COLON CANCER-SPECIFIC TARGET EXOSOME COMPOSITION AS DRUG DELIVERY MATERIAL, AND USE THEREOF

Abstract

The present invention relates to: a pharmaceutical composition comprising extracellular vesicles transformed with a recombinant expression vector in which a gene expressing a ligand that binds to receptors of colorectal cancer cells is inserted into a gene that is expressed in extracellular vesicles of stem cells; and a method for preparing an expression vector for preventing or treating colorectal cancer. The present invention has an advantage in that exosomes, as extracellular vesicles, can be utilized as a drug delivery material and target and specifically act on colorectal cancer by selectively binding to colorectal cancer cells which are target cells.

Claims

1. A method for treating colorectal cancer, comprising a step of administering a composition comprising extracellular vesicles transformed with a recombinant expression vector in which a gene expressing a ligand that binds to receptors of colorectal cancer cells is inserted into a gene that is expressed in extracellular vesicles of stem cells.

2. The method of claim 1, wherein the ligand that binds to receptors of colorectal cells is represented by the nucleic acid sequence of SEQ ID NO:1.

3. The method of claim 1, wherein the extracellular vesicles are exosomes.

4. The method of claim 3, wherein the exosomes are transfected with a nucleic acid capable of suppressing the expression of a gene inducing colorectal cancer.

5. The method of claim 4, wherein the nucleic acid is one or more nucleic acids selected from the group consisting of miRNA, siRNA, antisense RNA and ribozymes.

6. The method of claim 3, wherein the exosomes are included at a concentration of 1 to 50 ?L/ml

7. The method of claim 1, wherein the stem cells are bone marrow-derived stem cells, cord blood-derived stem cells or adipose-derived stem cells.

8. The method of claim 7, wherein the bone-marrow derived stem cells, cord blood-derived stem cells or adipose-derived stem cells are human- or animal-derived stem cells.

9. A method for preparing a pharmaceutical composition for treating colorectal cancer, the method comprising: (a) introducing a gene expressing a ligand that binds to receptors of colorectal cancer cells into a gene that is expressed in extracellular vesicles of adipose-derived stem cells; (b) obtaining extracellular vesicles by culturing the gene-introduced cells in a medium; and (c) transfecting the resulting extracellular vesicles with a nucleic acid capable of suppressing the expression of a gene inducing colorectal cancer.

10. The method of claim 9, wherein the extracellular vesicles are exosomes.

11. A pharmaceutical composition for treating colorectal cancer, comprising extracellular vesicles transformed with a recombinant expression vector in which a gene expressing a ligand that binds to receptors of colorectal cancer cells is inserted into a gene that is expressed in extracellular vesicles of stem cells.

Description

DESCRIPTION OF DRAWINGS

[0030] FIG. 1 is a schematic view illustrating how exosomes bind to cancer cells, which are target cells, by expressing a targeting ligand in exosomes through an expression vector. Upon treatment with exosomes targeting colorectal cancer, RNAs that suppress oncogenes can be inserted into the exosomes to be efficiently delivered to colorectal cancer cells rather than normal cells, thereby suppressing colorectal cancer cells.

[0031] FIG. 2A illustrates the vector configuration in pDisplay for the expression of the recombinant protein of the present invention, and FIG. 2B illustrates the pattern according to the expression of the vector.

[0032] FIG. 3 illustrates the results of analysis using transmission electron microscopy (TEM) and Western blot for the structural analysis of exosomes.

[0033] FIG. 4 illustrates the results of real-time PCR analysis of HCT29, which is a colorectal cancer cell line. The vertical axis of the graph means relative density.

[0034] FIG. 5 illustrates the results showing cell migration according to the wound healing assay of exosomes.

[0035] FIG. 6 illustrates the expression levels of E-cad, snail, and PIN1, which are factors involved in the epithelial to mesenchymal transition (EMT) mechanism, in HCT116, which is a colorectal cancer cell line.

[0036] FIG. 7 illustrates the expression levels of E-cad, N-cad, vimentin, snail, and PIN1, which are factors involved in the epithelial to mesenchymal transition (EMT) mechanism, in a colorectal cancer animal model in which HCT116, a colorectal cancer cell line, is transplanted into a BALB/C nude mouse.

