KR101585192B1 - Nanorod-based Nanocomposite Intracellular Delivery System - Google Patents

Abstract

The present invention relates to a nanorod-based nanocomposite cell carrier, comprising: (a) a nanorod comprising a multi-component inorganic metal portion comprising a gold component region and a nickel component region; The gold component portion and the nickel component portion are spatially distinguished from each other in the nanorods; (b) a cell-binding molecule bound to any one of a gold component region and a nickel component region of the nanorods; And (c) a carbohydrate bound to another of the gold component moiety and the nickel moiety of the nanorods. The nanorod-based nanocomposite intracellular delivery system comprises: The present invention can treat cancer through induction of cancer cell death by functionalizing miRNA capable of activating a cancer cell death killing protein, and it is also possible to display a fluorescent substance on a nanorod and to provide a cell imaging technique and a MRI contrast agent Can be used to diagnose cancer through simultaneous imaging of cells in vivo.

Description

Nanorod-based Nanocomposite Intracellular Delivery System < RTI ID = 0.0 >

The present invention relates to nanorod-based nanocomposite intracellular carriers.

Recently, as people's interest in healthy life has been increasing due to population aging and increasing chronic diseases, one out of four Koreans in Korea is currently suffering from cancer. Cancer is one of the most burdensome diseases of all existing human beings, and it is predicted that this trend will grow in the future. In order to reduce the burden of cancer, many efforts have been made at the national level, but above all, prevention of cancer through smoking cessation or preventive contact of individuals is important, and research on precise diagnosis and treatment of cancer that has once occurred is urgently needed Do.

The diagnosis of cancer can be made by a variety of methods including ultrasound, CT, blood, and biopsy. However, in the case of a blood test for a tumor marker whose numerical value can be elevated when the cancer is present, Due to the differences in the numerical values, the limitations of the test are implied and accurate diagnosis technology is difficult.

The method of medically treating cancer is artificially using a method of killing cancer cells in the human body, which can be classified into a resection surgery to remove a cancer mass, a method of inducing the death of cells through radiation, and an administration method of an anticancer drug. However, the above-mentioned conventional cancer treatment methods are urgently required to be improved because of the high cost of treatment, the risk of surgery, or the adverse health effects due to chemotherapy or radiation therapy.

Therefore, diagnosis and treatment technologies that can accurately diagnose cancer, which is one of the diseases that threaten humanity's health in the 21st century and cause great burdens, and that can significantly improve the risks and side effects of existing cancer treatment methods Research and development is urgently required.

Korean Patent Publication No. 10-2012-0035498 (April 16, 2012)

The present inventors have sought to develop nanodevice-based cell imaging and drug delivery vehicles. As a result, it was possible to image cells by binding cell-binding molecules and miRNA to nanorods containing gold and nickel components, and by selectively selecting miRNAs capable of inducing apoptosis, The present inventors have completed the present invention by developing a nanocomposite cell carrier capable of simultaneously treating the cell.

Accordingly, it is an object of the present invention to provide a nanorod-based nanocomposite intracellular delivery system.

It is another object of the present invention to provide a nanocomposite pharmaceutical composition for cancer diagnosis and treatment.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a nanorod-based nanocomposite intracellular delivery system comprising:

(a) a nanorod comprising a multi-component inorganic metal portion comprising a gold component and a nickel component; The gold component portion and the nickel component portion are spatially distinguished from each other in the nanorods;

(b) a cell-binding molecule bound to any one of a gold component region and a nickel component region of the nanorods; And

(c) a cargo bonded to the other of the gold component portion and the nickel component portion of the nanorod.

The present inventors have sought to develop nanodevice-based cell imaging and drug delivery vehicles. As a result, it was possible to image cells by binding cell-binding molecules and miRNA to nanorods containing gold and nickel components, and by selectively selecting miRNAs capable of inducing apoptosis, We have developed a carrier of nanocomposite cells capable of simultaneous treatment.

The nanorod-based nanocomposite intracellular carrier of the present invention comprises a nanorod comprising a multi-component inorganic metal portion comprising a gold component portion and a nickel component portion.

