌다. 현재 안구내 anti-VEGF 제제를 주사하는 것이 시력개선에 가장 효과적인 것으로 밝혀 졌으며 AMD의 표준 치료법으로 정착되었다. anti-VEGF 제제의 안구내 주사는 AMD 뿐 아니라 당뇨망막병증, 망막정맥폐쇄, 근시성황반변성 등 VEGF 와 관련된 다양한 망막 질환에서 효과가 있음이 입증되어 활발히 사용되고 있다. 안구내 주입한 anti-VEGF 제제의 반감기는 3-7일 정도이며 약효의 지속을 위해서는 매달 안구내 투여가 필요하다.Age-related macular degeneration (AMD) is the leading cause of adult blindness and is an ocular disease with increasing prevalence and increasing prevalence. It is characterized by an increase in intraocular vascular endodthelial 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 not been developed yet. Currently, photodynamic therapy and anti-VEGF (vascular endothelial growth factor) therapy are used. Photodynamic therapy involves injecting a light sensitizer (photosensitizer) that reacts to the light, Shooting selectively selectively accumulates light on diseased cells and has a therapeutic effect. In order to treat CNV associated with macular degeneration, the drug is injected intravenously and activated by the laser in the eye to prevent the formation of neovascularization. In addition, anti-VEGF treatment is usually performed using Avastin, Lucentis, and fusion protein Eylea, which are anti-VEGF antibodies. Photodynamic therapy has been shown to inhibit CNV activity but have no improvement in visual acuity. Currently, intraocular anti-VEGF injections have been found to be most effective for improving vision and have become the standard of care for AMD. Intraocular injections of anti-VEGF agents have been proven to be effective in a variety of VEGF-related retinal diseases, including AMD, diabetic retinopathy, retinal vein occlusion, and myopic macular degeneration. Intraocularly injected anti-VEGF preparations have a half-life of 3-7 days and require monthly intraocular administration to sustain their efficacy.
최근에는 황반변성 치료를 위해 유전자 치료가 새롭게 연구되고 있는데, mRNA, DNA가 여러 개의 단백질로 전사될 수 있으므로 RNA, DNA를 이용한 유전자 치료가 유효한 치료 방법이 될 수 있다.Recently, gene therapy has been newly studied for the treatment of macular degeneration. Since mRNA and DNA can be transcribed into several proteins, gene therapy using RNA and DNA may be an effective treatment method.
작은 간섭 RNA(small interfering RNA (siRNA))는 19 ~ 22개의 핵산으로 구성된 이중나선 구조의 짧은 RNA로서, 그의 센스 가닥과 염기 서열이 동일한 메신저 RNA(messenger RNA, mRNA)와 결합되면 RNA 유도 침묵 복합체(RNA induced silencing complex, RISC)가 결합된 mRNA를 분해시킴으로써 특정 단백질의 발현을 저해하는 것으로 알려져 있다(Elbashir, S. M. et al., 2001, Nature 411, 494-8). 적은 양으로도 효과적이고 정확하게 서열이 동일한 mRNA의 발현을 억제하는 이러한 siRNA의 성질을 이용하여 유전자 관련 질병 치료에 다양한 가능성이 연구되어 왔으며 siRNA를 이용한 치료약 개발 시장은 그 규모가 해마다 비약적으로 증가하고 있다.Small interfering RNA (siRNA) is a double-stranded short RNA consisting of 19 to 22 nucleic acids, which is an RNA-induced silencing complex when its sense strand and base sequence are combined with the same messenger RNA (mRNA) (RNA induced silencing complex (RISC)) is known to inhibit the expression of specific proteins by degrading the bound mRNA (Elbashir, SM et al., 2001, Nature 411, 494-8). Various possibilities have been studied for the treatment of gene-related diseases by using the siRNA properties that effectively and precisely suppress the expression of mRNAs having the same sequence, and the market for drug development using siRNAs is increasing rapidly each year. .
그러나, siRNA의 효소 불안정성, 열악한 세포 투과성은 실제 임상치료에 적용하는 데 있어서 큰 장애가 되어왔다. 또한 망막질병 치료에 있어서 siRNA 기반 약제의 큰 단점은 약효 지속 시간이 항체기반 단백질 약제들보다 짧아 더욱 빈번한 반복적인 유리체강내에 주입해야 할 필요가 있다. 이를 해결하기 위해 다양한 전달체의 개발 및 siRNA 자체에 다양한 분자의 접합 등이 연구되고 있다. 효소에 직접적으로 노출되는 것을 예방하고 세포 투과성을 증가시키기 위해 음이온성의 siRNA에 양이온성 고분자 또는 지질 분자, 및 펩타이드를 전기적 인력으로 결합시켜 전체적으로 양이온 성질을 가지는 나노 크기의 복합체를 형성하여 siRNA를 효소로부터 보호하고 동시에 음이온 성질을 가지는 세포막과의 상호작용을 향상시켜 세포 내 전달 효율을 증가시키는 연구가 대표적이라 할 수 있다. 좀 더 구체적으로는, 2-dioeloyl-3-trimethylammonium (DOTAP), poly ethylenimine (PEI), Chitosan groups, and poly L-lysine (PLL)은 양전하 특성이 있어 높은 RNA 수용성과 세포 침투 효율을 가진다. 유리체강의 98%가 물이지만 1-2%가 음이온을 가지는 proteoglycan filaments 연결된 collagen fibrils로 구성된 well-ordered, 3-dimensional network 구조를 가지고 있다. 이러한 유리체강내에 양이온 성질을 가지는 RNA 나노복합체를 주입하면 유리체강 구조와 결합하고 망막에 도달하지 못하는 것으로 알려져 있다. 또한, 망막은 여러 세포층으로 구성된 조직으로서 가장 안쪽은 Inner limiting membrane 이라고 하여 평균적으로 13.43 nm pore 구조를 가지고 있는 fine three-dimensional meshwork 구조로 이루어져 있으며, outer plexiform layer와 photorecepotr layer 사이에 external limiting membrane 이라는 pore 크기가 3-3.6 nm 의 tight junction 구조를 이루고 있는 구조가 있어, 일반적으로 나노전달체 (일반적으로 ~ 100 nm 이상)가 diffusion 기작으로 통과하여 맥락막 신생혈관을 치료하기 위한 Retinal pigment epithelium layer 와 맥락막까지 도달할 수 없는 구조이다.However, the enzymatic instability and poor cell permeability of siRNAs have been a major obstacle in their application in clinical practice. In addition, the major drawback of siRNA-based drugs in the treatment of retinal diseases is that the duration of drug efficacy is shorter than that of antibody-based protein drugs, requiring more frequent intravitreal injection. In order to solve this problem, development of various carriers and conjugation of various molecules to siRNA itself have been studied. In order to prevent direct exposure to enzymes and increase cell permeability, cationic polymers or lipid molecules and peptides are coupled to anionic siRNAs by electrical attraction to form nanoscale complexes with cationic properties as a whole. The study of increasing the intracellular delivery efficiency by protecting and improving the interaction with the anion cell membrane is representative. More specifically, 2-dioeloyl-3-trimethylammonium (DOTAP), poly ethylenimine (PEI), Chitosan groups, and poly L-lysine (PLL) are positively charged and have high RNA acceptability and cell penetration efficiency. It has a well-ordered, three-dimensional network structure consisting of collagen fibrils linked to proteoglycan filaments with 98% water in the vitreous steel but 1-2% with anions. Injecting RNA nanocomposites with cationic properties into the vitreous cavity is known to bind to the vitreous cavity structure and fail to reach the retina. In addition, the retina is a tissue composed of several cell layers, the innermost inner membrane is composed of fine three-dimensional meshwork structure with an average 13.43 nm pore structure, and the external limiting membrane pore between the outer plexiform layer and the photorecepotr layer. There is a structure of tight junction structure of 3-3.6 nm in size, in which nanotransmitters (usually ~ 100 nm or more) pass through diffusion mechanism to reach the Retinal pigment epithelium layer and choroid to treat choroidal neovascularization. It cannot be done.
본 발명자는 siRNA를 RCT로 복제하여 자기조립한 siRNA-마이크로스폰지에 linear PEI로 코팅한 전달체를 제시한 바 있다(self-assembled RNA interference microsponges for efficient siRNA delivery(nature material, 2012. 11. 26 공개). 하지만, linear PEI로 코팅된 siRNA-마이크로스폰지는 양이온성을 띄고 생체내 독성을 보여 망막 내에 주입이 어려운 문제점이 있었다.The present inventors have proposed a linear PEI-coated carrier in which a siRNA is cloned by RCT and coated with self-assembled siRNA-microsponges (self-assembled RNA interference microsponges for efficient siRNA delivery (nature material, published on Nov. 26, 2012) However, siRNA-microsponge coated with linear PEI showed cationic and in vivo toxicity, making it difficult to inject into the retina.
최근에는 글리콜 키토산이나 branched PEI와 같은 개질된 양이온 폴리머를 사용하여 양전하의 세기를 낮추어 세포 독성 문제를 해결하고자 하였으나, 양전하의 세기를 감소시키는 것은 RNA와 캐리어 사이의 결합력을 감소시켜 RNA가 혈액이나 혈청 내에서 효소들에 의해 쉽게 분해되는 또 다른 문제를 야기시킨다.Recently, modified cationic polymers such as glycol chitosan and branched PEI have been used to solve the cytotoxicity problem by lowering the strength of positive charges, but reducing the strength of positive charges reduces the binding force between RNA and the carriers, thereby reducing the amount of It causes another problem that is easily degraded by enzymes within.
[참고특허][Reference Patent]