[0037] FIG. 8 illustrates the appearance observation results of a colorectal cancer animal model constructed by transplanting HCT116, a colorectal cancer cell line, into a BALB/C nude mouse.

[0038] FIG. 9 illustrates the tumor volume and body weight in a colorectal cancer animal model constructed by transplanting the colorectal cancer cell line HCT116 into a BALB/C nude mouse.

MODES OF THE INVENTION

[0039] Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Production and Confirmation of Target Exosomes Derived from Adipose-Derived Stem Cells

[0040] Exosomes were extracted using adipose-derived stem cells to produce target exosomes that specifically act on colorectal cancer according to the present invention. For reference, in the present invention, adipose-derived stem cells were used as donor cells for producing target exosomes, but are not limited thereto because immune cells such as NK cells can also be used.

[0041] Adipose-derived stem cells were transduced with a base sequence encoding a colorectal cancer cell targeting ligand (ACG TGG TAT AAA ATC GCG TTT CAG CGC AAC CGA AAA) in a pDisplay vector (Invitrogen) using a transfection reagent (Lipofectamine, Invitrogen) according to the manufacturer's instructions. After cloning the vector, a cloned result of the corresponding vector was confirmed (FIG. 2), and target exosomes were produced by inserting the vector into adipose-derived stem cells.

[0042] To extract the produced target exosomes, adipose-derived stem cells were cultured in a DMEM low glucose medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) under conditions of 37? ? C. and 5% CO.sub.2.

[0043] After 24 hours, the medium was exchanged with an FBS-free DMEM low glucose medium, and after 24 hours after the medium exchange, the adipose-derived stem cell culture medium was harvested and centrifuged under 1000 rpm conditions by differential centrifugation to remove the cells.

[0044] Colorectal cancer target exosomes were obtained from the culture medium from which the cells were removed using a thermo exosome extraction kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0045] Here, the target exosomes obtained through the above process were transfected with PIN-siRNA using an Exo-Fect kit (SBI System Biosciences), and through this, exosomes that can target colorectal cancer receptors were additionally produced.

Example 2: Characterization of Target Exosomes Derived from Adipose-Derived Stem Cells

[0046] Transmission electron microscopy (TEM) and Western blot were used to analyze the structure of the target exosomes that specifically act on colorectal cancer according to the present invention. The TEM analysis and Western blot results for exosomes are illustrated in FIG. 3.

[0047] It could be confirmed that the exosomes isolated from the adipose-derived stem cell culture medium had a nanometer-sized spherical structure. In addition, as the expression of exosome surface markers CD81 and CD9 was confirmed using Western blot, it can be seen that the isolated exosomes have typical exosome characteristics (FIG. 3).

Example 3. Real-Time PCR Analysis of Target Exosomes Derived from Adipose-Derived Stem Cells

[0048] In order to confirm the difference in expression by colorectal cancer cell line, genes to be inserted into colorectal cancer cell lines and colorectal cancer target exosomes were selected. HCT116 and HCT29 were selected as colorectal cancer cell lines, and PIN1, which is known to promote EMT in colorectal cancer, was selected as a gene to be inserted into target exosomes. Meanwhile, cancer cells are known to undergo migration and infiltration by transforming epithelial cells characteristics into those of mesenchymal cells through the process of the epithelial to mesenchymal transition (EMT).

[0049] For real-time PCR analysis, 10 ng of RNA was converted into a first cDNA strand using specific primer using a cDNA kit (TOYOBO), and then using SYBR green (TOYOBO), real-time PCR was performed using a thermo PCR machine under the conditions of: 95?C for 20 sec, 95?C for 10 min (1cycle), 95? C. for 10 sec, 60? ? C. for 1 min (40 cycles), 95? C. for 15 sec, 60?C for 1 min, 95? C. for 15 sec (1cycle), and then the results were observed (FIG. 4).

[0050] As a result, as illustrated in FIG. 4, it could be confirmed that the relative density values of EMT cells (MET1) and PIN1 tended to significantly decrease in HCT29 as a colorectal cancer cell line.