According to an embodiment of the present invention, the gold component portion and the nickel component portion are spatially distinguished from each other in the nanorods, and the gold component parts and the nickel component parts may be gold-nickel nanorods, gold- , Nickel-gold-nickel nanorods or gold-nickel-gold-nickel nanorods, but are not limited thereto. According to another embodiment of the present invention, the nanorods have a spatial combination of gold-nickel having the same length.

Using a nickel having an MRI contrast agent of the present invention features vivo (in vivo . < / RTI >

According to one embodiment of the present invention, the nanorods have a diameter of 10 to 500 nm and a length of 100 to 1000 nm. The diameter of the nanorod is dependent on the pore size of the aluminum template, and the length of the nanorod can be arbitrarily controlled by electrodeposition of gold or nickel. According to another embodiment of the present invention, the nanorods have a diameter of 40 to 400 nm and a length of 400 to 800 nm. According to a specific embodiment of the present invention, the nanorods have a diameter of 50 to 250 nm and a diameter of 500 To 700 nm.

The nanorod-based nanocomposite carrier of the present invention comprises a cell-binding molecule at any one of a gold component portion and a nickel component portion of the nanorod, and is delivered to the cell through the nanorod.

According to an embodiment of the present invention, the cell-binding molecule is a peptide or a protein. According to another embodiment of the present invention, the cell-binding molecule is a peptide. According to a specific embodiment of the present invention, Luteinizing hormone-releasing hormone (LHR) peptide, folic acid, RGD peptide, chlorotoxin peptide, Erb B peptide, EGF (endothelial growth factor) peptide, adrenomedullin (ADM) peptide, an AMR (Autocrine Motility Factor) peptide, an APRIL peptide, an artemin peptide, a betacynline peptide, a bombesin peptide, a CTR (calcitonin receptor) peptide, an endothelin peptide, an erythropoietin peptide, an FGF Fibroblast Growth Factor) peptide, FSH (Follicle Stimulating Hormone) peptide, gastrin peptide, GRP (gastrin releasing p (G-CSF) peptide, growth hormone peptide, HB (Heparin Binding) -EGF peptide, HGF (human growth hormone) peptide, (Hepatocyte Growth Factor) peptide, IL (Interleukin) peptide, KGF (Keratinocyte Growth Factor) peptide, leptin peptide, Leukemia inhibitory factor (LIF) peptide, α-melanotropin peptide, MGSA (Melanoma Growth Stimulatory Activity) , NRG peptide, oxytocin peptide, osteopontin peptide, neuregulin-1 peptide, VIP (Vasoactive Intestinal Peptide), PACAP (Pituitary Adenylate Cyclase-Activating Peptide), PDGF (Platelet- Derived Growth Factor) peptide, prolactin peptide , SCF (Skp, Cullin, F-box containing complex) peptides, somatostatin peptides, Cortis tactin peptides, TNF (Tumor Necrosis Factor) peptides A cell growth factor (TGF) peptide, a Vascular Endothelial Growth Factor (VEGF) peptide, a vasopressin peptide, an angiopoietin peptide, a B-cell chronic lymphocytic leukemia (BCL) 12KD peptide, BAFF (B-cell Activating Factor) peptide, GalNAn peptide, GDNF peptide, GnRH peptide, Insulin-like Growth Factor- II peptide, Neurotropic peptide, SP Substance P) peptide, a TGF- alpha peptide, an IGF peptide, a CXC peptide or a CC chemokine ligand peptide, and according to another specific embodiment of the present invention, the peptide is a luteinizing hormone releasing hormone peptide. The amino acid sequence of the luteinizing hormone releasing hormone peptide is SEQ ID No. 1.

In the present invention, a peptide that can be used as a cell-binding molecule binds to a membrane protein such as a receptor on a cell surface and is delivered into the cell together with a nanoparticle-based nanocomposite carrier.

According to one embodiment of the present invention, the luteinizing hormone-releasing hormone peptide of the present invention binds to the luteinizing hormone-releasing hormone receptor (LHRHR) on the surface of a cancer cell, The carrier is transferred into the cell. It is delivered into the cell via a receptor-mediated endocytosis process.

The cell-binding molecules of the nanorod-based nanocomposite intracellular carriers of the present invention are associated with nanorod binding functional groups.

According to one embodiment of the present invention, the cell-binding molecule is associated with a functional group for nanorod binding. The nanorod binding functional group is an amino acid including an SH group or an immidazole group. According to another embodiment of the present invention, the amino acid comprising the SH group or the imidazole group is cysteine or histidine.