한국공개특허 10-2011-83919Korea Patent Publication 10-2011-83919
한국등록특허 10-806088Korea Patent Registration 10-806088
본 발명은 맥락막 신생혈관과 관련있는 노인성 황반변성을 포함하여 다양한 망막질병 치료를 위하여 유전자 치료제인 siRNA를 다량으로 유리체강내 조직 및 망막 조직에 의한 전달 장애물들을 극복함으로서 효과적으로 retinal pigment eipthelium 세포를 포함한 다양한 질병 관련 망막세포에 전달할 수 있는 망막질병 치료를 위한 유전자 약물 전달용 입자를 제공하는 것이다.DETAILED DESCRIPTION OF THE INVENTION The present invention effectively overcomes various diseases including retinal pigment eipthelium cells by overcoming the barriers of delivery by intravitreal and retinal tissues in large amounts of siRNA, a gene therapy, for the treatment of various retinal diseases including senile macular degeneration associated with choroidal neovascularization. It provides a particle for gene drug delivery for the treatment of retinal diseases that can be delivered to the relevant retinal cells.
본 발명은 망막 내로의 유전자 약물의 전달 효율이 높으면서 세포 독성이 낮은 유전체 약물 전달용 입자를 제공하는 것이다.The present invention provides a particle for the delivery of a genome drug having high cytotoxicity and low cytotoxicity.
본 발명은 RCT 공정에 의한 제작된 siRNA hydrogel 기반의 유전자 물질이 효소에 의해 분해되지 않고 또한 유리체 구조와 함께 망막구조를 투과하여 망막 내 세포에 도달할 수 있는 유전체 전달용 입자를 제공하는 것이다.The present invention provides a siRNA hydrogel-based genetic material produced by the RCT process, which is capable of reaching a cell in the retina without being degraded by an enzyme and penetrating the retina along with the vitreous structure.
본 발명의 하나의 양상은 유전자를 표적하는, 자기조립된 하나 이상의 핵산 분자를 포함하는 코어;One aspect of the invention provides a core comprising one or more nucleic acid molecules, self-assembled, that target a gene;
상기 코어를 둘러싸 응축시키는 양이온성 고분자층; 및A cationic polymer layer surrounding and condensing the core; And
상기 양이온성 고분자층 외면에 코팅된 음이온성 히알루론산 외곽층을 포함하는 유전체 약물 전달용 입자에 관계한다.It relates to dielectric drug delivery particles comprising an anionic hyaluronic acid outer layer coated on the outer surface of the cationic polymer layer.
다른 양상에서 본 발명은 안티센스 RNA와 이에 상보적인 센스 RNA가 포함된 환형 DNA를 증폭시켜 복수개의 이중 가닥 RNA를 제조하고, 상기 복수개의 이중 가닥 RNA들이 자기조립되어 구형 코어를 형성하는 단계;In another aspect, the present invention provides a method for producing a plurality of double-stranded RNA by amplifying a cyclic DNA including antisense RNA and a sense RNA complementary thereto, wherein the plurality of double-stranded RNAs are self-assembled to form a spherical core;
상기 구형 코어에 양이온성 고분자를 혼합하여 이온 상호 작용으로 상기 코어를 응축시키는 단계; 및Mixing a cationic polymer with the spherical core to condense the core by ion interaction; And
응축된 코어부 외면에 정전기적 인력으로 음이온성 히알루론산 외곽층을 코팅하는 단계를 포함하는 유전체 약물 전달용 입자 제조방법에 관계한다.It relates to a method for producing a particle for dielectric drug delivery comprising coating an outer layer of the anionic hyaluronic acid with an electrostatic attraction on the outer surface of the condensed core.
본 발명의 코어에는 1× 106 개 이상의 상기 작은 간섭 RNA(siRNA) 또는 안티센스 핵산(antisense nucleic acids)을 포함하므로 종래에 비해 많은 양의 siRNA를 전달할 수 있으며, 또한, siRNA를 장기간에 걸쳐 방출시킬 수 있으므로 약효의 지속성이 매우 크다.The core of the present invention includes at least 1 × 10 6 or more of these small interfering RNAs (siRNAs) or antisense nucleic acids, so that a larger amount of siRNAs can be delivered than in the prior art, and siRNAs can be released over a long period of time. It can be very long lasting efficacy.
본 발명은 외곽(쉘)을 (-) 전하를 띄는 히알루론산으로 코팅을 함으로서 (-) 전하를 띄고 있는 유리체 meshwork 구조물질들과 aggregation을 방지하고 망막의 inner limiting membrane 에 도달할 수 있다. 또한 히알루론산은 뮬러 세포의 CD44 receptor와 결합하여 망막의 구조를 트랜스시토시스 (transcytosis) 기작으로 sub-retinal space 에 통과한 후, 다시 CD44 receptor-mediated endocytosis 기작에 의하여 retinal pigment epithelium 세포내로 효과적으로 들어가서 특정 mRNA 의 translation 기작을 막는다.The present invention prevents aggregation with the vitreous meshwork structures having negative charges and reaches the inner limiting membrane of the retina by coating the outer shell with a negative charge hyaluronic acid. In addition, hyaluronic acid binds to the CD44 receptor of Muller cells and passes the retinal structure to the sub-retinal space through the transcytosis mechanism, and then effectively enters the retinal pigment epithelium cells by the CD44 receptor-mediated endocytosis mechanism. Block the translation mechanism of mRNA.
도 1은 본 발명의 일구현예에 의한 유전체 전달용 나노입지의 개념도를 나타낸다.Figure 1 shows a conceptual diagram of the nano-location for dielectric delivery according to an embodiment of the present invention.
도 2는 본 발명의 코어(siRNA-microsponge)를 제조하는 방법을 도시한다.Figure 2 shows a method of making a core (siRNA-microsponge) of the present invention.
도 3은 siRNA-microsponge 코어, siRNA-microsponge 코어-branched PEI (b-PEI), 그리고 마지막으로 외곽층을 히알루론산으로 코팅한 본 발명의 나노볼의 사이즈 분포(A)와 나노볼의 TEM 이미지(B)를 나타낸다.Figure 3 shows the siRNA-microsponge core, siRNA-microsponge core-branched PEI (b-PEI), and finally the size distribution (A) of the nanoball of the present invention coated with the hyaluronic acid outer layer and TEM image of the nanoball ( B) is shown.
도 4는 siRNA-microsponge 용액과 히알루론산이 코팅된 본 발명의 나노볼 용액을 촬영한 것이다.4 is a photograph of the nanoball solution of the present invention coated with siRNA-microsponge solution and hyaluronic acid.
도 5는 제조된 나노입자의 생체 외 조건하 망막세포내로 전달되는 시험과 나노입자에 의해 전달된 anti-VEGF-A siRNA 가 VEGF-A 발현을 선택적으로 억제하는 PCR 결과를 나타낸다.Figure 5 shows the test results delivered to the retinal cells under in vitro conditions of the prepared nanoparticles and PCR results that the anti-VEGF-A siRNA delivered by the nanoparticles selectively inhibit VEGF-A expression.
도 6은 제조된 나노입자를 laser를 이용하여 CNV를 유발한 mouse의 유리체강내 주입하여 2주와 6주동안 약효지속성을 확인한 결과이다. 주입된 나노입자가 망막내 세포를 통해 맥락막에 전달되어 VEGF-A 발현을 제한하여, CNV를 억제하게 됨을 보여준다.Figure 6 is a result of confirming the drug efficacy persistence for 2 weeks and 6 weeks by injecting the prepared nanoparticles into the vitreous cavity of the CNV-induced mouse using a laser. The injected nanoparticles are delivered to the choroid through intraretinal cells, limiting VEGF-A expression, inhibiting CNV.
도 7과 도8은 제조된 나노입자를 Oxygen-induced retinopathy 모델의 유리체강내 주입하여 망막내 세포에서 VEGF-A 발현을 제한하여 혈관생성을 억제함을 보여준다.7 and 8 show that the prepared nanoparticles are injected intravitreally in the Oxygen-induced retinopathy model to limit VEGF-A expression in intraretinal cells, thereby inhibiting angiogenesis.
도 9는 제조된 나노입자를 이용하여 독성 실험을 한 결과를 나타낸다.9 shows the results of toxicity experiments using the prepared nanoparticles.
본 발명은 하기의 설명에 의하여 모두 달성될 수 있다. 하기의 설명은 본 발명의 바람직한 구체 예를 기술하는 것으로 이해되어야 하며, 본 발명이 반드시 이에 한정되는 것은 아니다.The present invention can all be achieved by the following description. The following description is to be understood as describing preferred embodiments of the invention, but the invention is not necessarily limited thereto.
도 1은 본 발명의 일구현예에 의한 유전체 전달용 입자의 개념도를 나타낸다.1 is a conceptual diagram of a particle for dielectric delivery according to one embodiment of the present invention.
도 1을 참고하면, 본 발명의 유전체 전달용 나노입자는 코어(10), 양이온성 고분자층 (20) 및 히알루산으로 코팅된 외곽층 (30)을 포함한다.Referring to FIG. 1, the dielectric nanoparticle for delivery includes a core 10, a cationic polymer layer 20, and an outer layer 30 coated with hyaluronic acid.
상기 코어부(10)는 특정 유전자의 mRNA들을 표적하는 자기 조립된 하나 이상의 핵산(11) 분자를 포함한다. 상기 특정 유전자로는 망막 질병을 유발하는 모든 mRNA가 해당될 수 있다. 예를 들면, 상기 코어부는 황반형성(age-related macular degeneration, AMD), 녹내장 등 망막질환을 일으키는 유전자를 표적하는 핵산 분자를 포함한다.The core portion 10 includes one or more molecules of self-assembled nucleic acid 11 that target mRNAs of a particular gene. The specific gene may be any mRNA that causes retinal disease. For example, the core part includes a nucleic acid molecule that targets a gene causing retinal diseases such as age-related macular degeneration (AMD) and glaucoma.