Example 4. Evaluation of Tumorigenesis-Suppressing Ability of Target Exosomes Derived from Adipose-Derived Stem Cells

[0051] In order to observe the metastatic potential of the cells, a HCT116 colorectal cancer cell line was treated to perform wound healing assay. First, exosomes (TK-CT) as a control, target exosomes (TK), PIN-siRNA-loaded (siPIN) exosomes (siPIN TK-CT) and PIN-siRNA-loaded (siPIN) target exosomes (siPIN TK) were prepared.

[0052] As a result of the experiment, it could be confirmed that when wound healing assay (24H and 48H) was performed, cell migration was suppressed in the PIN-siRNA-loaded (siPIN) exosomes (siPIN TK-CT) and the PIN-siRNA-loaded (siPIN) target exosomes (siPIN TK) (FIG. 5).

Example 5. Efficacy Evaluation of Adipose-Derived Stem Cell-Derived Target Exosomes in Colorectal Cancer Cell Line HCT116

[0053] In order to evaluate the anti-cancer efficacy of colorectal cancer-targeting exosomes derived from adipose-derived stem cells cloned with the recombinant vector according to the present invention compared to exosomes derived from adipose-derived stem cells, HCT116, which is a colorectal cancer cell line, was treated with exosomes (TK-CT) as a control, target exosomes (TK), PIN-siRNA-loaded (si-pin) exosomes (si-pin TK-CT) and PIN-siRNA-loaded (si-pin) target exosomes (si-pin TK). Changes were observed by comparing the expression levels of E-cad, snail and PIN1, which are factors involved in the epithelial to mesenchymal transition (EMT) mechanism (FIG. 6).

[0054] As a result, as illustrated in FIG. 6, it could be confirmed that in PIN-siRNA-loaded (si-pin) exosomes (si-pin TK-CT) and PIN-siRNA-loaded (si-pin) target exosomes (si-pin TK), the expression levels of E-cad was increased and the expression levels of Snail and PIN1 were decreased to suppress EMT.

Example 6. Efficacy Evaluation of Target Exosomes Derived from Adipose-Derived Stem Cells Using Colorectal Cancer Animal Model Following Transplantation of Colorectal Cancer Cell Line HCT116

[0055] After colorectal cancer was induced by transplanting HCT116, a colorectal cancer cell line, into a BALB/C nude mouse (in vivo) at 5?10.sup.5, the present inventors attempted to confirm the efficacy of treating colorectal cancer using exosomes derived from adipose-derived stem cells.

[0056] After each of exosomes (tk-ct) as a control, target exosomes (tk), PIN-siRNA-loaded (si-PIN1) exosomes (si-PIN1tk-ct) and PIN-siRNA-loaded (si-PIN1) target exosomes (si-PIN1tk) was injected into the tumor model constructed above by a tail vein injection method for 2 weeks, mice were sacrificed to confirm the effect of the exosomes on the anti-tumor effect for colorectal cancer.

[0057] Changes were observed by comparing the expression levels of E-cad, N-cad, vimentin, snail and PIN1, which are factors involved in the epithelial to mesenchymal transition (EMT) mechanism (FIG. 7). As a result, as illustrated in FIG. 7, it could be confirmed that in PIN-siRNA-loaded (si-PIN1) exosomes (si-PIN1 tk-ct) and PIN-siRNA-loaded (si-PIN1) target exosomes (si-PIN1 tk), the expression levels of E-cad was increased and the expression levels of N-CAD, vimentin, snail and PIN1 were decreased to suppress EMT.

[0058] Furthermore, even by the appearance observation results in the colorectal cancer animal model constructed by transplanting the colorectal cancer cell line HCT116 into the BALB/C nude mouse (in vivo) at 5?10.sup.5, as illustrated in FIGS. 8 and 9, it was confirmed that the tumor size tended to be suppressed in the group treated with PIN-siRNA-loaded (siPIN) exosomes (TK-CT-Pin) and PIN-siRNA-loaded (siPIN) target exosomes (TK-Pin) (FIGS. 8 and 9).

[0059] The above-described description of the present invention is provided for illustrative purposes, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are only exemplary in all aspects and are not restrictive.