According to one embodiment of the present invention, 1 to 20 amino acids are bound continuously to the cell-binding molecule, and in another embodiment of the present invention, 2 to 15 amino acids are continuously connected According to a specific embodiment of the present invention, 3 to 10 amino acids are continuously connected to each other. According to another specific embodiment of the present invention, 4 to 8 amino acids are continuously connected.

As used herein, the term " bond " means having a physical and / or chemical link between the two elements to which it is attached. The physical connectivity is a physical coupling or a physical linking, and the chemical linkage is a chemical bond such as peptide bond, covalent bond, ionic bond, hydrogen bond, intermolecular force, van der Waals' force and electrostatic force. It also includes both direct bonds in which both moieties are directly bonded and indirect bonding in which they are bonded through another element (for example, a fluorescent dye).

According to one embodiment of the present invention, the cell-binding molecule of the present invention includes both a direct binding and an indirect binding in combination with a functional group for nanorod binding. According to another embodiment of the present invention, when the cell-binding molecule of the present invention and the functional group for nanorod binding are directly bound, the nanorod binding functional group is sequentially bonded to the 3 'of the cell-binding molecule, When the cell-binding molecule and the functional group for nanodevice binding are indirectly bonded, the fluorescent dye is bonded to the 3 'of the cell-binding molecule and the functional group for binding the nanodevice is functionally bound to the fluorescent dye.

The nanorod-based nanocomposite intracellular carrier of the present invention comprises a cargo in the gold moiety of the nanorod and the other of the nickel moiety.

According to one embodiment of the present invention, the cargo is at least one cargo selected from the group consisting of a siRNA, a drug, a contrast agent, a fluorescent marker, and a dye material. According to another embodiment of the present invention, siRNA is siRNA for cancer cell specific expression protein.

According to one embodiment of the present invention, the cancer cell-specific expression protein is selected from the group consisting of VEGF (Vascular Ephthalial Growth Factors), EGFR (Epidermal Growth Factor Receptors), estrogen receptors, progesterone receptors, fibrin, fibrinogen, HER2 (Human Epidermal Growth Factor 2) (AFP), carcinoembryonic actigen (CEA), CA-125, MUC-1, Epithelial tumor antigen, tyrosinase, (B-cell lymphoma) -2, cFLIP (b-cell lymphoma), and the like, as well as the β-2-microglobulin, thyroglobulin, β-human chorionic gonadotropin, uPA (urokinase plasminogen activator) cellular FLICE Inhibitory Protein, Myeloid cell leukemia-1, NR-13, centrin, fas apoptotic inhibitory molecule 3, caspase-activated nuclease, PED, DNA- 45), X-linked inhibitor of Apoptosis Protein (XIAP), cIAP-1 (cellular Inhibitor (B-cell lymphoma-extra large) and PED ((S) -1-phenylethanol dehydrogenase (Bcl-xL)), cIAP-2, ML-IAP (livin), BIRC5 (survivin), Apolon, ). According to another embodiment of the present invention, the cancer cell-specific expressing protein is VEGF. That is, the cargo bound to the nanorods of the nanorod-based nanocomposite cell of the present invention is siRNA for VEGF, and the siRNA is the second sequence of the sequence listing.

The nanorod-based nanocomposite intracellular carriers of the present invention can be imaged through a nanoparticle-coupled imaging agent.

According to an embodiment of the present invention, the nanorods are further combined with an imaging agent. According to another embodiment of the present invention, the imaging agent is a fluorescent dye, and according to a particular embodiment of the present invention, a Xanthene-based derivative (e.g., fluorescein, rhodamine, Oregon green, eosin and Texas red), cyanine derivatives (such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, Thiacarbocyanine and merocyanine], naphthalene derivatives, coumarin derivatives, oxadiazole derivatives (for example, pyridyloxazole, nitrobenzoxazine, Nitrobenzoxadiazole and benzodxadiazole], pyrene derivatives [such as cascade blue], oxazine derivatives [nile red, nile blue (for example, nile blue, cresyl violet, Acridine derivatives such as proflavin, acridine, orange, acridine yellow, etc., arylsulfonyl derivatives such as arylsulfonyl derivatives, For example, arylmethine-based derivatives (such as auramine, crystal violet, malachite green) and tetrapyrrole derivatives (e.g., porphin, phtalocyanine ), And bilirubin]. According to another specific embodiment of the present invention, the fluorescent dye is a xanthene derivative.