상기 코어는 복수개의 핵산분자를 포함하고, 상기 핵산분자들은 전부 동일한 핵산서열을 가지거나 적어도 하나 이상의 공통 핵산 서열을 가질 수 있다. 상기 핵산분자는 하나 이상의 서열 단위 (sequence elements)를 포함하는 뉴클레오티드 서열(nucleotide sequence)을 가질 수 있다. 상기 코어 내의 상기 핵산분자는 이중가닥 또는 삼중가닥 구조를 가지는 뉴클에오티드 서열 (nucleotide sequence)을 포함할 수 있다. 상기 코어 내의 상기 핵산분자는 두 개 이상의 상보적 단위를 가지는 뉴클에오티드 서열 (nucleotide sequence)을 포함할 수 있다. 상기 코어 내의 핵산 분자는 스템 루프 (STEM-LOOP) 또는 선형 구조를 포함할 수 있다. 예를 들면, 상기 상보적 단위는 하나 이상의 스템-루프 (STEM-LOOP) (예를 들면, 헤어핀 구조) 구조를 가질 수 있다. 또한 상기 코어 내의 상기 핵산분자는 단일 가닥으로 잔존하는 하나 이상의 뉴클레오티드 서열 (nucleotide sequence)을 포함할 수 있다.The core includes a plurality of nucleic acid molecules, the nucleic acid molecules may all have the same nucleic acid sequence or at least one consensus 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 include a nucleotide sequence having a double stranded or triple stranded structure. The nucleic acid molecule in the core may include a nucleotide sequence having two or more complementary units. Nucleic acid molecules in the core may comprise a stem loop (STEM-LOOP) or a linear structure. For example, the complementary unit can have one or more stem-loop (eg, hairpin structure) structures. In addition, the nucleic acid molecule in the core may include one or more nucleotide sequences remaining in a single strand.
상기 핵산(11) 분자는 세포내로의 전달을 목적으로 하는 핵산, 예컨대 작은 간접 RNA (siRNA), 안티센스 핵산 (antisense nucleic acids) 또는 마이크로 RNA (microRNA, miRNA)일 수 있다. siRNA는 그의 센서 (sense) 가닥과 염기 서열이 동일한 메신저 RNA (messenger RNA, mRNA)와 결합하여 RNA 유도 침묵 복합체 (RNA induced silencing complex, RISC)가 결합된 mRNA를 분해시킴으로써 특정 단백질의 발현을 저해할 수 있다.The nucleic acid 11 molecule may be a nucleic acid intended for delivery into a cell, such as small indirect RNA (siRNA), antisense nucleic acids or microRNA (microRNA, miRNA). siRNAs can inhibit the expression of certain proteins by binding to messenger RNAs (RNAs) that have the same bases as their sensor strands and break down mRNAs bound to RNA-induced silencing complexes (RISCs). Can be.
예를 들면, 상기 핵산 분자(11)는 망막의 혈관내피 성장인자(VEGF)를 표적하는 안티센서 RNA와 이에 상보적인 센스 RNA가 결합된 이중 가닥 RNA를 포함할 수 있으며, 이에 한정되는 것은 아니다.For example, the nucleic acid molecule 11 may include anti-sensor RNA that targets VEGF of the retina and double-stranded RNA combined with a sense RNA complementary thereto, but is not limited thereto.
상기 코어 내의 핵산 분자는 안정적인 3차원 구조로 접혀 자기조립이 될 수 있다. 전형적으로는, 핵산 내의 서로 다른 부분들이 비공유 상호 작용에 의해 자기조립될 수 있다. 상기 코어 내의 상기 핵산 분자는 층상 시트 (lamellar sheet)를 구비하는 결정 구조내에 배열될 수 있다.Nucleic acid molecules in the core can be folded into a stable three-dimensional structure to be self-assembled. Typically, different parts in a nucleic acid can self assemble by noncovalent interactions. The nucleic acid molecules in the core may be arranged in a crystal structure having a lamellar sheet.
코어는 하나 이상의 얽혀있는 핵산분자들로 이루어질 수 있다.The core may consist of one or more entangled nucleic acid molecules.
코어의 핵산분자는 분자량이 1×105 g/mol 이상일 수 있다.The nucleic acid molecule of the core may have a molecular weight of 1 × 10 5 g / mol or more.
상기 코어는 작은 간섭 RNA (siRNA) 단위 서열 또는 안티센서 핵산 (antisense nucleic acids) 단위 서열이 다수 개 복제(copy)된 핵산이 응집되어 형성될 수 있다.The core may be formed by aggregation of nucleic acids having a plurality of copies of small interfering RNA (siRNA) unit sequences or antisense nucleic acid unit sequences.
상기 코어에는 매우 많은 양의 핵산을 탑재할 수 있다. 즉, 상기 코어에는 작은 간섭 RNA(siRNA) 단위 서열 또는 안티센스 핵산(antisense nucleic acids) 단위 서열이 최소 1×103 이상 탑재될 수 있으며, 1×103 ~ 1×1010 개 범위 이상 복제되어 탑재될 수 있다.The core can carry a very large amount of nucleic acid. That is, a small interfering RNA (siRNA) unit sequence or antisense nucleic acid unit sequence may be mounted on the core at least 1 × 10 3 or more, and 1 × 10 3 to 1 × 10 10 or more ranges may be replicated and mounted. Can be.
예를 들면, 상기 핵산 분자는 망막 질환을 유도하는 특정 유전자( 예를 들면, 망막의 혈관내피 성장인자(VEGF))를 표적하는 작은 간섭 RNA(siRNA) 또는 안티센스 핵산 (antisense nucleic acids)로 분해될 수 있다.For example, the nucleic acid molecule can be broken down into small interfering RNAs (siRNAs) or antisense nucleic acids that target specific genes that lead to retinal disease (eg, retinal vascular endothelial growth factor (VEGF)). Can be.
상기 핵산 분자는 적어도 하나 이상의 절단 가능한 부위 (cleavage site)를 포함할 수 있다. 예를 들면, 상기 절단 가능 부위는 효소 (nuclease, dicer, DNAase, RNAase)에 의해 분리될 수 있다.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).
상기 양이온성 고분자(20)는 상기 코어를 둘러싸며 이온 상호 작용으로 상기 코어를 응축시킨다. 좀 더 구체적으로는, 강한 양전하를 가지는 branched PEI가 음전하의 코어를 둘러싸면서 응축 또는 압축하여 상기 코어부는 나노 파티클 형태가 될 수 있다. 상기 코어 외면에 강한 (+) 전하를 띄는 양이온성 고분자층이 코팅된다.The cationic polymer 20 surrounds the core and condenses the core by ionic interaction. More specifically, the branched PEI having a strong positive charge condenses or compresses while surrounding the core of the negative charge so that the core portion may be in the form of nanoparticles. A cationic polymer layer having a strong (+) charge is coated on the outer surface of the core.
상기 양이온성 고분자(20)는 폴리에틸렌이민, 폴리아민 및 폴리비닐아민으로 이루어진 군에서 선택된 어느 하나 이상일 수 있으며, 바람직하게는 독성이 상대적으로 낮거나 없는 branched PEI를 사용할 수 있다.The cationic polymer 20 may be any one or more selected from the group consisting of polyethyleneimine, polyamine, and polyvinylamine, and preferably branched PEI having relatively low or no toxicity may be used.
상기 코어에 상기 양이온성 고분자를 1 : 0.05 ~ 1 바람직하게는 1 : 0.1 ~ 0.5, 더욱 바람직하게는 1 : 0.2 ~ 0.25 중량비로 주입하여 양이온성 고분자층을 형성할 수 았다.The cationic polymer was injected into the core in a weight ratio of 1: 0.05 to 1, preferably 1: 0.1 to 0.5, and more preferably 1: 0.2 to 0.25, thereby forming a cationic polymer layer.
상기 외곽층(30)은 상기 양이온성 고분자층(20) 외면에 코팅된 히알루론산층이다.The outer layer 30 is a hyaluronic acid layer coated on the outer surface of the cationic polymer layer 20.
상기 히알루론산 외곽층(30)은 상기 양이온성 고분자층(20) 외면에 정전기적 인력으로 코팅된다.The hyaluronic acid outer layer 30 is coated with an electrostatic attraction on the outer surface of the cationic polymer layer 20.
상기 히알루론산 외곽층은 (-) 전하를 나타내고, 따라서, 본 발명의 코어를 포함한 입자도 그 외부가 (-)전하를 나타낸다.The outer layer of hyaluronic acid exhibits a negative charge, and therefore, the particles including the core of the present invention also exhibit a negative charge on the outside thereof.
본 발명의 목적에는 다양한 형태의 히알루론산이 가능하다. 특히, 분자량이 큰 것과 작은 히알루론산이 이용될 수 있으며, 바람직하게는 5~10K, 더욱 바람직하게는 5K 히알루론산을 사용할 수 있다.Various types of hyaluronic acid are possible for the purposes of the present invention. In particular, a large molecular weight and a small hyaluronic acid can be used, preferably 5-10K, more preferably 5K hyaluronic acid can be used.
히알루론산(HA)은 기본단위로 디사카라이드 글루쿠로닌산-β(1-3) N-아세틸글루코즈-아민β(1-4)이 최고 50,000쌍으로 구성된 큰 복합 올리고사카라이드이다. 이는 세포외매트릭스(extracellular matrix)의 주요 성분으로써 생체 내에서 발견된다. 이의 3차 구조는 직경이 약 50nm인 무작위(random) 코일 형태이다.Hyaluronic acid (HA) is a large complex oligosaccharide consisting of up to 50,000 pairs of disaccharide glucuronic acid-β (1-3) N-acetylglucose-amineβ (1-4) as the 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 about 50 nm in diameter.
안구 내 유리체(vitreous) 공간은 98%가 물이지만 1-2%가 음이온을 가지는 proteoglycan filaments 연결된 collagen fibrils로 구성된 well-ordered, 3-dimensional network 구조를 가지고 있다. 이러한 공간에 branched PEI로만 코팅된 입자는 (+) 전하 및 (-) 전하의 상호 인력에 의해 유리체 구조를 통과하지 못하고 유리체 공간 내에서 뭉치게 (aggregation)된다. 본원 발명의 나노입자는 히알루론산이 외곽층으로 코팅되어 있어 전체적으로 (-) 전하를 띄게 되므로 입자들이 유리체 형성 물질과 뭉쳐서 이동이 방해되는 것을 막을 수 있다.The intravitreal vitreous space has a well-ordered, 3-dimensional network structure consisting of collagen fibrils linked to proteoglycan filaments with 98% water but 1-2% anions. Particles coated with only PEI branched in these spaces do not pass through the vitreous structure by the mutual attraction of the positive and negative charges and are aggregated in the vitreous space. Since the nanoparticles of the present invention are coated with the hyaluronic acid as an outer layer and thus have a (-) charge as a whole, the particles may be prevented from moving together due to the aggregation of the vitreous forming material.