According to another aspect of the present invention, there is provided a nanocomposite pharmaceutical composition for diagnosing and treating cancer using a nanorod-based nanocomposite intracellular delivery system comprising:

(a) a nanorod comprising a multi-component inorganic metal portion comprising a gold component and a nickel component; The gold component portion and the nickel component portion are spatially distinguished from each other in the nanorods;

(b) a cancer cell-binding molecule bound to any one of a gold component site and a nickel component site of the nanorod; And

(c) an anticancer agent bound to the other of the gold component portion and the nickel component portion of the nanorod.

The nanorod-based nanocomposite intracellular carrier of the present invention can be used for treatment of a disease or a disease, detection of a specific cell, diagnosis of a disease (e.g., cancer), tracking of a specific cell, and in vivo imaging And the like.

In one embodiment of the present invention the cancer is selected from the group consisting of breast cancer, respiratory cancer, small cell lung cancer, non-small cell lung cancer, basal cell carcinoma, biliary cancer, bladder cancer, bone cancer, brain tumor, cervical cancer, Cancer, leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, leukemia, leukemia, acute myelogenous leukemia, cancer of the digestive system, endometrial cancer, esophagus cancer, Cancer of the stomach, cancer of the stomach, cancer of the prostate, retinoblastoma, rhabdomyosarcoma, rectal cancer, renal cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, Cancer of the uterus or urinary tract.

Since the nanocomposite pharmaceutical composition for diagnosing and treating cancer of the present invention is similar to the nanocomposite-based nanoparticle intracellular carrier, the description common to both is omitted in order to avoid the excessive complexity of the present specification .

The features and advantages of the present invention are summarized as follows:

(a) By controlling the size of the nanoparticles of the present invention, transfection into cells can be improved and gene transfer efficiency can be maximized.

(b) The present invention can maximize the selectivity of gene transfer by functioning inorganic nanomaterials as a ligand capable of selectively targeting cancer cells and bringing the gene transporter closer to the cancer cell surface only.

(c) The present invention can treat cancer through induction of cancer cell death by functionalizing miRNA capable of activating cancer cell death function protein.

(d) The present invention can diagnose cancer through simultaneous imaging of cells in vivo using nickel having a cell imaging technique and an MRI contrast agent function in vitro by displaying a fluorescent substance on a nanorod.

Fig. 1 shows a schematic view of the production of an aluminum mold plate.
2 shows an SEM (Scanning Electron Microscope) image of an aluminum mold plate. The image on the right is the purchased aluminum plate with a hole size of approximately 200 nm, and the left image is an aluminum plate manufactured by the present inventors, and the hole size is approximately 100 nm or less.
Figure 3 shows a schematic diagram of the production of inorganic nanorods.
FIG. 4 is a schematic view of an electrodeposition method for manufacturing nanorods using the aluminum plate.
FIG. 5 shows an SEM image and an optical microscope image of the nanorods prepared by the above-mentioned electrodeposition method. The image on the right is a gold nanorod made of an aluminum mold plate (pore size of 100 nm or less) manufactured by the present inventors, and a middle image and a left image represent gold-nickel nanorods made of purchased aluminum mold (pore size 200 nm).
FIG. 6 is a SEM image of nanorods of various sizes. The right image and the middle image are gold-nickel nanorods (diameter: about 200 nm) fabricated from the purchased aluminum template, and the left image is an aluminum template produced by the present inventors Manufactured nanorods (diameter of about 80 nm or less).
7 is a graph showing nanorod length versus time for electrodeposition. The left graph shows the length of the gold nanorods, and the right graph shows the length of the nickel nanorods.
Fig. 8 is a microscope image of a cancer cell. The left image shows A549 and the right image shows SK-OV-3.
Fig. 9 shows a schematic diagram of the peptide produced.
10 (a) to 10 (b) are microscope images of cancer cells, wherein (a) shows A549, (B) shows MCF-7 and (C) shows SK-OV-3.
11 (a) shows the nucleotide sequence of siVEGF, and Fig. 11 (b) shows the siVEGF expression suppression through the PCR result. Efficiency.
12 shows a schematic diagram of a nanocomposite.
13 (a) to 13 (d) are fluorescence images of a nanocomposite, wherein FIG. 13 (a) is an optical image, 13 (d) is an image obtained by combining Figs. 13 (a) to 13 (c). 13 is a nanorod formed at 10 [mu] m for fluorescence image identification, not actually used nanorods.
Figure 14 is an image showing nanocomposite delivery to MCF-7 cells. 14 (b) is a fluorescence (green) image, Fig. 14 (c) is a fluorescence (red) image, 14 (d) is an optical image.
15 is a graph comparing mRNA expression levels of VEGF (Vascular Endothelial Growth Factor).
16 is a graph comparing the degree of protein expression of VEGF treated with the nanocomposite.
17 is a graph comparing survival rates of cancer cells treated with the nanocomposite.

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

Example 1: Preparation of an aluminum mold plate for producing inorganic nanorods

Established aluminum plate manufacturing technology

Anodic Aluminum Oxide (AAO) is used to form uniform holes by using aluminum foil (AL000630 aluminum foil, 99.999%, 0.25 mm T × 100 mm W × 100 mm L, Future science). The aluminum foil was oxidized by applying a voltage of about 40 V with 0.3 M oxalic acid at 0 캜 for 12 hours, and then etching was performed at 60 캜 for 12 hours using a chromium oxide solution (1.8 wt%) . The same oxidation of oxalic acid was carried out at 0 ° C for 24 hours, then the aluminum was completely removed by soaking in a saturated mercury solution for 6 hours. Finally, in order to form the holes of the aluminum mold plate to have a constant size (100 nm or less), an aluminum mold plate for manufacturing nanorods was completed by reacting with a phosphoric acid solution (8.5%) for 30 minutes (FIG.

Obtain aluminum plate with various hole sizes

Two types of aluminum plates were prepared for the production of inorganic nanorods. An aluminum plate (Anodisc 13, whatman) used as a membrane filter was purchased. The hole size of the aluminum plate is about 200 nm and can be used for the production of inorganic nanorods having a diameter of 200 nm. In addition, the aluminum plate produced by the present inventors using an electro-oxidation method can produce a nanorod having a diameter of 100 nm or less (FIG. 2).

Example 2: Establishment of inorganic metal nanorod manufacturing technique

Gold? Obtained nanorod manufacturing technology using nickel

Inorganic nanorods were prepared using an aluminum template having holes of uniform size. Silver (thickness: 200 nm) was deposited on the opposite side of the aluminum template using a thermal evaporator. Prior to fabrication of the nanorods, electrodeposition (-0.95 ㎷, 1 C) was performed using a silver plating solution (1025 silver, technic inc.) For more accurate deposition. (TECHNI-GOLD 25, technic inc.) Or nickel (HANTAI PMC)] among the constituent components of the inorganic nanorods to be produced at a constant negative voltage (-0.95 ㎷) (E.g., gold: 0.15 C, 300 nm / nickel: 0.3 C, 300 nm). After making nanorods of the desired composition (e.g., gold-nickel or nickel-gold-nickel) and size, the silver deposited on one side of the aluminum template was completely removed using 50% nitric acid solution. The aluminum plate was rinsed with tertiary distilled water, and aluminum oxide was melted using 3M sodium hydroxide solution to obtain an inorganic nanorod (FIGS. 3 and 4).

Preparation of nanoparticles and identification of nanoparticles by scanning electron microscopy (SEM)

The prepared inorganic nanorods were confirmed by scanning electron microscope and optical microscope. The photo on the right is a gold nanorod made by directly manufacturing an aluminum plate with a hole size of 100 nm or less. The middle and left photographs are gold-nickel nanorods made by purchasing an aluminum plate with a hole size of about 200 nm. The number of nanorods that can be made in a single process is approximately 2 x 10 ⁹ (Fig. 5).

Example 3: Fabrication of nanorods of various sizes

Produce nanorods of various sizes

The nanorods of various diameters can be fabricated by using various hole size aluminum plates manufactured or purchased and the length of the nanorods can be controlled by controlling the electric deposition time. The middle and right photographs are gold-nickel nanorods made of purchased aluminum plate and have a diameter of about 200 nm. The photo on the left is a nanorod made of an aluminum plate manufactured by the present inventors and has a diameter of about 80 nm or less (FIG. 6).