망막은 여러 세포층으로 구성되어 있는데 가장 안쪽은 Inner limiting membrane 이라고 하여 평균적으로 13.43 nm pore 구조를 가지고 있는 fine three-dimensional meshwork 구조로 이루어져 있으며, outer plexiform layer와 photorecepotr layer 사이에 external limiting membrane 이라는 pore 크기가 3-3.6 nm 의 tight junction 구조를 이루고 있는 구조의 physical barrier들을 가진다.The retina is composed of several cell layers, the innermost of which is called the inner limiting membrane, consisting of a fine three-dimensional meshwork structure with an average of 13.43 nm pore structure, and the pore size of the external limiting membrane between the outer plexiform layer and the photorecepotr layer. The physical barriers have a tight junction structure of 3-3.6 nm.
본원 발명의 상기 입자는 사이즈가 50~350nm, 바람직하게는 200~300nm, 더욱 바람직하게는 250~260nm 이다.The particle of the present invention has a size of 50 to 350 nm, preferably 200 to 300 nm, more preferably 250 to 260 nm.
상기 나노입자는 크기만 고려하면 망막의 physical barrier들을 극복할 수 없지만, 나노입자의 외곽(쉘)에 코팅된 히알루론산에 의해 Muller 세포의 CD44 receptor와 반응하여 트랜스시토시스(transcytosis)하여 망막을 통과할 수 있다. 즉, 본원발명의 나노입자는 히알루론산 외곽층을 구비함에 따라 외측 망막 (outer retinal)에 효율적으로 도달할 수 있다.The nanoparticles cannot overcome the physical barriers of the retina by considering their size, but they react with the CD44 receptors of Muller cells by transluctosis through the retina by hyaluronic acid coated on the outer shell of the nanoparticles. can do. That is, the nanoparticles of the present invention can efficiently reach the outer retinal by having a hyaluronic acid outer layer.
상기 나노입자는 Muller 세포를 transcytosis 과정에서 일부 Muller 세포에 남아 Muller 세포에서의 표적 유전자 mRNA의 해석(translation)을 방해하여 표적 유전자 발현을 막을 뿐 아니라, 외측 망막에 도달한 나노입자는 다시 retinal pigment epithelium 세포내로 CD44 receptor 와 반응하여 엔도시토시스 (endocytosis)하여 세포내로 들어가 표적 유전자의 mRNA의 해석(translation)을 방해하여 표적 유전자 발현을 막는다.The nanoparticles remain in some Muller cells during transcytosis and prevent the translation of target gene mRNA in Muller cells to prevent the expression of target genes, and the nanoparticles that reach the outer retina are retinal pigment epithelium. Reacts with the CD44 receptor intracellularly, endocytosis enters the cell and interferes with the translation of the mRNA of the target gene, thereby preventing target gene expression.
다른 양상에서 본 발명은 유전체 약물 전달용 나노입자를 제조하는 방법에 관계한다. 상기 방법은 코어 형성 단계, 코어 응축 단계 및 외곽층 코팅 단계를 포함한다.In another aspect, the invention relates to a method of making nanoparticles for dielectric drug delivery. The method includes a core forming step, a core condensation step and an outer layer coating step.
상기 코어 형성 단계는 안티센스 RNA 와 이에 상보적인 센스 RNA가 포함된 환형 DNA를 증폭시켜 복수개의 이중 가닥 RNA를 제조하고, 상기 복수의 이중 가닥 RNA들이 자기 조립되어 구형 코어를 형성하는 단계이다. 상기 환형 DNA를 롤링 서클 증폭 (Rolling Cycle Amplification, RCA) 또는 롤링 서클 전사(Rolling Cycle Transcription, RCT)를 사용하여 복수개의 이중 가닥 RNA을 형성할 수 있다.The core forming step is to amplify a circular DNA containing antisense RNA and a sense RNA complementary thereto to prepare a plurality of double stranded RNA, and the plurality of double stranded RNAs are self-assembled to form a spherical core. The circular DNA may be formed using a rolling cycle amplification (RCA) or a rolling circle transcription (RCT) to form a plurality of double stranded RNAs.
도 2에 상기 코어를 제조하는 방법이 도시되어 있다. 도 2를 참고하면, anti-VEGF siRNA의 안티센스와 이에 상보적인 센스서열을 인코딩한 긴 선형 단일가닥 DNA를 준비한다. 선형 DNA의 말단이 T7 프로모터 서열에 부분적으로 상보적이기 때문에, 긴 선형 가닥은 T7 프로모터 서열을 포함하는 짧은 DNA 가닥과 혼성화되어 환형 DNA를 형성한다. 환형 DNA의 nick은 화학적으로 T4 DNA ligase로 폐쇄된다. 폐쇄된 환형 DNA는 RCT를 통해 RNA 전사체를 생산하기 위해 사용된다. RCT를 이용하여 상기 환형 DNA로부터 안티-혈관생성인자 siRNA의 안티센스, 센스 가닥의 인코딩한 헤어핀 RNA 구조를 반복 단위로 생성한다. 상기 복수개의 헤어핀 구조는 자기조립되어 스폰지 형태의 코어를 형성할 수 있다. 즉, 본 발명은 상기 환형 DNA로부터 RCT를 이용하여 수많은, 예를 들면, 최소 1×103 이상의 작은 간섭 RNA (siRNA) 단위 서열 또는 안티센스 단위 서열을 가진 구형 코어를 제조할 수 있다.2 shows a method of making the core. Referring to Figure 2, a long linear single-stranded DNA encoding the antisense and complementary sense sequence of the anti-VEGF siRNA is prepared. Since the ends of the linear DNA are partially complementary to the T7 promoter sequence, the long linear strand hybridizes with the short DNA strand containing the T7 promoter sequence to form a cyclic DNA. The nick of the circular DNA is chemically closed with a T4 DNA ligase. Closed circular DNA is used to produce RNA transcripts via RCT. RCT is used to generate an antisense and sense hair strand RNA structure of the anti-angiogenic factor siRNA from the circular DNA in repeat units. The plurality of hairpin structures may be self-assembled to form a sponge-shaped core. That is, the present invention can produce spherical cores having a large number of small interfering RNA (siRNA) unit sequences or antisense unit sequences, for example, at least 1 × 10 3 or more using RCT from the circular DNA.
응축단계는 상기 구형 코어에 양이온성 고분자를 혼합하여 이온 상호 작용으로 상기 코어를 응축시키는 단계이다.The condensation step is a step of condensing the core by ion interaction by mixing a cationic polymer in the spherical core.
상기 코어와 상기 양이온성 고분자의 중량비는 1 : 0.05 ~ 1 바람직하게는 1 : 0.1 ~ 0.5, 더욱 바람직하게는 1 : 0.2 ~ 0.25일 수 있다.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.
상기 응축단계는 상온에서 중성 pH 6.5~7.5 조건에서 반응시킨다.The condensation step is reacted at neutral pH 6.5 ~ 7.5 conditions at room temperature.
상기 코팅 단계는 양이온성 고분자 외면에 히알루론산 외곽층을 코팅하는 단계이다. 바람직하게는, 상기 방법은 양이온성 고분자의 (+)전하와 히알루론산의 (-) 전하와의 정전기적 인력으로 상기 외곽층을 코팅할 수 있다.The coating step is a step of coating the outer layer of hyaluronic acid on the cationic polymer outer surface. Preferably, the method may coat the outer layer with an electrostatic attraction between the positive charge of the cationic polymer and the negative charge of the hyaluronic acid.
상기 히알루론산은 상기 코어 대비 중량비로 1:0.1 ~ 0.5 (코어 : 히알루론산), 바람직하게는 1 : 0.2 ~ 0.25 일 수 있다.The hyaluronic acid may be 1: 0.1 to 0.5 (core: hyaluronic acid), preferably 1: 0.2 to 0.25 in a weight ratio to the core.
상기 코팅반응은 상온에서 중성pH(6.5~7.5) 조건에서 반응시킨다.The coating reaction is carried out at neutral pH (6.5 ~ 7.5) conditions at room temperature.
상기 코팅단계에 대해서는 앞에서 상술한 내용을 참고할 수 있다.For the coating step may refer to the above-described content.
이하, 본 발명의 이해를 돕기 위해 바람직한 실시예를 제시하지만, 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 본 발명이 이에 한정되는 것은 아니다.Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are provided only for easier understanding of the present invention, and the present invention is not limited thereto.
실시예Example 1 One
코어의 합성Synthesis of core
쥐의 혈관내피 성장 인자 (vascular endothelial growth factoer A (VEGF-A))의 센스는 5’-AUGUGAAUGCAGACCAAAGAA TT-3’이고, 안티센스는 5’-UUCUUUGGUCUGCAUUCA CAU TT-3’이다. 도 2와 같이, 상기 센스 및 안티센스를 인코딩한 긴 선형 단일가닥 DNA를 제조하였다.The sense of rat vascular endothelial growth factoer A (VEGF-A) is 5'-AUGUGAAUGCAGACCAAAGAA TT-3 'and the antisense is 5'-UUCUUUGGUCUGCAUUCA CAU TT-3'. As shown in Figure 2, a long linear single-stranded DNA encoding the sense and antisense was prepared.
Ligase된 원형 DNA 템플릿을 T7 RNA 폴리머레이즈와 같이 37℃에서 20 시간 동안 반응버퍼 (8mM Tris-HCl, 0.4mM spermidine, 1.2mM MgCl2, and 2mM dithiothreitol)에서 배양하였다. 생성된 용액을 여러 번 피펫하고 5분 동안 초음파처리해서 입자를 분해하였다. 용액을 6,000rpm에서 6분 동안 원심분리하고 상층액을 제거하였다. 그 후 RNase-free water를 첨가해 입자를 세척하였다. 용액을 1분 동안 다시 초음파처리하고 원심 분리하였다. 세척단계를 세 번 더 반복해서 RCT 시약을 제거하여 RNA-microsponge 코어를 수득하였다. RNA-microsponge 코어의 농도는 Quant-iT RNA BR assay kits (Invitrogen)를 사용하여 측정하였다. 코어 제조는 본 발명자의 선행 논문을 참고하였다.(Self-assembled RNA interference microsponges for efficient siRNA delivery (Nature Material, 2012. 11. 26 공개))Ligase-typed circular DNA templates were incubated in a reaction buffer (8 mM Tris-HCl, 0.4 mM spermidine, 1.2 mM MgCl 2 , and 2 mM dithiothreitol) for 20 hours at 37 ° C. like T7 RNA polymerase. The resulting solution was pipetted several times and sonicated for 5 minutes to decompose the particles. The solution was centrifuged at 6,000 rpm for 6 minutes and the supernatant was removed. RNase-free water was then added to wash the particles. The solution was sonicated again for 1 minute and centrifuged. The washing step was repeated three more times to remove the RCT reagent to obtain an RNA-microsponge core. The concentration of RNA-microsponge cores was measured using Quant-iT RNA BR assay kits (Invitrogen). Core preparation was referred to the inventor's prior papers. (Self-assembled RNA interference microsponges for efficient siRNA delivery (Nature Material, Nov. 26, 2012 publication))