Establishment of control technology of composition ratio by adjusting electric deposition time of gold and nickel

The length of the nanorods formed through the adjustment of the electrical deposition time can be controlled. In the preparation of gold nanorods using a gold solution with a monovalent ion state, nanorods of about 700 nm were formed per 0.2 C (Coulomb), and production of nickel nanorods using a bivalent ion nickel solution was about 0.2 C A nanorod of 230 nm was formed (Fig. 7).

Example  4: Selection of cancer cells to treat using inorganic nanocomposites

Selection of Cancer Cell Candidates and Receptor

It is an object of the present invention to induce the death of cancer cells through accurate cell imaging and treatment by selectively penetrating the nanocomposite into cancer cells. Recent studies in various papers have shown that different receptors are selectively expressed in various cancer cells.

Acquire cancer cells expressing specific receptors

Based on the data, A549 (lung adenocarcinoma epitherial cell), which is selectively expressing LHRR (LHRH), and ovarian cancer cells SK (LHRHR) -OV-3; ovarian cancer) were selected to observe the penetration of nanocomposites according to presence or absence of a receptor (FIG. 8).

certain Of peptide  making

A549 selected in this study expresses LHRHR, which specifically binds to luteinizing hormone-releasing hormone (LHRH) and induces receptor-mediated endocytosis. This binding of LHRH and LHRHR is made by a specific peptide sequence (Pyr-HWSYkLRPK; Sequence Listing 1 sequence), which is arbitrarily synthesized and prepared (Peptron Inc.). The synthetic peptides prepared in the present invention were designed by combining specific peptide sequences of Histidine, fluorescent dye and LHRH capable of binding to nanorods (Peptron Inc., FIG. 9).

Example  5: Cancer cells to be treated RNA  Candidate selection

Securing two or more cancer cells with the same receptor

In order to induce cell proliferation and apoptosis of the prepared multicomponent nanocomposite, it is important to secure the cell overexpressing the LHRH receptor. In addition to SK-OV-3 and A549, MCF-7 (breast cancer cells) was secured and the experiment was conducted (FIG. 10).

Cancer cell death induction is possible RNA  Material selection

VEGF (Vascular Endothelial Growth Factor) protein, which is related to cancer growth and metastasis, was selected as a target substance based on the research results of siRNA related research among the papers related to cancer cell death that have been studied so far. Although there is no direct relationship to cancer cell death, the selection of target substances that have been actively researched has led to a reduction in errors in the results of studies on target substances. In the field of cell research, there are many variables in terms of dealing with living biomaterials, so we can select the definite siRNA through selection of siRNA which regulates the mechanism of VEGF. We constructed candidate siRNA through http://sidirect2.rnai.jp/ and selected siRNA through experiment. FIG. 11 shows results of applying four candidate siRNAs to MCF-7. In this study, a second siRNA that inhibits the expression of VEGF was selected (Bioneer Inc., FIG. 11).

Example  6: The three components  Immobilized Versatile  Development of rod-shaped nanocomposite

3 components (Receptors, siRNA , Fluorescent material) Composition of nanocomposite

Three-component nanocomposites were prepared for induction of cancer cell death and cell imaging (diagnosis) in earnest. First, the gold-nickel nanorods were electrochemically synthesized to a size of 600 nm (gold: nickel = 300: 300). 1 mg / ml of LHRH-FITC-His previously designed on the washed nanorods 3 pM was reacted at 4 ° C for 8 hours. Histidine is selectively reacted with the nickel part of the nanorod. Then, the cells were washed three times with PBS (Phosphate Buffered Saline) containing 0.1% Tween 20 at 13000 rpm for 10 minutes using a centrifuge. The resulting 100 μM SH-siRNA-TAMRA (bioneer request, Korea) was reacted at 4 ° C for 8 hours on the nanorod so that the SH group selectively reacted with the gold portion (FIG. 12). This was confirmed by fluorescence microscopy to selectively immobilize the nanorods through a fluorescent image (FIG. 13).

Example  7: induction of cancer cell death using functional rod-shaped nanocomposite

3 components  Confirmation of cell transfer (diagnosis) using nanocomposite

Cell death of LHRH, siRNA and fluorescent substance immobilized multicomponent nanocomposites was confirmed using MCF-7 cells selected for cancer cell death. The prepared nanocomposite (3 pM, 100 μl) was incubated with the cells for 3 hours at room temperature and then washed three times with PBS solution. Since siRNA and LHRH each have red and green fluorescent materials, they can be used for the diagnosis of cancer cells by detecting fluorescence of two different colors. FIG. 14 is an image of the MCF-7 cell transferred with the confocal microscope, showing that the nanocomposite penetrated into the cell well (FIG. 14).

siRNA Induction of cancer cell death using

VEGF protein and siVEGF (one kind of siRNA) were selected for induction of cancer cell death. After the selected siVEGF (GAAGTTCATGGATGTCTAT; Sequence Listing 2) was immobilized on the prepared multi-component nanorods, it was transferred to cancer cells to study the induction of cancer cell death. Identification of the mechanism of cancer cells through siRNA (microRNA) should be examined in three aspects, such as mRNA stage, protein stage and cell proliferation as follows. First, in order to measure the expression level of VEGF mRNA, the expression level of siVEGF-treated cells was observed by RT-PCR (FIG. 15). As can be seen from the results, the expression level of mRNA of siRNA-treated cells was significantly reduced compared with the negative control cells which were not treated with anything, and compared with the nanorod-siRNA complex prepared in this study, siRNA alone penetrated The expression of mRNA was about 2-3 fold less than that of the control. This confirms that the siRNA mechanism works effectively through the nanocomposite. In addition, scrambled siRNAs that do not function as siRNAs and nanorods that are not functionalized as such penetrate into the cells to measure mRNA expression, resulting in the inhibition of mRNA expression through siRNA attached to nanorods I could confirm.

Secondly, in order to directly compare the expression level of VEGF protein, the degree of expression of VEGF was compared through an enzyme-linked immunosorbent assay (ELISA), which is widely used for protein quantification in the market (Fig. 16). The expression level of the protein was not greater than that of the mRNA. Even if mRNA expression is inhibited by siRNA, this phenomenon occurs because the protein that has been generated before is observed. This is a part that can be solved by regularly repeating intracellular penetration in the future. The graph shows that the degree of expression of the cells containing relatively nanocomposite is about 50% of that of the positive control cells, and that it is lower than the expression level of cells delivered by siRNA alone (FIG. 16).

Finally, MTT assay was performed after the cell delivery of the nanocomposite to confirm the death of cancer cells in the prepared nanocomposite. This technique is a simple method for examining the viability of cells. It can be confirmed that the nanocomposite has a survival rate of about 20% as compared with the positive control cells. However, the prepared nanocomposite also has a survival rate of about 70%. Therefore, it is necessary to study the method of coating the nanocomposite that does not affect the cells in the future. However, the cell survival rate of siRNA-nanocomposite is significantly lower than that of single-celled siRNA nanocomposite, and thus, the possibility of inducing cancer cell death through multicomponent nanocomposite used in this study was confirmed 17).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University <120> Nanorod-based Nanocomposite Intracellular Delivery System <130> PN130007 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> PRT <213> LHRH peptide <400> 1 His Trp Ser Tyr Lys Leu Arg Pro Lys   1 5 <210> 2 <211> 19 <212> RNA <213> siVEGF <400> 2 gaagttcatg gatgtctat 19

Claims (13)