나노볼Nano Ball (입자) 제조(Particle) manufacturing
siRNA microsponges 코어로부터 siRNA 나노볼(입자)을 제작하기 위해서, 합성된 1mg 의 siRNA microsponges를 100 마이크로리터의 nuclease-free water에 넣어 용해하고, 이를 branched PEI (bPEI) 용액 (1mg bPEI/ml 의 nuclease-free water)에 넣어 볼텍싱(vortexing)한 후, 상온에서 10분 동안 배양하였다. 이어서, 히알루론산(HA)용액 (1mg HA/ml 의 nuclease-free water)을 상기 용액에 넣어 혼합 한 후 5분 동안 배양하였다. 상기 용액을 13,200rpm으로 원심 분리하여 나노볼(입자)를 수득하였다.To prepare siRNA nanoballs (particles) from siRNA microsponges cores, the synthesized 1 mg of siRNA microsponges were dissolved in 100 microliters of nuclease-free water, which was then branched to a PEI (bPEI) solution (1 mg bPEI / ml of nuclease-). After vortexing in free water, the cells were incubated at room temperature for 10 minutes. Subsequently, hyaluronic acid (HA) solution (1 mg HA / ml of nuclease-free water) was added to the solution, followed by incubation for 5 minutes. The solution was centrifuged at 13,200 rpm to obtain nanoballs (particles).
제조된 나노입자의 특성 측정Characterization of the manufactured nanoparticles
최종적으로 제조된 나노볼, siRNA-microsponge 코어, siRNA-microsponge 코어-bPEI 의 사이즈 분포를 도 3A에 나타내었다. Finally, the size distribution of the prepared nanoball, siRNA-microsponge core, siRNA-microsponge core-bPEI is shown in Figure 3A.
siRNA-microsponge 코어는 659.92± 45.79nm (도 3A, 검정색), siRNA-microsponge-bPEI는 141.68 ± 19.38nm (도 3A, 빨간색)이고, 본 발명의 나노볼(입자)은 259.82 ± 33.35nm (도 3A, 파란색)이다. 도 3B는 최종적으로 제조된 나노볼(입자)이 구형이라는 것을 보여주는 전자현미경 사진이다.The siRNA-microsponge core is 659.92 ± 45.79nm (FIG. 3A, black), the siRNA-microsponge-bPEI is 141.68 ± 19.38nm (FIG. 3A, red), and the nanoballs (particles) of the present invention are 259.82 ± 33.35nm (FIG. 3A). , Blue). 3B is an electron micrograph showing that the finally prepared nanoballs (particles) are spherical.
도 4는 siRNA-microsponge 용액과 최종적으로 제조된 나노볼 용액을 촬영한 것이다. 도 4를 참고하면, siRNA-microsponge 용액은 sub-micro 또는 micro 사이즈이므로 탁도가 높지만, 본 발명의 나노볼 용액은 나노사이즈이기 때문에 매우 투명하다. 본 발명의 나노볼 용액은 매우 투명하다. 이것은 코팅에 의한 효과로, 입자의 size가 condense하여 투명도가 높아지는 것으로 코팅에 의한 입자 사이즈 축소를 간접적으로 확인할 수 있다.4 is a photograph of the siRNA-microsponge solution and finally prepared nanoball solution. Referring to FIG. 4, the siRNA-microsponge solution has a high turbidity because it is a sub-micro or micro size, but the nanoball solution of the present invention is very transparent because it is nanosize. The nanoball solution of the present invention is very transparent. This is the effect of the coating, the size of the particles is condense to increase the transparency can be indirectly confirmed to reduce the particle size by the coating.
세포배양Cell culture
B16F10 murine melanoma cells은 37℃ 5% 이산화탄소 인큐베이터에 DMEM media(Welgene, Dalseogu, Daegu, South Korea)(10% FBS와 1% penicillin/streptomycin 혼합됨)에서 유지하였다. Human retinal pigment epithelial (ARPE-19) cells (American Type Culture Collection, Manassas, VA)은 37℃ 5% 이산화탄소 인큐베이터의 Dulbecco's modified Eagle's 배지 F-12 (DMEM/F-12, Life Technologies Korea, Seoul, South Korea)(10% FBS와 1% penicillin/streptomycin 혼합됨)에서 유지하였다.B16F10 murine melanoma cells were maintained in DMEM media (Welgene, Dalseogu, Daegu, South Korea) (10% FBS and 1% penicillin / streptomycin mixed) in a 37% 5% carbon dioxide incubator. Human retinal pigment epithelial (ARPE-19) cells (American Type Culture Collection, Manassas, VA) were prepared using Dulbecco's modified Eagle's medium F-12 (DMEM / F-12, Life Technologies Korea, Seoul, South Korea) in a 37 ° C 5% carbon dioxide incubator. ) (10% FBS and 1% penicillin / streptomycin mixed).
세포 흡착 시험Cell adsorption test
Human ARPE-19 cell 2×104 개를 12 well plate에 seeding 한 후에 24시간 동안 인큐베이팅시킨다. 각각의 well에 SYBR®Green I로 염색된 나노볼을 serum free medium과 섞어 6㎍/㎕ 농도로 넣어주었다. 이어서, 3시간, 12시간 후에 4% paraformaldehyde(PFA)로 고정시켜 DAPI 염색 후 형광 현미경으로 관찰하여 도 5A에 나타내었다.2 x 10 4 human ARPE-19 cells are seeded in 12 well plates and incubated for 24 hours. In each well, SYBR® Green I stained nanoballs were mixed with serum free medium and placed at 6µg / µl concentration. Subsequently, after 3 hours and 12 hours, the cells were fixed with 4% paraformaldehyde (PFA) and observed with a fluorescence microscope after DAPI staining.
mRNAmRNA 발현 억제 시험 Expression inhibition test
3×105 개의 B16F10 mouse melanoma cell을 6-well plate에 seeding 한 후, mono-siRNA (Mono), siRNA-microsponge (Sponge), no treatment (CTL, C), 상용화된 transfection agents인 (lipofectamine, Lipo), 그리고 본 발명의 나노입자(NP)를 serum free medium과 섞어 처리하였다. 그 후에 cell들을 모두 걷어내어 mRNA를 추출하여 mouse VEGF-A 와 mouse beta actin 에 대하여 cDNA를 만든 후, PCR cycler로 증폭시켜 mRNA의 수준을 확인하였으며, 같은 방법으로 정량적인 분석을 위해 real-time PCR로 확인하여 이를 도 5B, 도5C에 나타내었다.After seeding 3 × 10 5 B16F10 mouse melanoma cells on a 6-well plate, mono-siRNA (Mono), siRNA-microsponge (Sponge), no treatment (CTL, C), commercialized transfection agents (lipofectamine, Lipo) ), And the nanoparticles (NP) of the present invention were treated with a serum free medium. After that, all cells were removed, mRNA was extracted, cDNA was generated for mouse VEGF-A and mouse beta actin, and amplified by PCR cycler to confirm mRNA levels. Real-time PCR was performed for quantitative analysis. This is confirmed as shown in Figure 5B, Figure 5C.
도 5A를 참고하면, CTL의 경우 나노볼을 처리하지 않았기 때문에 붉은색 파장의 현광이 전혀 검출되지 않음을 확인하였다. 나노볼을 처리한 경우 시간에 따라 세포내로 나노볼이 유입되어 DAPI(파랑)으로 염색된 핵 주위에 위치하고 있음을 확인하였고, 12시간 후에는 나노볼로부터 siRNA-microsponge가 방출되어 나오는 것을 붉은 현광이 번지는 것으로 확인하였다.Referring to FIG. 5A, in the case of CTL, since no nanoballs were processed, it was confirmed that no red light was detected. When the nanoballs were treated, it was confirmed that the nanoballs were introduced into the cells over time and located around the nucleus stained with DAPI (blue), and after 12 hours, the siRNA-microsponge was released from the nanoballs. It was confirmed to be smeared.
도 5B를 참고하면, 상용화된 agent인 Lipo와 나노볼(NP) 그룹에서만 mouse VEGF-A mRNA가 나오지 않고 분해되었음을 보여준다. 즉, 나노볼이 mouse melanoma B16F10 cell의 mouse VEGF-A의 발현을 효과적으로 억제함을 알 수 있다. mouse beta actin은 각 sample이 같은 cDNA농도로 PCR실험이 진행되었음을 확인하는 데이터이다.Referring to FIG. 5B, only the commercialized agent Lipo and the nanoball (NP) group showed that mouse VEGF-A mRNA was not released but degraded. In other words, it can be seen that the nanoball effectively inhibits the expression of mouse VEGF-A in mouse melanoma B16F10 cells. Mouse beta actin is the data confirming that the PCR experiment was carried out with the same cDNA concentration of each sample.
도 5C는 real-time PCR로 증폭시키면서 정량적인 분석을 한 데이터이다. 나노볼의 경우 mono, siRNA-microsponge가 VEGF-A mRNA를 거의 분해하지 못하는 것에 비해 26.5% 수준까지 VEGF-A 발현을 억제하는 것을 볼 수 있다.5C is data obtained by quantitative analysis while amplified by real-time PCR. In the case of nanoballs, mono and siRNA-microsponge inhibit VEGF-A expression by 26.5% compared with almost no degradation of VEGF-A mRNA.
CNVCNV 억제 시험과 정량화  Inhibition Testing and Quantification
6-8주의 C57BL/6J 쥐를 사용하였다. 마취와 산동제를 안구에 투여한 후, green Argon laser (532 nm) (장비명 : Lumenis Selecta Duet SLT (Lumenis, Santa Clara, CA, U.S.A)으로 RPE layer에 조사하였다.6-8 weeks of C57BL / 6J mice were used. After anesthesia and anesthetics were administered to the eye, the RPE layer was irradiated with a green Argon laser (532 nm) (device name: Lumenis Selecta Duet SLT (Lumenis, Santa Clara, CA, U.S.A).