Nanobarb-based nanocomplex intracellular delivery system comprising:
(a) a nanorod comprising a gold component portion and a nickel component portion, wherein the gold component portion and the nickel component portion are spatially separated from each other in the nanorods;
(b) a peptide that is a cell-binding molecule bound to a nickel moiety of the nanorod; the peptide binds to a receptor on the surface of the cell surface; And
(c) an siRNA that is a cargo bound to a gold component of the nanorod; And,
The peptide of (b) is selected from the group consisting of Luteinizing Hormone-Releasing Hormone (LHR) peptide, folic acid, RGD peptide, chlorotoxin peptide, Erb B peptide, EGF (Endothelial Growth Factor) , Adrenomedullin (ADM) peptides, AMR (Autocrine Motility Factor) peptides, amphiprienetic peptides, APRIL peptides, artemin peptides, betacyclin peptides, bombesin peptides, CTR (calcitonin receptor) peptides, endothelin Peptide, erythropoietin peptide, FGF (Fibroblast Growth Factor) peptide, FSH (Follicle Stimulating Hormone) peptide, gastrin peptide, gastrin releasing peptide (GRP), glial-derived neurotrophic factor (GDNF) peptide, ghrelin peptide, growth hormone releasing hormone ) Peptide, Granulocyte Colony Stimulating Factor (G-CSF) peptide, growth hormone peptide, Heparin Bin (leukemia inhibitory factor) peptide, alpha-melanotropin peptide, MGSA (melanoma, melanoma, melanoma, melanoma, etc.) peptide, Growth Stimulatory Activity / GRO peptide, NRG peptide, oxytocin peptide, osteopontin peptide, neuretin-1 peptide, VIP (Vasoactive Intestinal Peptide), PACAP (Pituitary Adenylate Cyclase-Activating Peptide), PDGF (TGF) peptide, TGF (Transforming Growth Factor) peptide, VEGF (TGF-β) peptide, progesterone peptide, progesterone peptide, peptidic peptide, Vascular Endothelial Growth Factor) peptide, vasopressin peptide, angiopoietin peptide, B-CLL (B-cell Chronic Lymphocytic Leukemia) peptide, a BCGF Growth Factor-12KD peptide, BAFF (B-cell Activating Factor) peptide, GalNAn peptide, GDNF peptide, GnRH peptide, Insulin-like Growth Factor- II peptide, Neurotropic Peptide, a SP (Substance P) peptide, a TGF- alpha peptide, an IGF peptide, a CXC peptide, or a CC (chemokine ligand) peptide.
delete delete delete 2. The nanodevice-based nanocomposite cell of claim 1, wherein the peptide is a luteinizing hormone-releasing hormone peptide.
2. The method of claim 1, wherein the cell-binding molecule or cargo is bound to a functional group for binding the nanorods, the functional group for binding nanorods is an amino acid comprising an SH group or an immidazole group, 20 &lt; / RTI &gt; of the nanoparticles are connected in series.
7. The nanodevice-based nanocomposite cell of claim 6, wherein the binding is directly or indirectly coupled to a cell-binding molecule or a functional group for binding cargo and nanorod.
delete The nano bar-based nanocomposite cell carrier according to claim 1, wherein the siRNA is siRNA for a cancer cell specific expression protein.
10. The method of claim 9, wherein the cancer cell specific expression protein is selected from the group consisting of VEGF (Vascular Ephthalial Growth Factors), EGFR (Epidermal Growth Factor Receptors), estrogen receptors, progesterone receptors, fibrin, fibrinogen, HER2 (human epidermal growth factor 2) ), CEA (carcinoembryonic actigen), CA-125, MUC-1, Epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), abnormal product of ras, abnormal product of p53 -2-microglobulin, thioglobulin, β-human chorionic gonadotropin, uPA (urokinase plasminogen activator), PAI-1 (Plasminogen activator inhibitor), Bcl-2, cFLIP Protein), Mcl-1 (Myeloid cell leukemia-1), NR-13, centrin, TOSO (fas apoptotic inhibitory molecule 3), CPAN (caspase-activated nuclease), PED, X-linked inhibitors of Apoptosis Protein (XIAP), cIAP-1 (Cellular Inhibitor of Apoptosis Protein (B-cell lymphoma-extra large) and PED ((S) -1-phenylethanol dehydrogenase), respectively. Wherein the carrier is one or more proteins selected from the group consisting of nanoparticle-based nanocomposite cells.
8. The method of claim 7, wherein the nanorods-based nanocomposite carrier further comprises an imaging agent between the cell-binding molecule or functional group for cargo and nanorod binding, Wherein the fluorescent dye is selected from the group consisting of fluorescein, FITC, rhodamine, TAMRA, Oregon green, eosin, Texas red, cyanine, But are not limited to, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, benzodiadiazole, benzodxadiazole, cascade blue, nile red, nile blue, cresyl violet, oxazine 170, proflavin, Acridine, orange, acry At least one member selected from the group consisting of acridine yellow, auramine, crystal violet, malachite green, porphin, phtalocyanine and bilirubin Nanoparticle-based nanocomposite cell carrier characterized by fluorescence.
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