laser를 조사한 쥐의 안구에서 CNV 현상이 일어나는데 laser 조사 하루 후에 나노볼을 해밀턴 주사기로 안구의 유리체강내 주입하여 치료효과를 본다. 2주후 안구적출 전에 쥐의 심장부분의 dextran-FITC를 주입하여 혈관을 염색하고 적출된 안구를 플랫마운팅(flat-mount)하여 현광현미경으로 관찰하였다. 또한 입자의 6주 동안의 지속효과를 알아보기 위하여, 나노볼을 해밀턴 주사기로 안구의 유리체강내 주입하고 4주후에 laser를 조사하여 쥐의 안구에 CNV를 유발 하였다. 2주후 안구적출 전에 쥐의 심장부분의 dextran-FITC를 주입하여 혈관을 염색하고 적출된 안구를 플랫마운팅하여 형광현미경으로 관찰하였다. 2주차 샘플과 6주차 샘플에서 생성된 CNV 부분을 관찰하여 대표적인 이미지를 도 6A에 나타내었다. 도 6B는 Image J program을 사용하여 CNV 면적 분석 측정한 결과이다.CNV phenomenon occurs in the eyes of rats irradiated with laser. One day after laser irradiation, nanoballs are injected into the vitreous cavity of the eye with a Hamilton syringe to see the therapeutic effect. Two weeks later, prior to ocular extraction, dextran-FITC was injected into the heart of rats to stain blood vessels, and the extracted eye was flat-mounted and examined under a light microscope. In addition, in order to investigate the sustained effect of the particles for 6 weeks, nanoballs were injected into the vitreous cavity of the eye with a Hamilton syringe and 4 weeks later, laser was irradiated to induce CNV in the rat eye. Two weeks later, prior to ocular extraction, dextran-FITC was injected into the heart of the rat to stain blood vessels, and the mounted eye was flat mounted for observation by fluorescence microscopy. A representative image is observed in FIG. 6A by observing the CNV portion generated in the 2nd and 6th sample. 6B is a result of CNV area analysis measurement using Image J program.
도 6A의 플랫마운트 CNV 이미지(좌측에서부터 control, 나노볼 주입후 2주차 샘플, 나노볼 주입후 6주차 샘플)를 보면 나노볼을 주입한 군들이 CNV가 control 군에 비하여 면적이 많이 줄었음을 정량적으로 알 수 있다. CNV를 일으키는 주요한 혈관이 모두 줄었음을 확인하였다. The flat mount CNV image of FIG. 6A (control from left, sample 2 weeks after nanoball injection, sample 6 weeks after nanoball injection) shows that the CNV group was significantly reduced in area compared to the control group. This can be seen. All major blood vessels causing CNV were confirmed to be reduced.
도 6B를 살펴보면, CNV 유발 후 나노볼 주입 후 2주차 샘플을 보면 대조군(CTL)에 비해서는 약 1/4정도로 CNV 면적이 줄었음을 확인하였다. 또한 나노볼 주입 후 6주차 샘플의 경우 역시 대조군(CTL)에 비해 약 1/2정도로 CNV 면적이 줄어들어, 나노볼의 약효가 최소한 6주까지 지속되는 것을 알 수 있다.Referring to FIG. 6B, after the nanoball injection after CNV induction, the CNV area was reduced to about 1/4 compared to the control (CTL). In addition, in the case of 6 weeks after the nano-ball injection, the CNV area is reduced by about 1/2 compared to the control (CTL), indicating that the efficacy of the nano-ball lasts at least 6 weeks.
Oxygen-induced Oxygen-induced retiopathyretiopathy (( OIROIR ) 모델에서 혈관억제 실험과 정량화Vascular suppression experiments and quantification
임신한 C57BL/6 mice를 사용하였다. 신생아 쥐가 태어난 후 7일부터 12일까지 75% Oxygen 탱크에 넣어 사육한다. 그 후, 신생아쥐에 마취와 산동제를 안구에 투여한 후, 나노볼을 해밀턴 주사기로 안구의 유리체강내에 주입하고 일반적인 조건의 동물 사육실에서 사육한다. 나노볼 주입 후 5일차(P17)와 12일차(P24)에 안구를 적출하고, 혈관을 염색할 수 있는 Lectin-FITC을 이용하여 staining 하고 플랫마운팅하여 형광현미경으로 관찰하였고 각 군의 대표적인 이미지를 도 7에 표시하였다.Pregnant C57BL / 6 mice were used. Newborn rats are kept in 75% Oxygen tanks from 7 to 12 days after birth. Subsequently, the newborn rats are anesthetized with anesthesia and acid, and then the nanoballs are injected into the vitreous cavity of the eye with a Hamilton syringe and bred in a normal animal breeding room. After injection of nanoballs, eyes were extracted on day 5 (P17) and day 12 (P24), stained and flat-mounted using Lectin-FITC to stain blood vessels, and observed by fluorescence microscopy. 7 is shown.
또한 이러한 관찰된 이미지를 정량화하여 나노볼의 효과를 확인하기 위해 optic nerve 로부터 1 mm 떨어진 부분에 500μm × 500μm을 4군데를 임의로 선택하여 branched point를 단위면적당 계산하여, 각 군마다 비교하였고 이를 도 8A에 막대그래프로 표시하였다. 또한 각 이미지별로 Neovascularization area (NA)를 계산하여 각 군마다 비교하였고 이를 도 8B의 막대그래프로 표시하였다. In addition, in order to quantify these observed images, 4 spots of 500μm × 500μm were randomly selected at 1 mm away from the optic nerve, and branched points were calculated per unit area, and compared with each group. Indicated by a bar graph. In addition, Neovascularization area (NA) was calculated for each image, and compared with each group, and the bar graph of FIG.
도 7을 관찰해보면, 대조군 (OIR CTL)에 비해 나노볼을 주입한 군이 혈관 생성이 억제되어 혈관의 굵기와 밀도가 줄어들고, NA 역시 줄어듦을 확인할 수 있다. Observing FIG. 7, compared to the control group (OIR CTL), the group injecting the nanoballs suppressed blood vessel generation, thereby decreasing the thickness and density of blood vessels and decreasing NA.
도 8은 도 7의 이미지를 정량화한 데이터로, 이를 통해 나노볼 주입 후 5일차와 12일차에 해당하는 군이 대조군 (OIR CTL)에 비해 혈관생성이 억제되고 NW가 억제됨을 확인할 수 있었다.FIG. 8 is a quantified data of FIG. 7, through which the group corresponding to the 5th and 12th days after the nanoball injection was inhibited from angiogenesis and suppressed NW compared to the control group (OIR CTL).
독성 실험Toxicity Experiment
MTT assay : Thiazolyl Blue Tetrazolium Bromide(MTT)를 PBS에 5mg/ml로 녹이여 solution 으로 만든다. MTT solution은 세포내의 미토콘드리아 내에서 반응하여 특정 파장대를 흡수는 발색물질을 만드는데 이를 통해 세포수로 간접적으로 알 수 있다. human ARPE-19 cell line은 CNV 억제를 통하여 황반변성을 치료하기 위하여 유전체입자가 전달해야 할 목표 세포 중 하나이다. 2 × 104개의 ARPE-19 cell을 96 well plate에 seeding 한 후 24시간 incubation 시킨다. 세포가 plate 내에 안착되면 serum free medium과 나노볼로 처리한 후에 24, 48, 72h 후에 DPBS로 washing 하고 MTT 시약으로 처리하여 6시간후에 DMSO 100㎕씩 처리하여 plate reader기로 cell viability를 측정하였다.MTT assay: Dissolve Thiazolyl Blue Tetrazolium Bromide (MTT) in PBS at 5mg / ml to make solution. The MTT solution reacts within the mitochondria in the cell to produce a chromophore that absorbs a specific wavelength band. The human ARPE-19 cell line is one of the target cells to be delivered by genomic particles to treat macular degeneration through CNV inhibition. 2 × 10 4 ARPE-19 cells are seeded in 96 well plate and incubated for 24 hours. When the cells were placed in the plate, the cells were treated with serum free medium and nanoballs, and then washed with DPBS after 24, 48, and 72 h, treated with MTT reagent, and treated with 100 μL of DMSO after 6 hours to measure cell viability with a plate reader.
Hematoxylin and eosin stain : Hematoxylin and eosin stain (H&E stain)Hematoxylin and eosin stain: Hematoxylin and eosin stain (H & E stain)
널리 쓰이는 조직분석 방법으로, 조직을 paraffin에 담아 블록으로 만든 후에 적당한 크기로 슬라이스를 만들어 xylene 3-5회 washing으로 paraffin을 제거하고 Hematoxylin(세포핵 염색)으로 염색한다. 그 후 1% HCL 로 남아있는 Hematoxylin를 제거한다. 그 후 Eosin(세포질 염색)으로 염색한다. 이를 통해 안구의 단면 슬라이스의 각각의 세포층들이 입자를 주입했을 때 변하는지 관찰하였다.A widely used tissue analysis method, the tissue is placed in a paraffin into blocks, and then slices are made into appropriate sizes, and the paraffin is removed by xylene 3-5 times washing and stained with hematoxylin (nucleus staining). Then remove the remaining Hematoxylin with 1% HCL. Then stain with Eosin (cytoplasmic staining). This was to observe whether each cell layer of the cross-sectional slice of the eye changes when the particles are injected.
도 9를 참고하면, mouse 안구 내에 나노볼을 주입한 경우와. 아무것도 주입하지 않은 군(negative, CTL) 에 비해 안구조직내에 cell layer의 변화나 macrophage등의 면역반응에 의한 세포들이 관찰되지 않아 독성이 없었음을 알 수 있다.Referring to Figure 9, and when the nano-ball injected into the mouse eye. Compared to the group injected with nothing (negative, CTL), cells were not observed due to the change of cell layer or the immune response such as macrophage.
본 발명의 단순한 변형 내지 변경은 모두 본 발명의 영역에 속하는 것으로, 본 발명의 구체적인 보호범위는 첨부된 특허청구범위에 의하여 명확해질 것이다.All simple modifications and variations of the present invention fall within the scope of the present invention, and the specific scope of the present invention will be apparent from the appended claims.
[부호의 설명][Description of the code]
10 : 코어부 20 : 양이온성 고분자층10 core portion 20 cationic polymer layer
30 : 히알루론산 외곽층30: hyaluronic acid outer layer
본 발명은 맥락막 신생혈관과 관련 있는 노인성 황반변성을 포함하여 다양한 망막질병 치료를 위하여 사용될 수 있는 유전자 치료제인 siRNA를 다량으로 함유한다.The present invention contains large amounts of siRNA, a gene therapy that can be used for the treatment of various retinal diseases, including senile macular degeneration associated with choroidal neovascularization.

Claims (17)

  1. 유전자를 표적하는, 자기조립된 하나 이상의 핵산 분자를 포함하는 코어;A core comprising one or more nucleic acid molecules self-assembled that target a gene;
    상기 코어를 둘러싸 응축시키는 양이온성 고분자층; 및A cationic polymer layer surrounding and condensing the core; And
    상기 양이온성 고분자층 외면에 코팅된 히알루론산 외곽층을 포함하는 것을 특징으로 하는 유전체 약물 전달용 입자.Particles for dielectric drug delivery, characterized in that it comprises a hyaluronic acid outer layer coated on the outer surface of the cationic polymer layer.
  2. 제 1 항에 있어서, 상기 입자는 크기가 50~350nm인 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the particle has a size of 50 to 350 nm.
  3. 제 1 항에 있어서, 상기 코어는 상기 양이온성 고분자층에 의해 직경이 500nm 이하로 수축되는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the core is contracted to a diameter of 500 nm or less by the cationic polymer layer.
  4. 제 1 항에 있어서, 상기 코어는 복수개의 핵산분자를 포함하고, 상기 핵산분자들은 전부 동일한 핵산서열을 가지거나 적어도 하나 이상의 공통 핵산 서열을 가지는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the core comprises a plurality of nucleic acid molecules, all of which have the same nucleic acid sequence or at least one consensus nucleic acid sequence.
  5. 제 1항에 있어서, 상기 핵산 분자는 망막 질환을 유도하는 특정 유전자의 mRNA들을 표적하는 안티센스 RNA와 이에 상보적인 센스 RNA가 결합된 이중 가닥 RNA를 포함하는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the nucleic acid molecule comprises an antisense RNA targeting mRNAs of a specific gene inducing retinal disease and a double stranded RNA complementary to the sense RNA.
  6. 제 1항에 있어서, 상기 핵산 분자는 망막 질환을 유도하는 특정 유전자의 mRNA를 표적하는 작은 간섭 RNA(siRNA) 또는 안티센스 핵산(antisense nucleic acids)로 분해되는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the nucleic acid molecule is degraded into small interfering RNA (siRNA) or antisense nucleic acids that target mRNA of a specific gene inducing retinal disease.
  7. 제 6항에 있어서, 상기 코어는 작은 간섭 RNA(siRNA) 단위 서열 또는 안티센스 핵산(antisense nucleic acids) 단위 서열이 복제(copy)된 1× 106 개 이상의 상기 작은 간섭 RNA(siRNA) 또는 안티센스 핵산(antisense nucleic acids)을 포함하는 것을 특징으로 하는 유전체 약물 전달용 입자.7. The method of claim 6, wherein the core comprises at least 1 × 10 6 or more of the small interfering RNA (siRNA) or antisense nucleic acid (siRNA) or copies of the antisense nucleic acid unit sequence. Particles for genome drug delivery, characterized in that it comprises antisense nucleic acids).
  8. 제 1항에 있어서, 상기 코어 내의 상기 핵산 분자는 층상 시트(lamellar sheet)를 구비하는 결정 구조 내에 배열되는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the nucleic acid molecules in the core are arranged in a crystal structure having a lamellar sheet.
  9. 제 1 항에 있어서, 상기 코어 내의 핵산 분자는 스템 루프(STEM-LOOP) 또는 선형 구조를 포함하는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the nucleic acid molecule in the core comprises a stem loop (STEM-LOOP) or a linear structure.
  10. 제 1항에 있어서, 상기 히알루론산 외곽층은 히알루론산이 상기 양이온성 고분자층 외면에 정전기적 인력으로 코팅되어 (-) 전하를 나타내는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the hyaluronic acid outer layer has a hyaluronic acid coated with an electrostatic attraction on the outer surface of the cationic polymer layer to exhibit a negative charge.
  11. 제 1항에 있어서, 상기 입자는 그 외부가 (-)전하를 나타내는 것을 특징으로 하는 유전체 약물 전달용 입자.The particle for dielectric drug delivery according to claim 1, wherein the particle has a negative charge outside thereof.
  12. 제 1항에 있어서, 상기 양이온성 고분자는 폴리에틸렌이민, 폴리아민 및 폴리비닐아민으로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 유전체 약물 전달용 입자.The particle of claim 1, wherein the cationic polymer is at least one selected from the group consisting of polyethyleneimine, polyamine, and polyvinylamine.
  13. 안티센스 RNA와 이에 상보적인 센스 RNA가 포함된 환형 DNA를 증폭시켜 복수개의 이중 가닥 RNA를 제조하고, 상기 복수개의 이중 가닥 RNA들이 자기조립되어 구형 코어를 형성하는 단계;Amplifying a circular DNA including antisense RNA and a sense RNA complementary thereto to prepare a plurality of double stranded RNAs, wherein the plurality of double stranded RNAs are self-assembled to form a spherical core;
    상기 구형 코어에 양이온성 고분자를 혼합하여 이온 상호 작용으로 상기 코어를 응축시키는 단계; 및Mixing a cationic polymer with the spherical core to condense the core by ion interaction; And
    상기 양이온성 고분자 외면에 정전기적 인력으로 히알루론산 외곽층을 코팅하는 단계를 포함하는 유전체 약물 전달용 입자 제조방법.Dielectric drug delivery particle manufacturing method comprising the step of coating the outer layer of hyaluronic acid electrostatic attraction on the outer surface of the cationic polymer.
  14. 제 13항에 있어서, 상기 방법은 상기 환형 DNA를 롤링 서클 증폭(RCA) 또는 롤링 서클 전사(RCT)를 사용하여 복수개의 이중 가닥 RNA을 형성하는 것을 특징으로 하는 유전체 약물 전달용 입자 제조방법.The method of claim 13, wherein the circular DNA is formed by rolling circle amplification (RCA) or rolling circle transcription (RCT) to form a plurality of double-stranded RNA.
  15. 제 13항에 있어서, 상기 코어와 상기 양이온성 고분자의 중량비는 1 : 0.05 ~ 1 인 것을 특징으로 하는 유전체 약물 전달용 입자 제조방법.15. The method of claim 13, wherein the weight ratio of the core and the cationic polymer is 1: 0.05 to 1.
  16. 제 13항에 있어서, 상기 히알루론산은 상기 응축된 코어 대비 중량비로 1 : 0.1 ~ 0.5(코어 : 히알루론산)인 것을 특징으로 하는 유전체 약물 전달용 입자 제조방법.14. The method of claim 13, wherein the hyaluronic acid is 1: 0.1 to 0.5 (core: hyaluronic acid) in the weight ratio of the condensed core (core: hyaluronic acid) manufacturing method of the particle for delivery of the drug.
  17. 제 13항에 있어서, 상기 안티센스 RNA는 망막 질환을 유도하는 특정 유전자의 mRNA를 표적하여 이의 발현을 억제하는 것을 특징으로 하는 유전체 약물 전달용 입자 제조방법.The method of claim 13, wherein the antisense RNA targets mRNA of a specific gene that induces retinal disease and inhibits expression thereof.
PCT/KR2016/003523 2015-04-07 2016-04-05 Sirna-based nanomedicine having sustained efficacy for retinal diseases and method for preparing same WO2016163718A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2015-0048982 2015-04-07
KR1020150048982A KR101880790B1 (en) 2015-04-07 2015-04-07 Nanoparticles for genes drug delivery with siRNA for the long -term treatment of retinal disorders and method for preparing the same

Publications (1)

Publication Number Publication Date
WO2016163718A1 true WO2016163718A1 (en) 2016-10-13

Family

ID=57072751

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2016/003523 WO2016163718A1 (en) 2015-04-07 2016-04-05 Sirna-based nanomedicine having sustained efficacy for retinal diseases and method for preparing same

Country Status (2)

Country Link
KR (1) KR101880790B1 (en)
WO (1) WO2016163718A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020074532A1 (en) * 2018-10-08 2020-04-16 Universiteit Gent Composition for treatment of vitreous disease or disorder
CN111803653A (en) * 2020-06-27 2020-10-23 苏州大学 Gene delivery system capable of removing mixed cell membrane coating and preparation method and application thereof
CN112568187A (en) * 2020-12-23 2021-03-30 上海谋始生物科技有限公司 Method for constructing novel mouse model with retinal vein occlusion

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102198900B1 (en) 2019-05-10 2021-01-07 서강대학교 산학협력단 Nanoparticle complex for treating disease and Method for manufacturing the same
KR102659310B1 (en) * 2021-08-23 2024-04-19 주식회사 코스메카코리아 Cosmetic composition containing acicular spicule powder and manufacturing method thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050056944A (en) * 2002-07-24 2005-06-16 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 Compositions and methods for sirna inhibition of angiogenesis
KR100806088B1 (en) * 2007-08-09 2008-02-21 고려대학교 산학협력단 Compositions for delivering nucleic acids using cationic polymer conjugates consisting of hyaluronic acid and polyethylenimine
KR20110027969A (en) * 2009-09-11 2011-03-17 포항공과대학교 산학협력단 Method of manufacturing polymer for delivering nucleic acids and compositions for delivering nucleic acids comprising the polymer
KR20110083919A (en) * 2010-01-15 2011-07-21 한국과학기술원 Multi-conjugate of sirna targeting multiple genes and preparing method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011526892A (en) * 2008-06-30 2011-10-20 アンジオブラスト システムズ，インコーポレーテッド Treatment of ocular diseases and hypervascularization using combination therapy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050056944A (en) * 2002-07-24 2005-06-16 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 Compositions and methods for sirna inhibition of angiogenesis
KR100806088B1 (en) * 2007-08-09 2008-02-21 고려대학교 산학협력단 Compositions for delivering nucleic acids using cationic polymer conjugates consisting of hyaluronic acid and polyethylenimine
KR20110027969A (en) * 2009-09-11 2011-03-17 포항공과대학교 산학협력단 Method of manufacturing polymer for delivering nucleic acids and compositions for delivering nucleic acids comprising the polymer
KR20110083919A (en) * 2010-01-15 2011-07-21 한국과학기술원 Multi-conjugate of sirna targeting multiple genes and preparing method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GANESH, S. ET AL.: "Hyaluronic Acid based Self-assembling Nanosystems for CD 44 Target Mediated siRNA Delivery to Solid Tumors", BIOMATERIALS, vol. 34, 2013, pages 3489 - 3502, XP028984671 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020074532A1 (en) * 2018-10-08 2020-04-16 Universiteit Gent Composition for treatment of vitreous disease or disorder
US20210338595A1 (en) * 2018-10-08 2021-11-04 Universiteit Gent Composition for treatment of vitreous disease or disorder
CN111803653A (en) * 2020-06-27 2020-10-23 苏州大学 Gene delivery system capable of removing mixed cell membrane coating and preparation method and application thereof
CN111803653B (en) * 2020-06-27 2022-04-15 苏州大学 Gene delivery system capable of removing mixed cell membrane coating and preparation method and application thereof
CN112568187A (en) * 2020-12-23 2021-03-30 上海谋始生物科技有限公司 Method for constructing novel mouse model with retinal vein occlusion

Also Published As

Publication number Publication date
KR20160120028A (en) 2016-10-17
KR101880790B1 (en) 2018-08-16

Similar Documents

Publication Publication Date Title
JP2024028909A (en) OLIGONUCLEOTIDE COMPOUNDS FOR TARGETING HUNTINGTIN mRNA
WO2016163718A1 (en) Sirna-based nanomedicine having sustained efficacy for retinal diseases and method for preparing same
US20230399659A1 (en) Exosomal loading using hydrophobically modified oligonucleotides
CN108165548A (en) Reduce the delivering RNAi compounds certainly of size
EP2205740A2 (en) Tripartite rnai constructs
US11839624B2 (en) Micellar nanoparticles and uses thereof
CN108175759A (en) A kind of antineoplastic target drug delivery system and preparation method and application
WO2015125147A9 (en) Anionic polyplexes for use in the delivery of nucleic acids
Comegna et al. Assisting PNA transport through cystic fibrosis human airway epithelia with biodegradable hybrid lipid-polymer nanoparticles
Xu et al. Efficient siRNA delivery using PEG-conjugated PAMAM dendrimers targeting vascular endothelial growth factor in a CoCl2-induced neovascularization model in retinal endothelial cells
Asakiya et al. Current progress of miRNA-derivative nucleotide drugs: modifications, delivery systems, applications
EP4183879A1 (en) Double-stranded oligonucleotide and composition for treating covid-19 containing same
WO2015174703A1 (en) Nanoparticles for gene drug delivery, and preparation method therefor
JP2010509401A (en) Efficient delivery of antisense oligonucleotides to the nucleus
JP7504482B2 (en) Oligonucleotide compounds targeting huntingtin mRNA
KR20170055012A (en) Nanoparticles for genes drug delivery with siRNA for the long -term treatment of retinal disorders and method for preparing the same
Wang et al. Compounding engineered mesenchymal stem cell-derived exosomes: A potential rescue strategy for retinal degeneration
Li et al. Tetrahedral DNA‐Based Functional MicroRNA‐21 Delivery System: Application to Corneal Epithelial Wound Healing
EP4388094A1 (en) Oligonucleotides for modulating synaptogyrin-3 expression
Mouro Identification of critical points in colloidal delivery systems for intravenous administration and intracellular delivery of nucleic acids as therapeutic agents

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16776821

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16776821

Country of ref document: EP

Kind code of ref document: A1